FUS-mediated BBB opening leads to transient perfusion decrease and inflammation without acute or chronic brain lesion.

Impact: The permeabilization of the BBB to deliver therapeutics with MR-guided FUS redefines therapeutic strategies as it improves patient outcomes. To ensure the best translation towards clinical treatment, the evaluation of hemodynamic modifications in the CNS is necessary to refine treatment parameters. Methods: MR-guided FUS was applied at 1.5 MHz with a 50 ms burst every 1 s to open the BBB. CBF, BVf and ADC parameters were monitored with MRI. Cavitation was monitored with a PCD during the FUS sequence and classified with the IUD index into three cavitation levels. We distinctly applied the FUS in the cortex or the striatum. After the BBB permeabilization, neuroinflammation markers were quantified longitudinally. Results: The BBB was successfully opened in all animals in this study and only one animal was classified as "hard" and excluded from the rest of the study. 30 min after FUS-induced BBB opening in the cortex, we measured a 54% drop in CBF and a 13% drop in BVf compared to the contralateral side. After permeabilization of the striatum, a 38% drop in CBF and a 15% drop in BVf were measured. CBF values rapidly returned to baseline, and 90 min after BBB opening, no significant differences were observed. We quantified the subsequent neuroinflammation, noting a significant increase in astrocytic recruitment at 2 days and microglial activation at 1 day after FUS. After 7 days, no more inflammation was visible in the brain. Conclusion: FUS-induced BBB opening transiently modifies hemodynamic parameters such as CBF and BVf, suggesting limited nutrients and oxygen supply to the CNS in the hour following the procedure.

Small, Fluorinated Mn2+ Chelate as an Efficient 1H and 19F MRI Probe.

We explore the potential of fluorine-containing small Mn2+ chelates as alternatives to perfluorinated nanoparticles, widely used as 19F MRI probes. In MnL1, the cyclohexanediamine skeleton and two piperidine rings, involving each a metal-coordinating amide group and an appended CF3 moiety, provide high rigidity to the complex. This allows for good control of the Mn-F distance (rMnF = 8.2±0.2 Å determined from 19F relaxation data), as well as for high kinetic inertness (a dissociation half-life of 1285 h is estimated for physiological conditions). The paramagnetic Mn2+ leads to a ~150-fold acceleration of the longitudinal 19F relaxation, with moderate line-broadening effect, resulting in T2/T1 ratios of 0.8 (9.4 T). Owing to its inner sphere water molecule, MnL1 is a good 1H relaxation agent as well (r1 = 5.36 mM-1s-1 at 298K, 20MHz). MnL1 could be readily visualized in 19F MRI by using fast acquisition techniques, both in phantom images and living mice following intramuscular injection, with remarkable signal-to-noise ratios and short acquisition times. While applications in targeted imaging or cell therapy monitoring require further optimisation of the molecular structure, these results argue for the potential of such small, monohydrated and fluorinated Mn2+ complexes for combined 19F and 1H MRI detection.

Messenger RNA Lipid-Based Nanoparticles: Optimization of Formulations in the Lab.

Among the large variety of messenger RNA (mRNA) delivery systems, those developed with lipid-based formulations were the most widely used and efficient. In our lab, we produced different mRNA formulations made with liposomes, hybrid lipid polymer, and lipid nanoparticles. Our formulations were made with lipids bearing imidazole groups that trigger the endosomal escape of nanoparticles once protonated inside the mild acidic milieu of endosomes upon their cell uptake. Herein, we describe protocols that we used to produce, optimize, and characterize those formulations. The transfection efficiency is influenced by various factors including the physicochemical parameters of the nanoparticles, their efficiency to be internalized in cells, and their intracellular routing as well as their capacity to induce immune system sensors. We provide details on how to quantify the amount of mRNA nanoparticles uptake by cells and evaluate the acidity of the intracellular compartments where they are located, to investigate the endosomal escape, and to assess the activation of innate immune sensors as phosphorylation of PKR hampering mRNA translation.

Therapeutic Vaccines for HPV-Associated Cervical Malignancies: A Systematic Review.

