704/DNA vaccines leverage cytoplasmic DNA stimulation to promote anti-HIV neutralizing antibody production in mice and strong immune response against alpha-fetoprotein in non-human primates.

Genetic immunization is an attractive approach for prophylactic and therapeutic vaccination using synthetic vectors to deliver antigen-encoding nucleic acids. Recently, DNA delivered by a physical means or RNA by liposomes consisting of four different lipids demonstrated good protection in human phase III clinical trials and received Drugs Controller General of India and US FDA approval to protect against COVID-19, respectively. However, the development of a system allowing for efficient and simple delivery of nucleic acids while improving immune response priming has the potential to unleash the full therapeutic potential of genetic immunization. DNA-based gene therapies and vaccines have the potential for rapid development, as exemplified by the recent approval of Collategene, a gene therapy to treat human critical limb ischemia, and ZyCoV, a DNA vaccine delivered by spring-powered jet injector to protect against SARS-CoV2 infection. Recently, we reported amphiphilic block copolymer 704 as a promising synthetic vector for DNA vaccination in various models of human diseases. This vector allows dose sparing of antigen-encoding plasmid DNA. Here, we report the capacity of 704-mediated HIV and anti-hepatocellular carcinoma DNA vaccines to induce the production of specific antibodies against gp120 HIV envelope proteins in mice and against alpha-fetoprotein antigen in non-human primates, respectively. An investigation of the underlying mechanisms showed that 704-mediated vaccination did trigger a strong immune response by (1) allowing a direct DNA delivery into the cytosol, (2) promoting an intracytoplasmic DNA sensing leading to both interferon and NF-κB cascade stimulation, and (3) inducing antigen expression by muscle cells and presentation by antigen-presenting cells, leading to the induction of a robust adaptive response. Overall, our findings suggest that the 704-mediated DNA vaccination platform is an attractive method to develop both prophylactic and therapeutic vaccines.

Respiratory mucosal vaccination of peptide-poloxamine-DNA nanoparticles provides complete protection against lethal SARS-CoV-2 challenge.

The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). The pSpike/PP-sNp not only displays superior gene transfection and favorable biocompatibility in the mouse airway, but also promotes a tripartite immunity consisting of systemic, cellular, and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, immunization with pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp is a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.

Two repeated motifs enriched within some enhancers and origins of replication are bound by SETMAR isoforms in human colon cells.

Setmar is a gene specific to simian genomes. The function(s) of its isoforms are poorly understood and their existence in healthy tissues remains to be validated. Here we profiled SETMAR expression and its genome-wide binding landscape in colon tissue. We found isoforms V3 and V6 in healthy and tumour colon tissues as well as incell lines. In two colorectal cell lines SETMAR binds to several thousand Hsmar1 and MADE1 terminal ends, transposons mostly located in non-genic regions of active chromatin including in enhancers. It also binds to a 12-bp motifs similar to an inner motif in Hsmar1 and MADE1 terminal ends. This motif is interspersed throughout the genome and is enriched in GC-rich regions as well as in CpG islands that contain constitutive replication origins. It is also found in enhancers other than those associated with Hsmar1 and MADE1. The role of SETMAR in the expression of genes, DNA replication and in DNA repair are discussed.

Aerosol-Mediated Non-Viral Lung Gene Therapy: The Potential of Aminoglycoside-Based Cationic Liposomes.

Aerosol lung gene therapy using non-viral delivery systems represents a credible therapeutic strategy for chronic respiratory diseases, such as cystic fibrosis (CF). Progress in CF clinical setting using the lipidic formulation GL67A has demonstrated the relevance of such a strategy while emphasizing the need for more potent gene transfer agents. In recent years, many novel non-viral gene delivery vehicles were proposed as potential alternatives to GL67 cationic lipid. However, they were usually evaluated using procedures difficult or even impossible to implement in clinical practice. In this study, a clinically-relevant administration protocol via aerosol in murine lungs was used to conduct a comparative study with GL67A. Diverse lipidic compounds were used to prepare a series of formulations inspired by the composition of GL67A. While some of these formulations were ineffective at transfecting murine lungs, others demonstrated modest-to-very-efficient activities and a series of structure-activity relationships were unveiled. Lipidic aminoglycoside derivative-based formulations were found to be at least as efficient as GL67A following aerosol delivery of a luciferase-encoding plasmid DNA. A single aerosol treatment with one such formulation was found to mediate long-term lung transgene expression, exceeding half the animal's lifetime. This study clearly supports the potential of aminoglycoside-based cationic lipids as potent GL67-alternative scaffolds for further enhanced aerosol non-viral lung gene therapy for diseases such as CF.

