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Accueil > Thèmes de recherche > Biologie cellulaire, Cibles moléculaires et Thérapies innovantes > Thérapies innovantes et nanomédecine

Thérapies innovantes et nanomédecine (Patrick Midoux & Jean-Marc Malinge)

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DESCRIPTIF DU TRAVAIL DE RECHERCHE

Our research activities encompass the development and optimization of new therapeutic nucleic acids. Overall, their formulations, delivery, targeting according to routes of administration constitute our main research activity and challenge. This is a multidisciplinary research field requiring chemistry, molecular and cell biology as well as animal models.

Our long-lasting expertise in plasmid DNA transfer by synthetic vectors is exploited to the delivery of other types of nucleic acids including oligonucleotides, DNA decoy, siRNA, microRNA and mRNA. Our formulation called Lipopolyplexes, a ternary complex between Liposomes, a Polymer and a Nucleic acid is used in the frame of pre-clinical experiments for therapeutic applications.

This is the case of mRNA-based vaccines against cancer. We wish to strengthen this axis by improving in vivo delivery for pre-clinical trials and hopefully clinical trials. The access to large quantities of therapeutic mRNA thanks to the RNA cell factories project will open new potent mRNA-based applications such as gene editing using CRISPR/Cas9 mRNA for gene therapy. For this purpose, more potent mRNA delivery vehicles are designed.

Regarding gene transfer, we have now identified several devices for designing a DNA Integrative Artificial virus that could be an alternative of recombinant virus. This is tackled by optimization of each known devices, their packaging as well as the co-assembly of DNA and mRNA for gene integration by transposon/transposase or gene editing. For this, bioengineering of plasmid DNA is developed by coupling specific intracellular signals as peptide mediated cytosolic transport and DNA sequence for nuclear import.

We also explore the co-delivery approach of siRNAs, anti-miRNAs and/or DNA decoys against oncogenic genes. DNA decoys are based on bioengineered small nucleic acids for cell and intracellular targeting.

Sonoporation based on ultrasound (US) and microbubbles (MB) provides significant results to deliver nucleic acids and drugs in vitro and in vivo on tendon repair and tumor regression. Current developments of cationic MB with smaller size and high capacity of encapsulation as well as applications are going-on.

US waves delivering physical forces on cells can lead to cell death, drug delivery or tissue regeneration depending on parameters and cell types. Thus potential mechanical sensors involved in mechanotransduction, Early Growth response-1, Adenosine monophosphate kinase and Yes-associated Protein /Transcriptional coactivator with PDZ motifs is currently under investigation. Since those molecules are involved in pathways related to either proliferation or apoptosis, getting a clear knowledge of activated process will allow to fine tune the use of US.


Compétences / savoir-faire

Synthèse et chimie fine de l’ADN
Vectorisation d’acides nucléiques
Endocytose et trafic intracellulaire
Microscopie confocale
Cytométrie en flux
Transfection
Expérimentation animale
Imagerie fonctionnelle in vivo


Partenaires


Principales publications

Thibault T., Degrouard J., Baril P., Pichon C., Midoux P. and Malinge J.-M. (2016) Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-ĸB inhibition activity. Nucleic Acids Research - accepted 21 octobre 2016 - doi : 10.1093/nar/gkw1034

Pigeon L., Gonçalves C., Pichon C., Midoux P. (2016) Evidence for plasmid DNA exchange after polyplex mixing. Soft Matter 12 (33) 7012-7019.

Delalande A., Gosselin M.-P., Suwalski A., Guilmain W., Leduc C., Berchel M., Jaffrès P. A., Baril P., Midoux P., Pichon C. (2015) Enhanced Achilles tendon healing by fibromodulin gene transfer. Nanomedicine 11 (7) 1735-1744.

Midoux P. and Pichon C. (2015) Lipid-based mRNA vaccine delivery systems. Expert Review of Vaccines 14, 221-234

Pigeon L., Gonçalves C., Gosset D., Pichon C. and Midoux P. (2013) An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA increases polyplexes transfection efficiency. Small 9, 3845-3851.

Billiet L., Gomez J.-P., Berchel M., Jaffrès P. A., Le Gall T., Montier T., Bertrand E., Cheradame H., Guégan P., Mével M., Pitard B., Benvegnu T., Lehn P., Pichon C. and Midoux P. (2012) Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line. Biomaterials 33, 2980-2990.


Publications

2016   Références trouvées : 9

Nadim W. D., Simion V., Bénédetti H., Pichon C., Baril P. and Morisset-Lopez S.  (2016)

MicroRNAs in Neurocognitive Dysfunctions : New Molecular Targets for Pharmacological Treatments ?

Curr Neuropharmacol (2016) sous presse - doi : 10.2174/1570159X14666160709001441
Neurodegenerative and cognitive disorders are multifactorial diseases (i.e., involving neurodevelopmental, genetic, age or environmental factors) that are characterized by an abnormal development affecting neuronal function and integrity. Recently, an increasing number of studies revealed that the dysregulation of microRNAs (miRNAs) may be involved in the etiology of cognitive disorders such as Alzheimer, Parkinson, and Huntington's diseases, Schizophrenia and Autism spectrum disorders. MiRNAs are a class of small non-coding RNAs that regulate gene expression through a base pairing mechanism with their target mRNAs, thereby inducing translational repression or mRNA degradation. Intriguingly, 70 % of known miRNAs are expressed in the brain while functional studies indicate that they play crucial roles in brain development by regulating key signaling pathways involved in synaptogenesis, neuronal plasticity, neurite outgrowth and memory processes. Further, the relevance of modulating miRNA expression levels to correct neurocognitive disorders is supported by the increasing number of studies demonstrating a link between miRNA dysregulation and the etiology and/or pathophysiology of cognitive dysfunction in several neurologic and neuropsychiatric disorders. This suggests that miRNAs might be used as diagnosis markers and/or could be exploited for therapeutic interventions. In this review, we first briefly summarize the basic molecular events involved in memory, a process inherent to cognitive disease, and then describe the role of miRNAs in neurodevelopment, synaptic plasticity and memory. Secondly, we provide an overview of the impact of miRNA dysregulation in the pathogenesis of different neurocognitive disorders, and lastly discuss the feasibility of miRNA-based therapeutics in the treatment of these cognitive disorders. This last part will be discussed in detail in the following review.

Neurodegenerative and cognitive disorders are multifactorial diseases (i.e., involving neurodevelopmental, genetic, age or environmental factors) that are characterized by an abnormal development affecting neuronal function and integrity. Recently, an increasing number of studies revealed that the dysregulation of microRNAs (miRNAs) may be involved in the etiology of cognitive disorders such as Alzheimer, Parkinson, and Huntington’s diseases, Schizophrenia and Autism spectrum disorders. MiRNAs are a class of small non-coding RNAs that regulate gene expression through a base pairing mechanism with their target mRNAs, thereby inducing translational repression or mRNA degradation. Intriguingly, 70 % of known miRNAs are expressed in the brain while functional studies indicate that they play crucial roles in brain development by regulating key signaling pathways involved in synaptogenesis, neuronal plasticity, neurite outgrowth and memory processes. Further, the relevance of modulating miRNA expression levels to correct neurocognitive disorders is supported by the increasing number of studies demonstrating a link between miRNA dysregulation and the etiology and/or pathophysiology of cognitive dysfunction in several neurologic and neuropsychiatric disorders. This suggests that miRNAs might be used as diagnosis markers and/or could be exploited for therapeutic interventions. In this review, we first briefly summarize the basic molecular events involved in memory, a process inherent to cognitive disease, and then describe the role of miRNAs in neurodevelopment, synaptic plasticity and memory. Secondly, we provide an overview of the impact of miRNA dysregulation in the pathogenesis of different neurocognitive disorders, and lastly discuss the feasibility of miRNA-based therapeutics in the treatment of these cognitive disorders. This last part will be discussed in detail in the following review.