IMPORTANCE: Despite widespread prophylactic vaccination, cervical cancer continues to be a major health problem with considerable mortality. Currently, therapeutic vaccines for HPV-associated cervical malignancies are being evaluated as a potential complement to the standard treatment. OBJECTIVE: The present systematic review was conducted on randomized controlled trials (RCTs) to investigate the effects of therapeutic vaccines on the treatment of patients with cervical cancer and cervical intraepithelial neoplasia (CIN) of Grades 2 and 3. EVIDENCE REVIEW: The PubMed, Embase, and Cochrane Central Register of Controlled Trials databases were searched. Only articles in English published up until 31 January 2024 were selected. Also, reference lists of the selected original papers and recent review articles were manually searched for additional sources. Data on study characteristics were extracted from the selected articles. Data on outcomes of interest were synthesized, and vaccine efficacy endpoints (histological lesion regression, clinical response, and overall survival) were selected as the basis for grouping the studies. FINDINGS: After screening 831 articles, nine RCTs with 800 participants were included, of which seven studies with 677 participants involved CIN2 and CIN3 and examined lesion regression to ≤CIN1 as the efficacy endpoint. Results of two of these studies were deemed to have a high risk of bias, and another one did not contain statistical analyses. Results of the other four studies were quantitively synthesized, and the pooling of p-values revealed a significant difference between the vaccine and placebo groups in terms of lesion regression (p-values of 0.135, 0.049, and 0.034 in RCTs, yielding a combined p-value of 0.010). The certainty of the evidence was rated as moderate. Patients with advanced cervical cancers were studied in two RCTs with 123 participants. Clinical response and overall survival were taken as endpoints, and the results were reported as not significant. The certainty of the evidence of these results was rated as very low, mainly due to the very small number of events. All studies reported good tolerance for the vaccines. CONCLUSIONS AND RELEVANCE: The results indicate the potential for therapeutic vaccines in the regression of CIN2 and CIN3 lesions. Moreover, a potential gap in evidence is identified regarding the very low number of RCTs in patients with advanced cervical cancer.

Neoantigen vaccine nanoformulations based on Chemically synthesized minimal mRNA (CmRNA): small molecules, big impact.

Recently, chemically synthesized minimal mRNA (CmRNA) has emerged as a promising alternative to in vitro transcribed mRNA (IVT-mRNA) for cancer therapy and immunotherapy. CmRNA lacking the untranslated regions and polyadenylation exhibits enhanced stability and efficiency. Encapsulation of CmRNA within lipid-polymer hybrid nanoparticles (LPPs) offers an effective approach for personalized neoantigen mRNA vaccines with improved control over tumor growth. LPP-based delivery systems provide superior pharmacokinetics, stability, and lower toxicity compared to viral vectors, naked mRNA, or lipid nanoparticles that are commonly used for mRNA delivery. Precise customization of LPPs in terms of size, surface charge, and composition allows for optimized cellular uptake, target specificity, and immune stimulation. CmRNA-encoded neo-antigens demonstrate high translational efficiency, enabling immune recognition by CD8+ T cells upon processing and presentation. This perspective highlights the potential benefits, challenges, and future directions of CmRNA neoantigen vaccines in cancer therapy compared to Circular RNAs and IVT-mRNA. Further research is needed to optimize vaccine design, delivery, and safety assessment in clinical trials. Nevertheless, personalized LPP-CmRNA vaccines hold great potential for advancing cancer immunotherapy, paving the way for personalized medicine.

Impact of net charge, targeting ligand amount and mRNA modification on the uptake, intracellular routing and the transfection efficiency of mRNA lipopolyplexes in dendritic cells.

Targeting mRNA formulations to achieve cell specificity is one of the challenges that must be tackled to mettle their therapeutic potential. Here, lipopolyplexes (LPR) bearing tri-mannose-lipid (TM) are used to target mannose receptor on dendritic cells. We investigated the impact of the net charge and percentage of TM units on the binding, uptake, transfection efficiency (TE) and RNA sensors activation. Binding and uptake capacities of naked and targeted LPR increase with the percent of cationic lipid, but the latter are 2-fold more up taken by the cells. Cationic LPR bearing 5 % and 10 % TM were localized in acidic compartments in contrast to naked LPR and 2.5 % TM-LPR. The drawback is the dramatic decrease of TE as the number of TM-units increases. Cationic LPR bearing 5 % and 10 % TM strongly induced NF-κB and PKR phosphorylation at 6 h. Conversely, mTOR is less activated in line with their low TE. Those side effects are overcome by using 5-methoxyuridine mRNA resulting in an improved TE due to non-phosphorylation of NF-κB and PKR and mTOR activation. Our results point out that targeting DC via mannose receptor triggers a higher uptake of cationic LPRs and fast routing to acidic compartments, and that efficient TE requires low number of TM units use or modified mRNA to escape RNA sensors activation to enhance the translation.