Lipidic Aminoglycoside Derivatives: A New Class of Immunomodulators Inducing a Potent Innate Immune Stimulation.

Development of simple and fully characterized immunomodulatory molecules is an active area of research to enhance current immunotherapies. Monophosphoryl lipid A (MPL), a nontoxic lipidic derivative from bacteria, is the first and currently only adjuvant approved in humans. However, its capacity to induce a potent response against weak immunogenic tumoral-associated antigens remains limited. Herein, a new generation of lipidic immunomodulators to conduct a structure-activity relationship study to determine the minimal structural elements conferring immunomodulatory properties is introduced. Two lead molecules characterized by a short succinyl linker between two oleyl chains and a polar headgroup consisting of either naturally occurring tobramycin (DOST) or kanamycin (DOSK) are identified. These two lipoaminoglycosides self-assemble in very small vesicles. In a wide variety of cells including 3D human cell culture, DOST and DOSK induce the upregulation of proinflammatory cytokines and interferon-inducible proteins in a dose and time-dependent manner via a caveolae-dependent proinflammatory mechanism and phosphatidylinositol phospholipase C activation. Furthermore, after intratumoral administration, these lipoaminoglycosides induce an efficient immune response leading to significant antitumor activity in a mouse breast cancer model. Altogether, these findings indicate that DOST and DOSK are two groundbreaking synthetic lipid immunostimulators that can be used as adjuvants to enhance current immunotherapeutic treatments.

Tetrafunctional Block Copolymers Promote Lung Gene Transfer in Newborn Piglets.

Tetrafunctional block copolymers are molecules capable of complexing DNA. Although ineffective in vitro, studies in mice have shown that the tetrafunctional block copolymer 704 is a more efficient lung gene transfer agent than the cationic liposome GL67A, previously used in a phase II clinical trial in cystic fibrosis patients. In the present study, we compared the gene transfer capacity of the 704-DNA formulation and a cationic liposome-DNA formulation equivalent to GL67A in a larger-animal model, the newborn piglet. Our results indicate an efficacy of the 704-DNA formulation well above one order of magnitude higher than that of the cationic liposome-DNA formulation, with no elevated levels of interleukin-6 (IL-6), taken as a marker of inflammation. Transgene expression was heterogeneous within lung lobes, with expression levels that were below the detection threshold in some samples, while high in other samples. This heterogeneity is likely to be due to the bolus injection procedure as well as to the small volume of injection. The present study highlights the potential of tetrafunctional block copolymers as non-viral vectors for lung gene therapy.

MgtC as a Host-Induced Factor and Vaccine Candidate against Mycobacterium abscessus Infection.

Mycobacterium abscessus is an emerging pathogenic mycobacterium involved in pulmonary and mucocutaneous infections, presenting a serious threat for patients with cystic fibrosis (CF). The lack of an efficient treatment regimen and the emergence of multidrug resistance in clinical isolates require the development of new therapeutic strategies against this pathogen. Reverse genetics has revealed genes that are present in M. abscessus but absent from saprophytic mycobacteria and that are potentially involved in pathogenicity. Among them, MAB_3593 encodes MgtC, a known virulence factor involved in intramacrophage survival and adaptation to Mg(2+) deprivation in several major bacterial pathogens. Here, we demonstrated a strong induction of M. abscessus MgtC at both the transcriptional and translational levels when bacteria reside inside macrophages or upon Mg(2+) deprivation. Moreover, we showed that M. abscessus MgtC was recognized by sera from M. abscessus-infected CF patients. The intramacrophage growth (J774 or THP1 cells) of a M. abscessus knockout mgtC mutant was, however, not significantly impeded. Importantly, our results indicated that inhibition of MgtC in vivo through immunization with M. abscessus mgtC DNA, formulated with a tetrafunctional amphiphilic block copolymer, exerted a protective effect against an aerosolized M. abscessus challenge in CF (ΔF508 FVB) mice. The formulated DNA immunization was likely associated with the production of specific MgtC antibodies, which may stimulate a protective effect by counteracting MgtC activity during M. abscessus infection. These results emphasize the importance of M. abscessus MgtC in vivo and provide a basis for the development of novel therapeutic tools against pulmonary M. abscessus infections in CF patients.