Gaut L., Robert N., Delalande A., Bonnin M.-A., Pichon C. and Duprez D.  (2016)

EGR1 Regulates Transcription Downstream of Mechanical Signals during Tendon Formation and Healing

PLoS ONE (2016) 11 (11) e0166237 - doi : 10.1371/journal.pone.0166237.t001
BackgroundTendon is a mechanical tissue that transmits forces generated by muscle to bone in order to allow body motion. The molecular pathways that sense mechanical forces during tendon formation, homeostasis and repair are not known. EGR1 is a mechanosensitive transcription factor involved in tendon formation, homeostasis and repair. We hypothesized that EGR1 senses mechanical signals to promote tendon gene expression.Methodology/Principal findingsUsing in vitro and in vivo models, we show that the expression of Egr1 and tendon genes is downregulated in 3D-engineered tendons made of mesenchymal stem cells when tension is released as well as in tendon homeostasis and healing when mechanical signals are reduced. We further demonstrate that EGR1 overexpression prevents tendon gene downregulation in 3D-engineered tendons when tension is released. Lastly, ultrasound and microbubbles mediated EGR1 overexpression prevents the downregulation of tendon gene expression during tendon healing in reduced load conditions.Conclusion/SignificanceThese results show that Egr1 expression is sensitive to mechanical signals in tendon cells. Moreover, EGR1 overexpression prevents the downregulation of tendon gene expression in the absence of mechanical signals in 3D-engineered tendons and tendon healing. These results show that EGR1 induces a transcriptional response downstream of mechanical signals in tendon cells and open new avenues to use EGR1 to promote tendon healing in reduced load conditions.

BackgroundTendon is a mechanical tissue that transmits forces generated by muscle to bone in order to allow body motion. The molecular pathways that sense mechanical forces during tendon formation, homeostasis and repair are not known. EGR1 is a mechanosensitive transcription factor involved in tendon formation, homeostasis and repair. We hypothesized that EGR1 senses mechanical signals to promote tendon gene expression.Methodology/Principal findingsUsing in vitro and in vivo models, we show that the expression of Egr1 and tendon genes is downregulated in 3D-engineered tendons made of mesenchymal stem cells when tension is released as well as in tendon homeostasis and healing when mechanical signals are reduced. We further demonstrate that EGR1 overexpression prevents tendon gene downregulation in 3D-engineered tendons when tension is released. Lastly, ultrasound and microbubbles mediated EGR1 overexpression prevents the downregulation of tendon gene expression during tendon healing in reduced load conditions.Conclusion/SignificanceThese results show that Egr1 expression is sensitive to mechanical signals in tendon cells. Moreover, EGR1 overexpression prevents the downregulation of tendon gene expression in the absence of mechanical signals in 3D-engineered tendons and tendon healing. These results show that EGR1 induces a transcriptional response downstream of mechanical signals in tendon cells and open new avenues to use EGR1 to promote tendon healing in reduced load conditions.

Leduc C., Sobilo L., Toumi H., Mondon P., Lespessailles E., Ossant F., Kurfürst R. and Pichon C.  (2016)

TGF-beta induced early gene-1 overexpression promotes oxidative stress protection and actin cytoskeleton rearrangement in human skin fibroblasts

Biochimica Et Biophysica Acta (2016) 1860 (6) 1071-1078 - doi : 10.1016/j.bbagen.2016.02.009
BACKGROUND : Transforming growth factor beta inducible early gene-1 (TIEG-1), a member of the Kruppel-like factor, was identified as a primary response gene for TGF-beta. The role of TIEG-1 in skin repair has been mainly addressed in vivo on TIEG-1 null mice model and the mechanism remains unexplored. METHODS : We investigated the modulation of TIEG-1 expression in normal human skin fibroblasts by either down-expressing or overexpressing the gene. We evaluated reactive oxygen species production and the cell viability of treated cells. The effect of TIEG-1 overexpression was monitored by wound healing assay and immunofluorescence staining of actin fibers organization and alpha-smooth muscle actin (alpha-SMA). Western blots were carried out to identify the level of expression or phosphorylation of key proteins such as cofilin, Rho GTPases, and p38 mitogen-activated protein kinase (p38 MAPK).

RESULTS : TIEG-1 down-regulation had a deleterious effect on the cell viability. It was significantly reduced (65+/-5%) and exposure to ultraviolet further increased this effect (47+/-3%). By contrast, cells overexpressing TIEG-1 had a reduced reactive oxygen species production (75%) compared to control and mock-transfected cells. This overexpression also resulted in formation of actin stress fibers and increased alpha-SMA expression and an enhanced wound healing feature. RhoB GTPase was upregulated and phosphorylation of cofilin and p38 MAPK was observed.

CONCLUSION : TIEG-1 overexpression in normal human skin fibroblasts results in improved resistance to oxidative stress, myofibroblast-like conversion that involved RhoB signaling pathway with cofilin and p38 MAPK proteins activation. GENERAL SIGNIFICANCE : This study enlightens the role of TIEG-1 role in skin biology.

BACKGROUND : Transforming growth factor beta inducible early gene-1 (TIEG-1), a member of the Kruppel-like factor, was identified as a primary response gene for TGF-beta. The role of TIEG-1 in skin repair has been mainly addressed in vivo on TIEG-1 null mice model and the mechanism remains unexplored. METHODS : We investigated the modulation of TIEG-1 expression in normal human skin fibroblasts by either down-expressing or overexpressing the gene. We evaluated reactive oxygen species production and the cell viability of treated cells. The effect of TIEG-1 overexpression was monitored by wound healing assay and immunofluorescence staining of actin fibers organization and alpha-smooth muscle actin (alpha-SMA). Western blots were carried out to identify the level of expression or phosphorylation of key proteins such as cofilin, Rho GTPases, and p38 mitogen-activated protein kinase (p38 MAPK).
RESULTS : TIEG-1 down-regulation had a deleterious effect on the cell viability. It was significantly reduced (65+/-5%) and exposure to ultraviolet further increased this effect (47+/-3%). By contrast, cells overexpressing TIEG-1 had a reduced reactive oxygen species production (75%) compared to control and mock-transfected cells. This overexpression also resulted in formation of actin stress fibers and increased alpha-SMA expression and an enhanced wound healing feature. RhoB GTPase was upregulated and phosphorylation of cofilin and p38 MAPK was observed.
CONCLUSION : TIEG-1 overexpression in normal human skin fibroblasts results in improved resistance to oxidative stress, myofibroblast-like conversion that involved RhoB signaling pathway with cofilin and p38 MAPK proteins activation. GENERAL SIGNIFICANCE : This study enlightens the role of TIEG-1 role in skin biology.

Chaveroux C., Bruhat A., Carraro V., Jousse C., Averous J., Maurin A. C., Parry L., Mesclon F., Muranishi Y., Meulle A., Baril P., Thi A. D., Ravassard P., Mallet J. and Fafournoux P.  (2016)

Tuning transgene expression with an artificial diet : a compelling resource in gene therapy

Nature Biotechnology (sous presse)

Demoulins T., Milona P., Englezou P. C., Ebensen T., Schulze K., Suter R., Pichon C., Midoux P., Guzman C. A., Ruggli N. and McCullough K. C.  (2016)

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines

Nanomedicine-Nanotechnology, Biology and Medicine (2016) 12 (3) 711-722 - doi : 10.1016/j.nano.2015.11.001
Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb) ; their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity ; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. FROM THE CLINICAL EDITOR : The use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb) ; their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity ; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. FROM THE CLINICAL EDITOR : The use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

Vassaux G., Angelova A. L., Rommelaere J., Baril P., Midoux P. and Cordelier P.  (2016)

The promise of gene therapy for pancreatic cancer

Human Gene Therapy Methods (2016) 27 (2) 127-133 - doi : 10.1089/hum.2015.141
Unlike other digestive-cancer entities, chemotherapy, radiotherapy and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for PDAC patients. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using non-viral therapeutic gene delivery, targeted transgene expression or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC and may also largely influence the field of gene-based molecular treatment of cancer.

Unlike other digestive-cancer entities, chemotherapy, radiotherapy and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for PDAC patients. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using non-viral therapeutic gene delivery, targeted transgene expression or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC and may also largely influence the field of gene-based molecular treatment of cancer.