Murine Breast Cancer Radiosensitization Using Oxygen Microbubbles and Metformin: Vessels Are the Key.

Radiotherapy is a cornerstone of cancer treatment, but tumor hypoxia and resistance to radiation remain significant challenges. Vascular normalization has emerged as a strategy to improve oxygenation and enhance therapeutic outcomes. In this study, we examine the radiosensitization potential of vascular normalization using metformin, a widely used anti-diabetic drug, and oxygen microbubbles (OMBs). We investigated the synergistic action of metformin and OMBs and the impact of this therapeutic combination on the vasculature, oxygenation, invasiveness, and radiosensitivity of murine 4T1 breast cancer. We employed in vivo Doppler ultrasonographic imaging for vasculature analysis, electron paramagnetic resonance oximetry, and immunohistochemical assessment of microvessels, perfusion, and invasiveness markers. Our findings demonstrate that both two-week metformin therapy and oxygen microbubble treatment normalize abnormal cancer vasculature. The combination of metformin and OMB yielded more pronounced and sustained effects than either treatment alone. The investigated therapy protocols led to nearly twice the radiosensitivity of 4T1 tumors; however, no significant differences in radiosensitivity were observed between the various treatment groups. Despite these improvements, resistance to treatment inevitably emerged, leading to the recurrence of hypoxia and an increased incidence of metastasis.

Recent Advances Using Genetic Therapies Against Infectious Diseases and for Vaccination.

The development of prophylatic or therapeutic medicines for infectious diseases is one of the priorities for health organizations worldwide. Innovative solutions are required to achieve effective, safe, and accessible treatments for most if not all infectious diseases, particularly those that are chronic in nature or that emerge unexpectedly over time. Genetic technologies offer versatile possibilities to design therapies against pathogens. Recent developments such as mRNA vaccines, CRISPR gene editing, and immunotherapies provide unprecedented hope to achieve significant results in the field of infectious diseases. This review will focus on advances in this domain, showcasing the cross-fertilization with other fields (e.g., oncology), and addressing some of the logistical and economic concerns important to consider when making these advances accessible to diverse populations around the world.

Two-Photon Light Trigger siRNA Transfection of Cancer Cells Using Non-Toxic Porous Silicon Nanoparticles.

The concept of using two-photon excitation in the NIR for the spatiotemporal control of biological processes holds great promise. However, its use for the delivery of nucleic acids has been very scarcely described and the reported procedures are not optimal as they often involve potentially toxic materials and irradiation conditions. This work prepares a simple system made of biocompatible porous silicon nanoparticles (pSiNP) for the safe siRNA photocontrolled delivery and gene silencing in cells upon two-photon excitation. PSiNP are linked to an azobenzene moiety, which possesses a lysine group (pSiNP@ICPES-azo@Lys) to efficiently complex siRNA. Non-linear excitation of the two-photon absorber system (pSiNP) followed by intermolecular energy transfer (FRET) to trans azobenzene moiety, result in the photoisomerization of the azobenzene from trans to cis and in the destabilization of the azobenzene-siRNA complex, thus inducing the delivery of the cargo siRNA to the cytoplasm of cells. Efficient silencing in MCF-7 expressing stable firefly luciferase with siRNAluc against luciferase is observed. Furthermore, siRNA against inhibitory apoptotic protein (IAP) leads to over 70% of MCF-7 cancer cell death. The developed technique using two-photon light allows a unique high spatiotemporally controlled and safe siRNA delivery in cells in few seconds of irradiation.

CFTR and dystrophin encoding plasmids carrying both luciferase reporter gene, nuclear import specific sequences and triple helix sites.