Liposome-based Formulation for Intracellular Delivery of Functional Proteins.

The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the ß-galactosidase (ß-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the ß-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/ß-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

Bacterial phospholipases C as vaccine candidate antigens against cystic fibrosis respiratory pathogens: the Mycobacterium abscessus model.

BACKGROUND: Vaccine strategies represent one of the fighting answers against multiresistant bacteria in a number of clinical settings like cystic fibrosis (CF). Mycobacterium abscessus, an emerging CF pathogen, raises difficult therapeutic problems due to its intrinsic antibiotic multiresistance. METHODS: By reverse vaccinology, we identified M. abscessus phospholipase C (MA-PLC) as a potential vaccine target. We deciphered here the protective response generated by vaccination with plasmid DNA encoding the MA-PLC formulated with a tetra functional block copolymer 704, in CF (ΔF508) mice. Protection was tested against aerosolized smooth and rough (hypervirulent) variants of M. abscessus. RESULTS: MA-PLC DNA vaccination (days 0, 21, 42) elicited a strong antibody response. A significant protective effect was obtained against aerosolized M. abscessus (S variant) in ΔF508 mice, but not in wild-type FVB littermates; similar results were observed when: (i) challenging mice with the "hypervirulent" R variant, and; (ii) immunizing mice with purified MA-PLC protein. High IgG titers against MA-PLC protein were measured in CF patients with M. abscessus infection; interestingly, significant titers were also detected in CF patients positive for Pseudomonas aeruginosa versus P. aeruginosa-negative controls. CONCLUSIONS: MA-PLC DNA- and PLC protein-vaccinated mice cleared more rapidly M. abscessus than ß-galactosidase DNA- or PBS- vaccinated mice in the context of CF. PLCs could constitute interesting vaccine targets against common PLC-producing CF pathogens like P. aeruginosa.

Evaluation of tetrafunctional block copolymers as synthetic vectors for lung gene transfer.

In the present study, we evaluated, in mice, the efficacy of the tetrafunctional block copolymer 704 as a nonviral gene delivery vector to the lungs. SPECT/CT molecular imaging of gene expression, biochemical assays, and immunohistochemistry were used. Our dataset shows that the formulation 704 resulted in higher levels of reporter gene expression than the GL67A formulation currently being used in a clinical trial in cystic fibrosis patients. The inflammatory response associated with this gene transfer was lower than that induced by the GL67A formulation, and the 704 formulation was amenable to repeated administrations. The cell types transfected by the 704 formulation were type I and type II pneumocytes, and transgene expression could not be detected in macrophages. These results emphasize the relevance of the 704 formulation as a nonviral gene delivery vector for lung gene therapy. Further studies will be required to validate this vector in larger animals, in which the lungs are more similar to human lungs.

Block copolymer/DNA vaccination induces a strong allergen-specific local response in a mouse model of house dust mite asthma.

BACKGROUND: Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity. OBJECTIVE: The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice. METHODS: Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements. RESULTS: Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted. CONCLUSIONS & CLINICAL RELEVANCE: DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.

Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteins.

The molecular domestication of several DNA transposons that occurred during the evolution of the mammalian lineage, has led to the emergence of at least 43 genes, known as neogenes. To date, the limited availability of efficient commercial antibodies directed against most of their protein isoforms hampers investigation of their expression in vitro and in situ. Since immunization protocols using peptides or recombinant proteins have revealed that it is difficult to recover antibodies, we planned to produce antisera in mice using a new technique of nanopheres/DNA immunization, the ICANtibodies™ technology. Here, we investigate the possibilities of obtaining polyclonal antibodies for 24 proteins or protein domains using this immunization strategy. We successfully obtained 13 antisera that were able to detect neogenic proteins by Western blotting and ELISA in protein extracts of transiently-transfected cells and various cancer cell lines, plus another two that only detected the in ELISA and in in situ hybridizations. The features required for the production of these antibodies are analyzed and discussed, and examples are given of the advantages they offer for the study of neogenic proteins.

Osteoprotegerin inhibits bone resorption and prevents tumor development in a xenogenic model of Ewing's sarcoma by inhibiting RANKL.