Manta S., Delalande A., Bessodes M., Bureau M. F., Scherman D., Pichon C. and Mignet N.  (2016)

Shell Microbubbles with Tunable Resistive Pulse Sensing (TRPS) Method : A Technical Note

Ultrasound in medicine & biology (2016) 42 (2) 624-630 - doi : 10.1016/j.ultrasmedbio.2015.10.010
Microbubbles are polydisperse microparticles. Their size distribution cannot be accurately measured from the current methods used, such as optical microscopy, electrical sensing or light scattering. Indeed, these techniques present some limitations when applied to microbubbles, which prompted us to investigate the use of an alternative technique : tunable resistive pulse sensing (TRPS). This technique is based on the principle of the Coulter counter with the advantage of being more flexible compared to other methods using this principle, since the flow rate, the potential difference and the pore size can be modulated. The main limitation of TRPS is that more than one size of nanopore membrane is required to obtain the full size distribution of polydisperse microparticles. To evaluate this technique, the concentration and the size distribution of positively charged microbubbles were studied using TRPS and compared to data obtained using optical microscopy. We describe herein the parameters required for the accurate measurement of microbubble concentration and size distribution by TRPS and present a statistical comparison of the data obtained by TRPS and optical microscopy.

Microbubbles are polydisperse microparticles. Their size distribution cannot be accurately measured from the current methods used, such as optical microscopy, electrical sensing or light scattering. Indeed, these techniques present some limitations when applied to microbubbles, which prompted us to investigate the use of an alternative technique : tunable resistive pulse sensing (TRPS). This technique is based on the principle of the Coulter counter with the advantage of being more flexible compared to other methods using this principle, since the flow rate, the potential difference and the pore size can be modulated. The main limitation of TRPS is that more than one size of nanopore membrane is required to obtain the full size distribution of polydisperse microparticles. To evaluate this technique, the concentration and the size distribution of positively charged microbubbles were studied using TRPS and compared to data obtained using optical microscopy. We describe herein the parameters required for the accurate measurement of microbubble concentration and size distribution by TRPS and present a statistical comparison of the data obtained by TRPS and optical microscopy.

Ben Halima, N. Khemakhem, B. Fendri, I. Ogata, H. Baril, P. Pichon, C. and Abdelkafi, S.  (2016)

Identification of a new oat beta-amylase by functional proteomics

Biochimica Et Biophysica Acta - Biomembranes (2016) 1864, 52-61 - doi : 10.1016/j.bbapap.2015.10.001
Oat (Avena sativa L.) seed extracts exhibited a high degree of catalytic activity including amylase activities. Proteins in the oat seed extracts were optimized for their amylolytic activities. Oat extract with amylolytic activity was separated by SDS-PAGE and a major protein band with an apparent molecular mass of 53 kDa was subjected to tryptic digestion. The generated amino acid sequences were analyzed by liquid chromatography–tandem mass spectrometry (LC/ESI/MS/MS) and database searches. These sequences were used to identify a partial cDNA from expressed sequence tags (ESTs) of A. sativa L. Based upon EST sequences, a predicted full-length gene was identified, with an open reading frame of 1464 bp encoding a protein of 488 amino acid residues (AsBAMY), with a theoretical molecular mass of 55 kDa identified as a β-amylase belonging to the plant β-amylase family. Primary structure of oat β-amylase (AsBAMY) protein indicated high similarity with other β-amylase from other cereals such as wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale) with two conserved Glu residues (E184 and E378) assigned as the “putative” catalytic residues which would act as an acid and base pair in the catalytic process. In addition, a 3D-model of AsBAMY was built from known X-ray structures and sequence alignments. A similar core (β/α)8-barrel architecture was found in AsBAMY like the other cereal β-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (β/α)8-barrel domain suggesting an accessibility of the non-reducing end of the substrate and thus confirming the results of AsBAMY exo-acting hydrolase.

Oat (Avena sativa L.) seed extracts exhibited a high degree of catalytic activity including amylase activities. Proteins in the oat seed extracts were optimized for their amylolytic activities. Oat extract with amylolytic activity was separated by SDS-PAGE and a major protein band with an apparent molecular mass of 53 kDa was subjected to tryptic digestion. The generated amino acid sequences were analyzed by liquid chromatography–tandem mass spectrometry (LC/ESI/MS/MS) and database searches. These sequences were used to identify a partial cDNA from expressed sequence tags (ESTs) of A. sativa L. Based upon EST sequences, a predicted full-length gene was identified, with an open reading frame of 1464 bp encoding a protein of 488 amino acid residues (AsBAMY), with a theoretical molecular mass of 55 kDa identified as a β-amylase belonging to the plant β-amylase family. Primary structure of oat β-amylase (AsBAMY) protein indicated high similarity with other β-amylase from other cereals such as wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale) with two conserved Glu residues (E184 and E378) assigned as the “putative” catalytic residues which would act as an acid and base pair in the catalytic process. In addition, a 3D-model of AsBAMY was built from known X-ray structures and sequence alignments. A similar core (β/α)8-barrel architecture was found in AsBAMY like the other cereal β-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (β/α)8-barrel domain suggesting an accessibility of the non-reducing end of the substrate and thus confirming the results of AsBAMY exo-acting hydrolase.

Baril, P. and Pichon, C.  (2016)

Positive Bioluminescence Imaging of MicroRNA Expression in Small Animal Models Using an Engineered Genetic-Switch Expression System, RILES

Methods in molecular biology (2016) 1372, 193-208 - doi : 10.1007/978-1-4939-3148-4_15
MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, monitoring of miRNAs is difficult due to their small size, low abundance, high degree of sequence similarity, and their dynamic expression pattern which is subjected to tight transcriptional and post-transcriptional controls. Recently, we developed a miRNA monitoring system called RILES, standing for RNAi-inducible expression system, which relies on an engineered regulatable expression system, to switch on the expression of the luciferase gene when the targeted miRNA is expressed in cells. We demonstrated that RILES is a specific, sensitive, and robust method to determine the fine-tuning of miRNA expression during the development of an experimental pathological process in mice. Because RILES offers the possibility for longitudinal studies on individual subjects, sharper insights into miRNA regulation can be generated, with applications in physiology, pathophysiology and development of RNAi-based therapies. This chapter describes methods and protocols to monitor the expression of myomiR-206, -1, and -133 in the tibialis anterior muscle of mice. These protocols can be used and adapted to monitor the expression of other miRNAs in other biological processes.

MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, monitoring of miRNAs is difficult due to their small size, low abundance, high degree of sequence similarity, and their dynamic expression pattern which is subjected to tight transcriptional and post-transcriptional controls. Recently, we developed a miRNA monitoring system called RILES, standing for RNAi-inducible expression system, which relies on an engineered regulatable expression system, to switch on the expression of the luciferase gene when the targeted miRNA is expressed in cells. We demonstrated that RILES is a specific, sensitive, and robust method to determine the fine-tuning of miRNA expression during the development of an experimental pathological process in mice. Because RILES offers the possibility for longitudinal studies on individual subjects, sharper insights into miRNA regulation can be generated, with applications in physiology, pathophysiology and development of RNAi-based therapies. This chapter describes methods and protocols to monitor the expression of myomiR-206, -1, and -133 in the tibialis anterior muscle of mice. These protocols can be used and adapted to monitor the expression of other miRNAs in other biological processes.


2015   Références trouvées : 14

Rahman M., Akhter S., Ahmad M. Z., Ahmad J., Addo R. T., Ahmad F. J. and Pichon C.  (2015)

Emerging advances in cancer nanotheranostics with graphene nanocomposites : opportunities and challenges

Nanomedicine (2015) 10 (15) 2405-2422 - doi : 10.2217/nnm.15.68
As an inorganic nanomaterial, graphene nanocomposites have gained much attention in cancer nanotechnology compared with the other inorganic nanomaterial in recent times. Although a relatively new drug carrier, it has been extensively explored as a potential chemotherapeutic carrier and theranostic because of its numerous physicochemical properties, including, capability of multiple pay load, functionalization for drug targeting and photothermal effect. Despite potential benefit, its translation from bench to bed-side in cancer therapy is challenged due to its toxicity concern. Here, we discussed the present progress and future possibilities of graphene nanocomposites as a cancer theranostic. Moreover, the paper also exemplifies the effects of graphene/graphene oxide on tissues and organ functions in order to understand the extent and mechanism of toxicity.