Duchenne Muscular Dystrophy and Cystic Fibrosis are two major monogenetic diseases which could be treated by non-viral gene therapy. For this purpose, plasmid DNA (pDNA) coding for the functional genes requires its equipment with signal molecules favouring its intracellular trafficking and delivery in the nucleus of the target cells. Here, two novel constructions of large pDNAs encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and full-length dystrophin (DYS) genes are reported. The expression of CFTR and DYS genes are driven respectively by the hCEF1 airway epithelial cells and spc5-12 muscle cells specific promoter. Those pDNAs encode also the luciferase reporter gene driven by the CMV promoter to evaluate gene delivery in animals by bioluminescence. In addition, oligopurine • oligopyrimidine sequences are inserted to enable equipment of pDNAs with peptides conjugated with a triple helix forming oligonucleotide (TFO). Furthermore, specific κB sequences are also inserted to promote their NFκB-mediated nuclear import. pDNA constructions are reported; transfection efficiency, tissue specific expression of CFTR and dystrophin in target cells, and triple helix formation are demonstrated. These plasmids are tools of interest to develop non-viral gene therapy of Cystic Fibrosis and Duchenne Muscular Dystrophy.

Intrahippocampal Inoculation of Aß1-42 Peptide in Rat as a Model of Alzheimer's Disease Identified MicroRNA-146a-5p as Blood Marker with Anti-Inflammatory Function in Astrocyte Cells.

Circulating microRNAs (miRNAs) have aroused a lot of interest as reliable blood diagnostic biomarkers of Alzheimer's disease (AD). Here, we investigated the panel of expressed blood miRNAs in response to aggregated Aß1-42 peptides infused in the hippocampus of adult rats to mimic events of the early onset of non-familial AD disorder. Aß1-42 peptides in the hippocampus led to cognitive impairments associated with an astrogliosis and downregulation of circulating miRNA-146a-5p, -29a-3p, -29c-3p, -125b-5p, and-191-5p. We established the kinetics of expression of selected miRNAs and found differences with those detected in the APPswe/PS1dE9 transgenic mouse model. Of note, miRNA-146a-5p was exclusively dysregulated in the Aß-induced AD model. The treatment of primary astrocytes with Aß1-42 peptides led to miRNA-146a-5p upregulation though the activation of the NF-κB signaling pathway, which in turn downregulated IRAK-1 but not TRAF-6 expression. As a consequence, no induction of IL-1ß, IL-6, or TNF-α was detected. Astrocytes treated with a miRNA-146-5p inhibitor rescued IRAK-1 and changed TRAF-6 steady-state levels that correlated with the induction of IL-6, IL-1ß, and CXCL1 production, indicating that miRNA-146a-5p operates anti-inflammatory functions through a NF-κB pathway negative feedback loop. Overall, we report a panel of circulating miRNAs that correlated with Aß1-42 peptides' presence in the hippocampus and provide mechanistic insights into miRNA-146a-5p biological function in the development of the early stage of sporadic AD.

Ultrasound sensitive O2 microbubbles radiosensitize murine breast cancer but lead to higher metastatic spread.

The inadequate level of oxygenation in tumors has been shown to correlate not only with greater invasiveness of cancer cells, but also with a reduction in their sensitivity to anticancer therapies. Over the years, many attempts have been made to increase the oxygenation level of cancer, but most of them have been ineffective. We investigated the heterogeneous response of tumor tissue to phospholipid-coated oxygen microbubbles (OMB) in murine tumors in vivo using oxygen and hemoglobin saturation mapping and the influence of OMB treatment on microvasculature, perfusion, and radiotherapy effectiveness. Intravenous administration of OMB followed by ultrasound pulse leads to increased oxygenation of a tumor, found mainly in the vicinity of tumor vessels, while intratumoral delivery resulted in areas of increased pO2 more evenly distributed within the tumor. Furthermore, hemoglobin contributes little to the increase in tumor oxygenation caused by oxygen microbubbles. Extensive vasculature disruption was observed in the groups treated with both oxygen/nitrogen microbubbles and ultrasound pulse. This therapy also led to a reduction in the coverage of the vessels by pericytes, while the density of the microvessels was unchanged. Radiotherapy with a single dose of 12Gy reduced tumor growth by 50% in all treated groups. Unfortunately, at the same time, the number of macroscopic metastases in the lungs increased significantly after intravenous administration of oxygen/nitrogen microbubbles and the application of an ultrasound pulse. In conclusion, ultrasound-sensitive oxygen microbubbles are effective in delivering oxygen to tumor tissue, thus increasing the effectiveness of radiotherapy. However, cavitation effects and destruction of the integrity of tumor vessels result in greater spread of cancer cells in the host organism.