Ewing's sarcoma (ES) associated with high osyeolytic lesions typically arises in the bones of children and adolescents. The development of multi-disciplinary therapy has increased current long-term survival rates to greater than 50% but only 20% for high risk group patients (relapse, metastases, etc.). Among new therapeutic approaches, osteoprotegerin (OPG), an anti-bone resorption molecule may represent a promising candidate to inhibit RANKL-mediated osteolytic component of ES and consequently to limit the tumor development. Xenogenic orthotopic models of Ewing's sarcoma were induced by intra-osseous injection of human TC-71 ES cells. OPG was administered in vivo by non-viral gene transfer using an amphiphilic non ionic block copolymer. ES bearing mice were assigned to controls (no treatment, synthetic vector alone or F68/empty pcDNA3.1 plasmid) and hOPG treated groups. A substantial but not significant inhibition of tumor development was observed in the hOPG group as compared to control groups. Marked bone lesions were revealed by micro-computed tomography analyses in control groups whereas a normal bone micro-architecture was preserved in the hOPG treated group. RANKL over-expressed in ES animal model was expressed by tumor cells rather than by host cells. However, TRAIL present in the tumor microenvironment may interfere with OPG effect on tumor development and bone remodeling via RANKL inhibition. In conclusion, the use of a xenogenic model of Ewing's sarcoma allowed discriminating between the tumor and host cells responsible for the elevation of RANKL production observed in this tumor and demonstrated the relevance of blocking RANKL by OPG as a promising therapy in ES.

Treatment efficacy of DNA lipid nanocapsules and DNA multimodular systems after systemic administration in a human glioma model.

BACKGROUND: We previously developed different types of DNA nanocarriers for systemic administration. Recently, the biodistribution profiles of these intravenously administered nanocarriers, DNA lipid nanocapsules (LNCs) and different multimodular systems (MMS), were analysed in healthy mice using in vivo biofluorescence imaging. METHODS: In the present study, the experiments were performed in an ectopic human U87MG glioma model in nude mice. First, the biodistribution profiles of intravenously administered multimodular systems delivering a plasmid DNA with a luciferase cassette were analysed using in vivo biofluorescence imaging. Afterwards, a systemic treatment with two long circulating DNA nanocarriers, poly(ethylene glycol) (PEG) DNA LNCs and galactose (GAL) DNA MMS dioleylamin-succinyl paromomycin (DOSP) was performed on this glioma model using a plasmid encoding the herpes simplex virus thymidine kinase (HSV-tk) and subsequent ganciclovir (GCV) treatment. RESULTS: The biodistribution profiles of the different DNA nanocarriers on this glioma model were similar to those observed on healthy animals and varied in function of their cationic lipid composition and their surface characteristics. Furthermore, PEG DNA LNCs and GAL DNA MMS DOSP showed a specific accumulation and some luciferase expression in the tumour tissue. The systemic treatment using the HSV-tk/GCV approach showed a tumour growth reduction compared to the nontreated mice cohort. CONCLUSIONS: These results are in good accordance with those obtained previously with PEG DNA LNCs in a human melanoma mouse model and highlight the potential use of GAL DNA MMS DOSP and PEG DNA LNCs as future therapeutics in glioma and other cancers.

DNA/amphiphilic block copolymer nanospheres reduce asthmatic response in a mouse model of allergic asthma.

Asthma is a chronic, inflammatory, respiratory disease caused by an abnormal reactivity against allergens. The most promising treatments for asthma are based on specific immunotherapies, but they lack efficiency and can induce deleterious side effects. Among new modalities of immunotherapy currently in development, DNA vaccination presents a promising approach, as it enables targeted immunotherapy in association with reduced allergenicity. We have developed an innovative, DNA-based vaccine against Dermatophagoides farinae 1 allergen (Der f 1), one of the allergens most commonly encountered by asthma patients in Europe. Intramuscular administration of a Der f 1-encoding plasmid formulated with the block copolymer 704 in healthy mice induced a strong humoral and cellular response with a pro-helper T cell type 1 bias. Administration of the same formulation in asthmatic mice, according to an early vaccination protocol, led to a reduction of airway hyperresponsiveness and a significant decrease in the level of inflammatory cytokines in the bronchoalveolar lavage of Der f 1-vaccinated mice.

siRNA LNCs--a novel platform of lipid nanocapsules for systemic siRNA administration.