As an inorganic nanomaterial, graphene nanocomposites have gained much attention in cancer nanotechnology compared with the other inorganic nanomaterial in recent times. Although a relatively new drug carrier, it has been extensively explored as a potential chemotherapeutic carrier and theranostic because of its numerous physicochemical properties, including, capability of multiple pay load, functionalization for drug targeting and photothermal effect. Despite potential benefit, its translation from bench to bed-side in cancer therapy is challenged due to its toxicity concern. Here, we discussed the present progress and future possibilities of graphene nanocomposites as a cancer theranostic. Moreover, the paper also exemplifies the effects of graphene/graphene oxide on tissues and organ functions in order to understand the extent and mechanism of toxicity.

Jazwa A., Stoszko M., Tomczyk M., Bukowska-Strakova K., Pichon C., Jozkowicz A. and Dulak J.  (2015)

HIF-regulated HO-1 gene transfer improves the post-ischemic limb recovery and diminishes TLR-triggered immune responses - Effects modified by concomitant VEGF overexpression

Vascular Pharmacology (2015) 71, 127-138 - doi : 10.1016/j.vph.2015.02.011
Heme oxygenase-1 (HO-1) mitigates cellular injury by antioxidant, anti-apoptotic, anti-inflammatory and proangiogenic effects. Vascular endothelial growth factor (VEGF) is a critical regulator of blood vessel growth. Their coordinated action was analyzed in a model of femoral artery ligation (FAL) in mice lacking HO-1 gene (HO-1 KO). Gastrocnemius skeletal muscles of HO-1 KO mice were preemptively injected with plasmids containing hypoxia-response element (HRE) driving the expression of only HO-1 (pHRE-HO1) or both HO-1 and VEGF (pHRE-HO1 VEGF). At day 14th the pHRE-HO1 vector increased an impaired post-ischemic blood flow recovery in HO-1 KO mice to the level observed in wild-type (WT) mice subjected to FAL and pHRE-HO1 VEGF restored it already at day 7. The pHRE-HO1 gene therapy diminished, when compared to control pHRE-empty-treated HO-1 KO mice, the expression of toll-like receptors (TLR4 and TLR9) and inflammatory cytokines (IL-1 beta, IL-6 and TNF alpha) at day 3, whereas opposite effects were observed following concomitant HO-1 and VEGF gene transfer. Moreover, HO-1 diminished ischemia-induced expression of MyoD involved in satellite cell differentiation in HO-1 KO mice. Our results confirm the therapeutic potential of HO-1 and VEGF against critical limb ischemia although, their concomitant delivery may have contradictory actions on the resolution of inflammation.

Heme oxygenase-1 (HO-1) mitigates cellular injury by antioxidant, anti-apoptotic, anti-inflammatory and proangiogenic effects. Vascular endothelial growth factor (VEGF) is a critical regulator of blood vessel growth. Their coordinated action was analyzed in a model of femoral artery ligation (FAL) in mice lacking HO-1 gene (HO-1 KO). Gastrocnemius skeletal muscles of HO-1 KO mice were preemptively injected with plasmids containing hypoxia-response element (HRE) driving the expression of only HO-1 (pHRE-HO1) or both HO-1 and VEGF (pHRE-HO1 VEGF). At day 14th the pHRE-HO1 vector increased an impaired post-ischemic blood flow recovery in HO-1 KO mice to the level observed in wild-type (WT) mice subjected to FAL and pHRE-HO1 VEGF restored it already at day 7. The pHRE-HO1 gene therapy diminished, when compared to control pHRE-empty-treated HO-1 KO mice, the expression of toll-like receptors (TLR4 and TLR9) and inflammatory cytokines (IL-1 beta, IL-6 and TNF alpha) at day 3, whereas opposite effects were observed following concomitant HO-1 and VEGF gene transfer. Moreover, HO-1 diminished ischemia-induced expression of MyoD involved in satellite cell differentiation in HO-1 KO mice. Our results confirm the therapeutic potential of HO-1 and VEGF against critical limb ischemia although, their concomitant delivery may have contradictory actions on the resolution of inflammation.

Gaspar V. M., Moreira A. F., Costa E. C., Queiroz J. A., Sousa F., Pichon C. and Correi I. J.  (2015)

Gas-generating TPGS-PLGA microspheres loaded with nanoparticles (NIMPS) for co-delivery of minicircle DNA and anti-tumoral drugs

Colloids and Surfaces B-Biointerfaces (2015) 134, 287-294 - doi : 10.1016/j.colsurfb.2015.07.004
Drug-DNA combination therapies are receiving an ever growing focus due to their potential for improving cancer treatment. However, such approaches are still limited by the lack of multipurpose delivery systems that encapsulate drugs and condense DNA simultaneously. In this study, we describe the successful formulation of gas-generating pH-responsive D-ot-tocopherol PEG succinate-poly(D,L-lacticco-glycolic acid) (TPGS-PLGA) hollow microspheres loaded with both Doxorubicin (Dox) and minicircle DNA (mcDNA) nanoparticles as a strategy to co-deliver these therapeutics. For this study mcDNA vectors were chosen due to their increased therapeutic efficiency in comparison to standard plasmid DNA. The results demonstrate that TPGS-PLGA microcarriers can encapsulate Dox and chitosan nanoparticles completely condense mcDNA. The loading of mcDNA-nanoparticles into microspheres was confirmed by 3D confocal microscopy and co-localization analysis. The resulting TPGS-PLGA-DoxmcDNA nanoparticle-in-microsphere hybrid carriers exhibit a well-defined spherical shape and neutral surface charge. Microcarriers incubation in acidic pH produced a gas-mediated Dox release, corroborating the microcarriers stimuli-responsive character. Also, the dual-loaded TPGS-PLGA particles achieved 5.2-fold higher cellular internalization in comparison with non-pegylated microspheres. This increased intracellular concentration resulted in a higher cytotoxic effect. Successful transgene expression was obtained after nanoparticle-mcDNA co-delivery in the microspheres. Overall these findings support the concept of using nanoparticle-microsphere multipart systems to achieve efficient co-delivery of various drug-mcDNA combinations. (C) 2015 Elsevier B.V. All rights reserved.

Drug-DNA combination therapies are receiving an ever growing focus due to their potential for improving cancer treatment. However, such approaches are still limited by the lack of multipurpose delivery systems that encapsulate drugs and condense DNA simultaneously. In this study, we describe the successful formulation of gas-generating pH-responsive D-ot-tocopherol PEG succinate-poly(D,L-lacticco-glycolic acid) (TPGS-PLGA) hollow microspheres loaded with both Doxorubicin (Dox) and minicircle DNA (mcDNA) nanoparticles as a strategy to co-deliver these therapeutics. For this study mcDNA vectors were chosen due to their increased therapeutic efficiency in comparison to standard plasmid DNA. The results demonstrate that TPGS-PLGA microcarriers can encapsulate Dox and chitosan nanoparticles completely condense mcDNA. The loading of mcDNA-nanoparticles into microspheres was confirmed by 3D confocal microscopy and co-localization analysis. The resulting TPGS-PLGA-DoxmcDNA nanoparticle-in-microsphere hybrid carriers exhibit a well-defined spherical shape and neutral surface charge. Microcarriers incubation in acidic pH produced a gas-mediated Dox release, corroborating the microcarriers stimuli-responsive character. Also, the dual-loaded TPGS-PLGA particles achieved 5.2-fold higher cellular internalization in comparison with non-pegylated microspheres. This increased intracellular concentration resulted in a higher cytotoxic effect. Successful transgene expression was obtained after nanoparticle-mcDNA co-delivery in the microspheres. Overall these findings support the concept of using nanoparticle-microsphere multipart systems to achieve efficient co-delivery of various drug-mcDNA combinations. (C) 2015 Elsevier B.V. All rights reserved.