Targeting TGF-ß1/miR-21 Pathway in Keratinocytes Reveals Protective Effects of Silymarin on Imiquimod-Induced Psoriasis Mouse Model.

Epidermal cells integrate multiple signals that activate the signaling pathways involved in skin homeostasis. TGF-ß1 signaling pathway upregulates microRNA (miR)-21-5p in keratinocytes and is often deregulated in skin diseases. To identify the bioactive compounds that enable to modulate the TGF-ß1/miR-21-5p signaling pathway, we screened a library of medicinal plant extracts using our miR-ON RILES luciferase reporter system placed under the control of the miR-21-5p in keratinocytes treated with TGF-ß1. We identified silymarin, a mixture of flavonolignans extracted from Silybum marianum (L.) Gaertn., as the most potent regulator of miR-21-5p expression. Using Argonaute 2 immunoprecipitation and RT-qPCR, we showed that silymarin regulates the expression of miR-21-5p through a noncanonical TGF-ß1 signaling pathway, whereas RNA-sequencing analysis revealed three unexpected transcriptomic signatures associated with keratinocyte differentiation, cell cycle, and lipid metabolism. Mechanistically, we demonstrated that SM blocks cell cycle progression, inhibits keratinocyte differentiation through repression of Notch3 expression, stimulates lipid synthesis via activation of PPARγ signaling and inhibits inflammatory responses by suppressing the transcriptional activity of NF-κB. We finally showed that topical application of silymarin alleviates the development of imiquimod-induced psoriasiform lesions in mice by abrogating the altered expression levels of markers involved in inflammation, proliferation, differentiation, and lipid metabolism.

Reversible stabilization of DNA/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity.

Nonviral transfection of mammalian cells can be performed with electrostatic complexes (polyplexes) between a plasmid DNA (pDNA) encoding a foreign gene and a cationic polymer. However, an excess of the cationic polymer is required for pDNA condensation and polyplexes formation, which generate in vivo toxicity. Here, we present a new concept of polyplexes preparation aiming to reduce the polymer quantity. pDNA was functionalized with 3,6,9-trioxaundecan-1- {4 - [(2-chloroethyl) ethylamino)] - benzylamino}, 11-azide, and polyethyleneimine (lPEI) with reducible dibenzocyclooctyl (SS-DBCO) groups allowing azide-alkyne cycloaddition between pDNA and lPEI after condensation. The size of polyplexes with DBCO-SS-lPEI was smaller than with lPEI due to a stronger DNA condensation thanks to linkages between polymer and pDNA preventing dissociation until disulfide bridges reduction. In vitro transfection showed that the amount of DBCO-SS-lPEI leading to the most efficient polyplexes was three times lower than lPEI. As expected, toxicity in mice was significantly reduced upon intravenous injection of DBCO-SS-lPEI polyplexes at doses where the lPEI polyplexes killed mice. This is probably due to the high stability of the DBCO-SS-lPEI polyplexes which prevented their aggregation in the pulmonary capillaries. Overall, this new concept of polyplexes with DBCO-SS-lPEI offering the possibility of administering higher doses of polyplexes than lPEI and their ability to pass the pulmonary barrier could be favorably exploited for transfection of distant organs or tissues, such as tumors.

Selective attachment of a microtubule interacting peptide to plasmid DNA via a triplex forming oligonucleotide for transfection improvement.

In nonviral gene therapy approaches, the linkage of signal molecules to plasmid DNA (pDNA) is of interest for guiding its delivery to the nucleus. Here, we report its linkage to a peptide (P79-98) mediating migration on microtubules by using a triplex-forming oligonucleotide (TFO). pDNA of 5 kbp and 21 kbp containing 6 and 36 oligopurine • oligopyrimidine sites (TH), respectively, inserted outside the luciferase gene sequence were used. TFO with a dibenzocyclooctyl (DBCO) group in 3' end comprising some Bridged Nucleic Acid bases was conjugated by click chemistry with the peptide carrying an azide function in the C-terminal end. We found the formation of 6 and 18 triplex with pDNA of 5 kbp and 21 kbp, respectively. A twofold increase of the transfection efficiency was observed in the hind-limbs upon Hydrodynamic Limb Vein (HLV) injection in mice of naked P79-98 -pDNA of 21 kbp. This work paves the way for the selective equipping of pDNA with intracellular targeting molecules while preserving the full expression of the encoded gene.