Several siRNA (small interfering RNA) therapeutics are undergoing clinical trials for cancer, respiratory diseases or macular degeneration, but most are administrated locally. In order to overcome the different barriers to attain an efficient siRNA action after systemic administration, nanocarriers able to carry and protect siRNA are awaited. With this aim, we developed a new platform of siRNA lipid nanocapsules (LNCs) using different cationic lipids, combining the properties of LNCs (siRNA protection and targeting) and lipoplexes (efficient siRNA delivery into the cell). The formulation was revealed to contain different compartments. A siRNA quantification method based on UV spectroscopy was developed to locate and quantify siRNA in each compartment. All in all, these novel siRNA LNCs presented sizes of about 55 nm with a neutral surface charge and siRNA encapsulation efficiencies up to 65% representing appropriate characteristics for systemic administration.

Novel approach for the development of new antibodies directed against transposase-derived proteins encoded by human neogenes.

Molecular domestication of several DNA transposons has occurred during the evolution of the primate lineage, and has led to the emergence of at least 42 new genes known as neogenes. Because these genes are derived from transposons, they encode proteins that are related to certain recombinases, known as transposases. Consequently, they may make an important contribution to the genetic instability of some human cells. In order to investigate the role of these neogenes, we need to be able to study their expression as proteins, for example in tumours, which often provide good models of genetic instability. In order to perform such studies, polyclonal antibodies directed against the proteins expressed by neogenes are obtained using a recently developed new method of Nanospheres/DNA immunisation in laboratory mammals. In this chapter, we describe a fully integrated process of producing antibodies that consists of a series of steps starting with the preparation and synthetic formulation of plasmids encoding neogenes, and culminating in the final production and confirmation of the quality of these polyclonal antibodies.

Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line.

Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.

In vivo imaging of DNA lipid nanocapsules after systemic administration in a melanoma mouse model.

The biodistribution of intravenously injected DNA lipid nanocapsules (DNA LNCs), encapsulating pHSV-tk, was analysed by in vivo imaging on an orthotopic melanoma mouse model and by a subsequent treatment with ganciclovir (GCV), using the gene-directed enzyme prodrug therapy (GDEPT) approach. Luminescent melanoma cells, implanted subcutaneously in the right flank of the mice, allowed us to follow tumour growth and tumour localisation with in vivo bioluminescence imaging (BLI). In parallel, DNA LNCs or PEG DNA LNCs (DNA LNCs recovered with PEG(2000)) encapsulating a fluorescent probe, DiD, allowed us to follow their biodistribution with in vivo biofluorescence imaging (BFI). The BF-images confirmed a prolonged circulation-time for PEG DNA LNCs as was previously observed on an ectotopic model of glioma; comparison with BL-images evidenced the colocalisation of PEG DNA LNCs and melanoma cells. After these promising results, treatment with PEG DNA LNCs and GCV on a few animals was performed and the treatment efficacy measured by BLI. The first results showed tumour growth reduction tendency and, once optimised, this therapy strategy could become a new option for melanoma treatment.

CCL5 neutralization restricts cancer growth and potentiates the targeting of PDGFRß in colorectal carcinoma.

Increased CCL5 levels are markers of an unfavourable outcome in patients with melanoma, breast, cervical, prostate, gastric or pancreatic cancer. Here, we have assessed the role played by CCL5/CCR5 interactions in the development of colon cancer. To do so, we have examined a number of human colorectal carcinoma clinical specimens and found CCL5 and its receptors over-expressed within primary as well as liver and pulmonary metastases of patients compared to healthy tissues. In vitro, CCL5 increased the growth and migratory responses of colon cancer cells from both human and mouse origins. In addition, systemic treatment of mice with CCL5-directed antibodies reduced the extent of development of subcutaneous colon tumors, of liver metastases and of peritoneal carcinosis. Consistently, we found increased numbers of CD45-immunoreactive cells within the stroma of the remaining lesions as well as at the interface with the healthy tissue. In contrast, selective targeting of CCR5 through administration of TAK-779, a CCR5 antagonist, only partially compromised colon cancer progression. Furthermore, CCL5 neutralization rendered the tumors more sensitive to a PDGFRß-directed strategy in mice, this combination regimen offering the greatest protection against liver metastases and suppressing macroscopic peritoneal carcinosis. Collectively, our data demonstrate the involvement of CCL5 in the pathogenesis of colorectal carcinoma and point to its potential value as a therapeutic target.