Gaspar V. M., Baril P., Costa E. C., de Melo-Diogo D., Foucher F., Queiroz J. A., Sousa F., Pichon C. and Correia I. J.  (2015)

Bioreducible poly(2-ethyl-2-oxazoline)-PLA-PEI-SS triblock copolymer micelles for co-delivery of DNA minicircles and Doxorubicin

Journal of Controlled Release (2015) 213, 175-191 - doi : 10.1016/j.jconrel.2015.07.011
The co-delivery of minicircle DNA (mcDNA) and small anti-cancer drugs via stimuli-sensitive nanocarriers is a promising approach for combinatorial cancer therapy. However, the simultaneous loading of drugs and DNA in nanosized delivery systems is remarkably challenging. In this study we describe the synthesis of triblock copolymer micelles based on poly(2-ethyl-2-oxazoline)-poly(L-lactide) grafted with bioreducible polyethylenimine (PEOz-PLA-g-PEI-SS) for co-delivery of supercoiled (sc) mcDNA vectors and Doxorubicin (Dox). These amphiphilic carriers take advantage of non-fouling oxazolines to confer biological stability, of PLA to provide a hydrophobic core for drug encapsulation and of bioreducible PEI-SS to provide mcDNA complexation and an on-demand stimuli-responsive release. The obtained results show that mcDNA-loaded micelleplexes penetrate into in vitro tumor spheroid models with specific kinetics and exhibit a higher gene expression when compared to non-bioreducible nanocarriers. Moreover, in vivo bioluminescence imaging showed that gene expression is detected up to 8days following mcDNA-micelles intratumoral administration. Furthermore, drug-gene co-delivery in PEOz-PLA-g-PEI-SS carriers was verified by successful encapsulation of both Dox and mcDNA with high efficacy. Moreover, dual-loaded micelleplexes presented significant uptake and a cytotoxic effect in 2D cultures of cancer cells. The co-delivery of mcDNA-Dox to B16F10-Luciferase tumor bearing mice resulted in a reduction in tumor volume and cancer cells viability. Overall, such findings indicate that bioreducible triblock micelles are efficient for focal delivery in vivo and have potential for future application in combinatorial DNA-drug therapy.

The co-delivery of minicircle DNA (mcDNA) and small anti-cancer drugs via stimuli-sensitive nanocarriers is a promising approach for combinatorial cancer therapy. However, the simultaneous loading of drugs and DNA in nanosized delivery systems is remarkably challenging. In this study we describe the synthesis of triblock copolymer micelles based on poly(2-ethyl-2-oxazoline)-poly(L-lactide) grafted with bioreducible polyethylenimine (PEOz-PLA-g-PEI-SS) for co-delivery of supercoiled (sc) mcDNA vectors and Doxorubicin (Dox). These amphiphilic carriers take advantage of non-fouling oxazolines to confer biological stability, of PLA to provide a hydrophobic core for drug encapsulation and of bioreducible PEI-SS to provide mcDNA complexation and an on-demand stimuli-responsive release. The obtained results show that mcDNA-loaded micelleplexes penetrate into in vitro tumor spheroid models with specific kinetics and exhibit a higher gene expression when compared to non-bioreducible nanocarriers. Moreover, in vivo bioluminescence imaging showed that gene expression is detected up to 8days following mcDNA-micelles intratumoral administration. Furthermore, drug-gene co-delivery in PEOz-PLA-g-PEI-SS carriers was verified by successful encapsulation of both Dox and mcDNA with high efficacy. Moreover, dual-loaded micelleplexes presented significant uptake and a cytotoxic effect in 2D cultures of cancer cells. The co-delivery of mcDNA-Dox to B16F10-Luciferase tumor bearing mice resulted in a reduction in tumor volume and cancer cells viability. Overall, such findings indicate that bioreducible triblock micelles are efficient for focal delivery in vivo and have potential for future application in combinatorial DNA-drug therapy.

Delalande A., Leduc C., Midoux P., Postema M. and Pichon C.  (2015)

Efficient Gene Delivery by Sonoporation Is Associated with Microbubble Entry into Cells and the Clathrin-Dependent Endocytosis Pathway

Ultrasound in medicine & biology (2015) 41 (7) 1913-1926 - doi : 10.1016/j.ultrasmedbio.2015.03.010
Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble-cell interactions do not occur randomly during sonoporation ; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings ; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

Microbubble oscillation at specific ultrasound settings leads to permeabilization of surrounding cells. This phenomenon, referred to as sonoporation, allows for the in vitro and in vivo delivery of extracellular molecules, including plasmid DNA. To date, the biological and physical mechanisms underlying this phenomenon are not fully understood. The aim of this study was to investigate the interactions between microbubbles and cells, as well as the intracellular routing of plasmid DNA and microbubbles, during and after sonoporation. High-speed imaging and fluorescence confocal microscopy of HeLa cells stably expressing enhanced green fluorescent protein fused with markers of cellular compartments were used for this investigation. Soft-shelled microbubbles were observed to enter cells during sonoporation using experimental parameters that led to optimal gene transfer. They interacted with the plasma membrane in a specific area stained with fluorescent cholera subunit B, a marker of lipid rafts. This process was not observed with hard-shelled microbubbles, which were not efficient in gene delivery under our conditions. The plasmid DNA was delivered to late endosomes after 3 h post-sonoporation, and a few were found in the nucleus after 6 h. Gene transfer efficacy was greatly inhibited when cells were treated with chlorpromazine, an inhibitor of the clathrin-dependent endocytosis pathway. In contrast, no significant alteration was observed when cells were treated with filipin III or genistein, both inhibitors of the caveolin-dependent pathway. This study emphasizes that microbubble-cell interactions do not occur randomly during sonoporation ; microbubble penetration inside cells affects the efficacy of gene transfer at specific ultrasound settings ; and plasmid DNA uptake is an active mechanism that involves the clathrin-dependent pathway.

Delalande A., Gosselin M.-P., Suwalski A., Guilmain W., Leduc C., Berchel M., Jaffres P.-A., Baril P., Midoux P. and Pichon C.  (2015)

Enhanced Achilles tendon healing by fibromodulin gene transfer

Nanomedicine (2016) 11 (7) 1735-1744 - doi : 10.1016/j.nano.2015.05.004
Tendon injury is a major musculoskeletal disorder with a high public health impact. We propose a non-viral based strategy of gene therapy for the treatment of tendon injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed. In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed. These results suggest that fibromodulin non-viral gene therapy could be proposed as a new therapeutic strategy to accelerate tendon healing. FROM THE CLINICAL EDITOR : Tendon injury is relatively common and healing remains unsatisfactory. In this study, the effects of liposomal-based delivery of fibromodulin gene were investigated in a rat Achilles tendon injury model. The positive results observed would provide a new therapeutic strategy in clinical setting in the future.

Tendon injury is a major musculoskeletal disorder with a high public health impact. We propose a non-viral based strategy of gene therapy for the treatment of tendon injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed. In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed. These results suggest that fibromodulin non-viral gene therapy could be proposed as a new therapeutic strategy to accelerate tendon healing. FROM THE CLINICAL EDITOR : Tendon injury is relatively common and healing remains unsatisfactory. In this study, the effects of liposomal-based delivery of fibromodulin gene were investigated in a rat Achilles tendon injury model. The positive results observed would provide a new therapeutic strategy in clinical setting in the future.

Belmadi N., Midoux P., Loyer P., Passirani C., Pichon C., Le Gall T., Jaffres P.-A., Lehn P., Montier T.  (2015)

Synthetic vectors for gene delivery : An overview of their evolution depending on routes of administration

Biotechnology Journal (2015) first published online : Jun 3, 2015 - doi : 10.1002/biot.201400841
Nucleic acid delivery constitutes an emerging therapeutic strategy to cure various human pathologies. This therapy consists of introducing genetic material into the whole body or isolated cells to correct a cellular abnormality or disfunction. As with any drug, the main objective of nucleic acid delivery is to establish optimal balance between efficacy and tolerance. The methods of administration and the vectors used are selected depending on whether the goal of treatment is the production of an active protein ; the replacement of a missing or inactive gene ; or the combat of acquired diseases, such as cancer or AIDS. In that sense, synthetic vectors represent a valuable solution because they are well characterized, their structure can be fine tuned, and their potential toxicity can be reduced, since toxicity depends on the composition of the formulations. Here we review various synthetic vectors for gene delivery and address the question of their biodistribution as a function of the route of administration. We highlight the modifications to vectors structure and formulations necessary to overcome the major hurdles limiting the effectiveness of nucleic acid therapies.