Increasing oxygen tension in tumor tissue using ultrasound sensitive O2 microbubbles.

Low tissue oxygenation significantly impairs the effectiveness of cancer therapy and promotes a more aggressive phenotype. Many strategies to improve tissue oxygenation have been proposed throughout the years, but only a few showed significant effects in clinical settings. We investigated stability and ultrasound pulse (UP) triggered oxygen release from phospholipid coated oxygen microbubbles (OMB) in vitro and in murine tumors in vivo using EPR oximetry. In solution, the investigated microbubbles are stable and responsive to ultrasound pulse. The addition of the OMB solution alone resulted in an increase in pO2 of approximately 70 mmHg which was further increased for an additional 80 mmHg after the application of UP. The in vivo kinetic study revealed a substantial, up to 120 mmHg, increase in tumor pO2 after UP application and then pO2 was decreasing for 20 min for intravenous injection and 15 min for intratumoral injection. A significant increase was also observed in groups that received microbubbles filled with nitrogen and ultrasound pulse and OMB without UP, but the effect was much lower. Oxygen microbubbles lead to a decrease in HIF-1a and VEGF-A both at the level of mRNA and protein. Toxicity analysis showed that intravenous injection of OMB does not cause oxidative damage to the heart, liver, or kidneys. However, elevated levels of oxidative damage to lipids and proteins were observed short-term in tumor tissue. In conclusion, we have demonstrated the feasibility of oxygen microbubbles in delivering oxygen effectively and safely to the tumor in living animals. Such treatment might enhance the effectiveness of other anticancer therapies.

Role of Muscle LIM Protein in Mechanotransduction Process.

The induction of protein synthesis is crucial to counteract the deconditioning of neuromuscular system and its atrophy. In the past, hormones and cytokines acting as growth factors involved in the intracellular events of these processes have been identified, while the implications of signaling pathways associated with the anabolism/catabolism ratio in reference to the molecular mechanism of skeletal muscle hypertrophy have been recently identified. Among them, the mechanotransduction resulting from a mechanical stress applied to the cell appears increasingly interesting as a potential pathway for therapeutic intervention. At present, there is an open question regarding the type of stress to apply in order to induce anabolic events or the type of mechanical strain with respect to the possible mechanosensing and mechanotransduction processes involved in muscle cells protein synthesis. This review is focused on the muscle LIM protein (MLP), a structural and mechanosensing protein with a LIM domain, which is expressed in the sarcomere and costamere of striated muscle cells. It acts as a transcriptional cofactor during cell proliferation after its nuclear translocation during the anabolic process of differentiation and rebuilding. Moreover, we discuss the possible opportunity of stimulating this mechanotransduction process to counteract the muscle atrophy induced by anabolic versus catabolic disorders coming from the environment, aging or myopathies.

DNA minicircles as novel STAT3 decoy oligodeoxynucleotides endowed with anticancer activity in triple-negative breast cancer.

Decoy technology is a versatile and specific DNA oligonucleotide-based targeting strategy of pathogenic transcription factors (TFs). Chemical modifications of linear decoy oligonucleotides have been made to decrease nuclease sensitivity because of the presence of free ends but at the cost of new limitations that affect their use as therapeutic drugs. Although a short DNA minicircle is a phosphodiester nucleic acid without free ends, its potential therapeutic activity as a TF decoy oligonucleotide has not yet been investigated. Here we describe the in vitro and in vivo activity of formulated 95-bp minicircles bearing one or several STAT3 binding sequences in triple-negative breast cancer (TNBC). Minicircles bearing one STAT3 binding site interacted specifically with the active form of STAT3 and inhibited proliferation, induced apoptosis, slowed down cell cycle progression, and decreased STAT3 target gene expression in human and murine TNBC cells. Intratumoral injection of STAT3 minicircles inhibited tumor growth and metastasis in a murine model of TNBC. Increasing the number of STAT3 binding sites resulted in improved anticancer activity, opening the way for a TF multitargeting strategy. Our data provide the first demonstration of minicircles acting as STAT3 decoys and show that they could be an effective therapeutic drug for TNBC treatment.