Nucleic acid delivery constitutes an emerging therapeutic strategy to cure various human pathologies. This therapy consists of introducing genetic material into the whole body or isolated cells to correct a cellular abnormality or disfunction. As with any drug, the main objective of nucleic acid delivery is to establish optimal balance between efficacy and tolerance. The methods of administration and the vectors used are selected depending on whether the goal of treatment is the production of an active protein ; the replacement of a missing or inactive gene ; or the combat of acquired diseases, such as cancer or AIDS. In that sense, synthetic vectors represent a valuable solution because they are well characterized, their structure can be fine tuned, and their potential toxicity can be reduced, since toxicity depends on the composition of the formulations. Here we review various synthetic vectors for gene delivery and address the question of their biodistribution as a function of the route of administration. We highlight the modifications to vectors structure and formulations necessary to overcome the major hurdles limiting the effectiveness of nucleic acid therapies.

Wytrwal M., Leduc C., Sarna M., Goncalves C., Kepczynski M., Midoux P., Nowakowska M., Pichon C.  (2015)

Gene delivery efficiency and intracellular trafficking of novel poly(allylamine) derivatives

International Journal of Pharmaceutics (2015) 478 (1) 372-382 - doi : 10.1016/j.ijpharm.2014.11.053
Non-viral gene carriers for safe and efficient gene transfection have become of particular interest among researchers of different disciplines ranging from physical chemistry to biotechnology. Recently polymeric vectors have been extensively studied as potentially new gene transfer agents. Until now most of the research efforts were made to optimize the gene-to-polymer weight ratio of polyplexes for safe and efficient gene transfection. In this work, we report on the development of novel poly(allylamine) derivatives with different balance of the primary, secondary, tertiary, and quaternary amino groups. All derivatives were able to complex pDNA into polyplexes at low gene-to-polymer weight ratios i.e., 1:1 or 1:2. Moreover, the examined polyplexes were less cytotoxic and showed better transfection efficiency when compared to linear poly(ethyleneimine). These results indicate that the presence of quaternary ammonium groups is important in the formation of stable polyplexes. Polymers with all types of amino groups showed large potential for gene delivery. Furthermore, polyplexes with such derivatives were well internalized by cells and ended up into acidic late endosomes. (C) 2014 Elsevier B.V. All rights reserved.

Non-viral gene carriers for safe and efficient gene transfection have become of particular interest among researchers of different disciplines ranging from physical chemistry to biotechnology. Recently polymeric vectors have been extensively studied as potentially new gene transfer agents. Until now most of the research efforts were made to optimize the gene-to-polymer weight ratio of polyplexes for safe and efficient gene transfection. In this work, we report on the development of novel poly(allylamine) derivatives with different balance of the primary, secondary, tertiary, and quaternary amino groups. All derivatives were able to complex pDNA into polyplexes at low gene-to-polymer weight ratios i.e., 1:1 or 1:2. Moreover, the examined polyplexes were less cytotoxic and showed better transfection efficiency when compared to linear poly(ethyleneimine). These results indicate that the presence of quaternary ammonium groups is important in the formation of stable polyplexes. Polymers with all types of amino groups showed large potential for gene delivery. Furthermore, polyplexes with such derivatives were well internalized by cells and ended up into acidic late endosomes. (C) 2014 Elsevier B.V. All rights reserved.

Rasolonjatovo B., Gomez J. P., Même W., Goncalves C., Huin C., Bennevault-Celton V., Le Gall T., Montier T., Lehn P., Cheradame H., Midoux P., Guegan P.  (2015)

Poly(2-methyl-2-oxazoline)-b-poly(tetrahydrofuran)-b-poly(2-methyl-2-oxazoline) Amphiphilic Triblock Copolymers : Synthesis, Physicochemical Characterizations, and Hydrosolubilizing Properties

Biomacromolecules (2015) 16 (3) 748-756 - doi : 10.1021/bm5016656
Block copolymers assembled into micelles have gained a lot of attention to improve drug delivery. The recent drawbacks of the poly(ethylene oxide) blocks (PEO) contained in amphiphilic pluronics derivatives made of a central poly(propylene oxide) block surrounded by two PEO blocks were recently revealed, opening the way to the design of new amphiphilic block copolymers able to self-assemble in water and to entrap molecules of interest. Here, a family of p(methyloxazoline)-b-p(tetrahydrofuran)-b-p(methyloxazoline) triblock copolymers (called TBCP) is synthesized using cationic ring opening polymerization. Studies of micelle formation using dynamic light scattering, isothermal titration calorimetry (ITC), NMR diffusion-ordered spectroscopy (DOSY), and fluorescence experiments lead us to draw a relationship between copolymer structure and the physicochemical properties of the block copolymers (critical micellar concentration (CMC), Nagg, core diameter, shell thickness, etc.). The packing parameter of the block copolymers indicates the formation of a core-corona structure. Hydrosolubilizing properties of TBCPs were exemplified with curcumin selected as a highly insoluble drug model. Curcumin, a natural polyphenolic compound, has shown a large spectrum of biological and pharmacological activity, including anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic activities. An optimized formulation process reveals that the aggregation number is the parameter affecting drug encapsulation. Patch clamp experiments carried out to study the interaction of TBCP with the cell membrane demonstrate their permeation property suitable to promote the cellular internalization of curcumin.

Block copolymers assembled into micelles have gained a lot of attention to improve drug delivery. The recent drawbacks of the poly(ethylene oxide) blocks (PEO) contained in amphiphilic pluronics derivatives made of a central poly(propylene oxide) block surrounded by two PEO blocks were recently revealed, opening the way to the design of new amphiphilic block copolymers able to self-assemble in water and to entrap molecules of interest. Here, a family of p(methyloxazoline)-b-p(tetrahydrofuran)-b-p(methyloxazoline) triblock copolymers (called TBCP) is synthesized using cationic ring opening polymerization. Studies of micelle formation using dynamic light scattering, isothermal titration calorimetry (ITC), NMR diffusion-ordered spectroscopy (DOSY), and fluorescence experiments lead us to draw a relationship between copolymer structure and the physicochemical properties of the block copolymers (critical micellar concentration (CMC), Nagg, core diameter, shell thickness, etc.). The packing parameter of the block copolymers indicates the formation of a core-corona structure. Hydrosolubilizing properties of TBCPs were exemplified with curcumin selected as a highly insoluble drug model. Curcumin, a natural polyphenolic compound, has shown a large spectrum of biological and pharmacological activity, including anti-inflammatory, antimicrobial, antioxidant, and anticarcinogenic activities. An optimized formulation process reveals that the aggregation number is the parameter affecting drug encapsulation. Patch clamp experiments carried out to study the interaction of TBCP with the cell membrane demonstrate their permeation property suitable to promote the cellular internalization of curcumin.

Midoux P., Pichon C.  (2015)

Lipid-based mRNA vaccine delivery systems

Expert Review of Vaccines (2015) 14 (2) 221-234 - doi : 10.1586/14760584.2015.986104
Synthetic mRNAs can become biopharmaceutics allowing vaccination against cancer, bacterial and virus infections. Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Although immune responses are generated by naked mRNAs, their formulations with chemical carriers are expected to provide more specificity and internalization in dendritic cells (DCs) for better immune responses and dose reduction. This review reports lipid-based formulations (LBFs) that have proved preclinical efficacy. The selective delivery of mRNA LBFs to favor intracellular accumulation in DCs and reduction of the effective doses is discussed, notably to decorate LBFs with carbohydrates or glycomimetics allowing endocytosis in DCs. We also report how smart intracellular delivery is achieved using pH-sensitive lipids or polymers for an efficient mRNA escape from endosomes and limitations regarding cytosolic mRNA location for translation.

Synthetic mRNAs can become biopharmaceutics allowing vaccination against cancer, bacterial and virus infections. Clinical trials with direct administration of synthetic mRNAs encoding tumor antigens demonstrated safety and induction of tumor-specific immune responses. Although immune responses are generated by naked mRNAs, their formulations with chemical carriers are expected to provide more specificity and internalization in dendritic cells (DCs) for better immune responses and dose reduction. This review reports lipid-based formulations (LBFs) that have proved preclinical efficacy. The selective delivery of mRNA LBFs to favor intracellular accumulation in DCs and reduction of the effective doses is discussed, notably to decorate LBFs with carbohydrates or glycomimetics allowing endocytosis in DCs. We also report how smart intracellular delivery is achieved using pH-sensitive lipids or polymers for an efficient mRNA escape from endosomes and limitations regarding cytosolic mRNA location for translation.

Gaspar V., de Melo-Diogo D., Costa E., Moreira, A., Queiroz J., Pichon C., Correia I., Sousa F.  (2015)

Minicircle DNA vectors for gene therapy : advances and applications

Expert opinion on biological therapy (2015) 15 (3) 353-379 - doi : 10.1517/14712598.2015.996544
INTRODUCTION : Nucleic-acid-based biopharmaceuticals enclose a remarkable potential for treating debilitating or life-threatening diseases that currently remain incurable. This promising area of research envisages the creation of state-of-the-art DNA vaccines, pluripotent cells or gene-based therapies, which can be used to overcome current issues. To achieve this goal, DNA minicircles are emerging as ideal nonviral vectors due to their safety and persistent transgene expression in either quiescent or actively dividing cells.

AREAS COVERED : This review focuses on the characteristics of minicircle DNA (mcDNA) technology and the current advances in their production. The possible modifications to further improve minicircle efficacy are also emphasized and discussed in light of recent advances. As a final point, the main therapeutic applications of mcDNA are summarized, with a special focus on pluripotent stem cells production and cancer therapy.

EXPERT OPINION : Achieving in-target and persistent transgene expression is a challenging issue that is of critical importance for a successful therapeutic outcome. The use of miniaturized mcDNA cassettes with additional modifications that increase and prolong expression may contribute to an improved generation of biopharmaceuticals. The unique features of mcDNA render it an attractive alternative to overcome current technical issues and to bridge the significant gap that exists between basic research and clinical applications.

INTRODUCTION : Nucleic-acid-based biopharmaceuticals enclose a remarkable potential for treating debilitating or life-threatening diseases that currently remain incurable. This promising area of research envisages the creation of state-of-the-art DNA vaccines, pluripotent cells or gene-based therapies, which can be used to overcome current issues. To achieve this goal, DNA minicircles are emerging as ideal nonviral vectors due to their safety and persistent transgene expression in either quiescent or actively dividing cells.
AREAS COVERED : This review focuses on the characteristics of minicircle DNA (mcDNA) technology and the current advances in their production. The possible modifications to further improve minicircle efficacy are also emphasized and discussed in light of recent advances. As a final point, the main therapeutic applications of mcDNA are summarized, with a special focus on pluripotent stem cells production and cancer therapy.
EXPERT OPINION : Achieving in-target and persistent transgene expression is a challenging issue that is of critical importance for a successful therapeutic outcome. The use of miniaturized mcDNA cassettes with additional modifications that increase and prolong expression may contribute to an improved generation of biopharmaceuticals. The unique features of mcDNA render it an attractive alternative to overcome current technical issues and to bridge the significant gap that exists between basic research and clinical applications.

Ben Halima, N. Borchani, M. Fendri, I. Khemakhem, B. Gosset, D. Baril, P. Pichon, C. Ayadi, M. A. Abdelkafi, S  (2015)

Optimised amylases extraction from oat seeds and its impact on bread properties

International journal of biological macromolecules (2015) 72 1213-1221 - doi : 10.1016/j.ijbiomac.2014.10.018
Statistical approaches were employed for the optimisation of the extraction of amylolytic activity from oat (Avena sativa) seeds. The application of the response surface methodology allows us to determine a set of optimal conditions (ratio seed weight/buffer volume 0.1, germination days 10 days, temperature 20 degrees C and pH 5.6). Experiments carried out under these conditions led to amylase production yield of 91 U/g. Its maximal activity was in the pH 5.6 and at 55 degrees C. Study of the incorporation of the optimised oat extract into the bread formulation revealed an improvement of the sensory quality and the textural properties of fresh and stored bread. Three-dimensional elaborations of Confocal Laser Scanning Microscopy (CLSM) images were performed on crumb of the different breads to evaluate the influence of amylase activity on microstructure. The result showed improved baking characteristics as well as overall microscopic and macroscopic appearance.

Statistical approaches were employed for the optimisation of the extraction of amylolytic activity from oat (Avena sativa) seeds. The application of the response surface methodology allows us to determine a set of optimal conditions (ratio seed weight/buffer volume 0.1, germination days 10 days, temperature 20 degrees C and pH 5.6). Experiments carried out under these conditions led to amylase production yield of 91 U/g. Its maximal activity was in the pH 5.6 and at 55 degrees C. Study of the incorporation of the optimised oat extract into the bread formulation revealed an improvement of the sensory quality and the textural properties of fresh and stored bread. Three-dimensional elaborations of Confocal Laser Scanning Microscopy (CLSM) images were performed on crumb of the different breads to evaluate the influence of amylase activity on microstructure. The result showed improved baking characteristics as well as overall microscopic and macroscopic appearance.

Baril P., Ezzine S., Pichon C.  (2015)

Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities

International Journal of Molecular Sciences (2015) 16 (3) 4947-4972 - doi : 10.3390/ijms16034947
MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.

Gaspar, V. M., Costa, E. C., Queiroz, J. A., Pichon, C., Sousa, F. and Correia, I. J.  (2015)

Folate-Targeted Multifunctional Amino Acid-Chitosan Nanoparticles for Improved Cancer Therapy

Pharmaceutical Research (2015) 32 (2) 562-577 - doi :10.1007/s11095-014-1486-0
PURPOSE :

Tumor targeting nanomaterials have potential for improving the efficiency of anti-tumoral therapeutics. However, the evaluation of their biological performance remains highly challenging. In this study we describe the synthesis of multifunctional nanoparticles decorated with folic acid-PEG and dual amino acid-modified chitosan (CM-PFA) complexed with DNA and their evaluation in organotypic 2D co-cultures of cancer-normal cells and also on 3D multicellular tumor spheroids models. 

METHODS : The physicochemical characterization of CM-PFA multifunctional carriers was performed by FTIR, 1H NMR and DLS. 2D co-culture models were established by using a 1:2 cancer-to-normal cell ratio. 3D organotypic tumor spheroids were assembled using micromolding technology for high throughput screening. Nanoparticle efficiency was evaluated by flow cytometry and confocal microscopy. 

RESULTS : The CM-PFA nanocarriers (126-176 nm) showed hemocompatibility and were internalized by target cells, achieving a 3.7 fold increase in gene expression. In vivo-mimicking 2D co-cultures confirmed a real affinity towards cancer cells and a negligible uptake in normal cells. The targeted nanoparticles penetrated into 3D spheroids to a higher extent than non-targeted nanocarriers. Also, CM-PFA-mediated delivery of p53 tumor suppressor promoted a decrease in tumor-spheroids volume. 

CONCLUSION : These findings corroborate the improved efficiency of this delivery system and demonstrate its potential for application in cancer therapy.

PURPOSE :
Tumor targeting nanomaterials have potential for improving the efficiency of anti-tumoral therapeutics. However, the evaluation of their biological performance remains highly challenging. In this study we describe the synthesis of multifunctional nanoparticles decorated with folic acid-PEG and dual amino acid-modified chitosan (CM-PFA) complexed with DNA and their evaluation in organotypic 2D co-cultures of cancer-normal cells and also on 3D multicellular tumor spheroids models.

METHODS : The physicochemical characterization of CM-PFA multifunctional carriers was performed by FTIR, 1H NMR and DLS. 2D co-culture models were established by using a 1:2 cancer-to-normal cell ratio. 3D organotypic tumor spheroids were assembled using micromolding technology for high throughput screening. Nanoparticle efficiency was evaluated by flow cytometry and confocal microscopy.

RESULTS : The CM-PFA nanocarriers (126-176 nm) showed hemocompatibility and were internalized by target cells, achieving a 3.7 fold increase in gene expression. In vivo-mimicking 2D co-cultures confirmed a real affinity towards cancer cells and a negligible uptake in normal cells. The targeted nanoparticles penetrated into 3D spheroids to a higher extent than non-targeted nanocarriers. Also, CM-PFA-mediated delivery of p53 tumor suppressor promoted a decrease in tumor-spheroids volume.

CONCLUSION : These findings corroborate the improved efficiency of this delivery system and demonstrate its potential for application in cancer therapy.


2014   Références trouvées : 14

Goncalves C., Gross F., Guegan P., Cheradame H., Midoux P.  (2014)

A robust transfection reagent for the transfection of CHO and HEK293 cells and production of recombinant proteins and lentiviral particles - PTG1

Biotechnology journal (2014) 9 (11) 1380-1388 - doi : 10.1002/biot.201400324
Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 x 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 mug/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000, and gene expression is higher than observed with FreeStyle and JetPEI(R). Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.

Bioproduction of recombinant proteins (r-proteins) and recombinant lentiviral particles (r-lentiviral particles) requires robust transfections consisting of efficient protocols that are easy to implement, with good reproducibility for a maximum production of proteins and lentiviral particles in a short time with low cytotoxicity. This study evaluates the capacity of histidinylated polyethyleneimine I (PTG1) to facilitate robust DNA transfection, with low cytotoxicity, of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells for the production of r-proteins and r-lentiviral particles. We report that PTG1 transfection of cells in suspension with a plasmid DNA encoding enhanced green fluorescent protein leads to 72 and 97% of transfected CHO and HEK293T cells respectively, and does not significantly affect cell viability. PTG1 transfection of 100 mL of CHO-S cell culture in suspension at a cell density of 2 x 10(6) cells /mL resulted in a high level of transfected cells and protein expression after transfection with 0.75 mug/mL plasmid DNA. Transfection with PTG1 is more efficient than LipofectAmine2000, and gene expression is higher than observed with FreeStyle and JetPEI(R). Tri-transfection of HEK293T packaging cells leads to the production of a higher level of r-lentiviral particles compared to the calcium phosphate method, and permits two harvests of viral particles within three days. These results show that PTG1 is a powerful new transfection reagent for cell lines frequently used for recombinant protein and lentiviral particle production. PTG1 could be used in protocols for bioproduction of therapeutic proteins such as antibodies for cancer treatments and viral vectors for gene therapy applications.

Montier, T., Pichon, C., Blondel, M. and Midoux, P.  (2014)

Editorial : Current status and prospects on nucleic acid transfer

Biotechnology Journal (2014à 9 (11) 1363-1364 - doi : 10.1002/biot.201300510

Ben Halima, N., Ben Slima, A., Moalla, I., Fetoui, H., Pichon, C., Gdoura, R. and Abdelkafi, S.  (2014)

Protective effects of oat oil on deltamethrin-induced reprotoxicity in male mice

Food Funct (2014) 5 (9) 2070 - 2077 - doi : 10.1039/c4fo00190g
Oats (Avena sativa L.), which are used in foods, are a potential economically viable source of oil. The purpose of this study was to determine the efficiency of oats oil to alleviate oxidative damage of testis induced by deltamethrin, which is a pyrethroid pesticide that exerts a wide range of effects on non-targeted organisms. The reprotoxicity caused by orally administered deltamethrin (DEL) to mice can be effectively antagonized by the beneficial effects of oats oil (OO) as an antioxidant. Thirty-two male albino mice were divided into four equal groups : a control group, a group of mice given deltamethrin (5 mg per kg b.w.), a group administered deltamethrin after receiving oats oil (6 g per kg b.w.), and a group receiving only OO. Exposure to deltamethrin at a dose of 5 mg per kg b.w. per day caused oxidative stress in testis, proven by a decrease in the epididymal sperm count and motility, an increase in the number of abnormal morphologies in spermatozoa and a significant increase of lipid peroxidation (LP) in the testis when compared to control animals. Co-administration of oats oil to the DEL-treated mice ameliorated the testicular biochemical parameters as well as the histological impairments in testis. We concluded that oats oil ameliorated the toxic effects of deltamethrin in testis explored by reduced LP and improved total sperm density, motility and morphology in mice spermatozoa, suggesting its role as a potential antioxidant.

Oats (Avena sativa L.), which are used in foods, are a potential economically viable source of oil. The purpose of this study was to determine the efficiency of oats oil to alleviate oxidative damage of testis induced by deltamethrin, which is a pyrethroid pesticide that exerts a wide range of effects on non-targeted organisms. The reprotoxicity caused by orally administered deltamethrin (DEL) to mice can be effectively antagonized by the beneficial effects of oats oil (OO) as an antioxidant. Thirty-two male albino mice were divided into four equal groups : a control group, a group of mice given deltamethrin (5 mg per kg b.w.), a group administered deltamethrin after receiving oats oil (6 g per kg b.w.), and a group receiving only OO. Exposure to deltamethrin at a dose of 5 mg per kg b.w. per day caused oxidative stress in testis, proven by a decrease in the epididymal sperm count and motility, an increase in the number of abnormal morphologies in spermatozoa and a significant increase of lipid peroxidation (LP) in the testis when compared to control animals. Co-administration of oats oil to the DEL-treated mice ameliorated the testicular biochemical parameters as well as the histological impairments in testis. We concluded that oats oil ameliorated the toxic effects of deltamethrin in testis explored by reduced LP and improved total sperm density, motility and morphology in mice spermatozoa, suggesting its role as a potential antioxidant.

Gaspar, V. M., Goncalves, C., de Melo-Diogo, D., Costa, E. C., Queiroz, J. A., Pichon, C., Sousa, F. and Correia, I. J.  (2014)

Poly(2-ethyl-2-oxazoline)-PLA-g-PEI amphiphilic triblock micelles for co-delivery of minicircle DNA and chemotherapeutics

J Control Release (2014) 189 90-104 - doi : 10.1016/j.jconrel.2014.06.040
The design of nanocarriers for the delivery of drugs and nucleic-acids remains a very challenging goal due to their physicochemical differences. In addition, the reported accelerated clearance and immune response of pegylated nanomedicines highlight the necessity to develop carriers using new materials. Herein, we describe the synthesis of amphiphilic triblock poly(2-ethyl-2-oxazoline)-PLA-g-PEI (PEOz-PLA-g-PEI) micelles for the delivery of minicircle DNA (mcDNA) vectors. In this copolymer the generally used PEG moieties are replaced by the biocompatible PEOz polymer backbone that assembles the hydrophilic shell. The obtained results show that amphiphilic micelles have low critical micellar concentration, are hemocompatible and exhibit stability upon incubation in serum. The uptake in MCF-7 cells was efficient and the nanocarriers achieved 2.7 fold higher expression than control particles. Moreover, mcDNA-loaded micelleplexes penetrated into 3D multicellular spheroids and promoted widespread gene expression. Additionally, to prove the concept of co-delivery, mcDNA and doxorubicin (Dox) were simultaneously encapsulated in PEOz-PLA-g-PEI carriers, with high efficiency. Dox-mcDNA micelleplexes exhibited extensive cellular uptake and demonstrated anti-tumoral activity. These findings led us to conclude that this system has a potential not only for the delivery of novel mcDNA vectors, but also for the co-delivery of drug-mcDNA combinations without PEG functionalization.

The design of nanocarriers for the delivery of drugs and nucleic-acids remains a very challenging goal due to their physicochemical differences. In addition, the reported accelerated clearance and immune response of pegylated nanomedicines highlight the necessity to develop carriers using new materials. Herein, we describe the synthesis of amphiphilic triblock poly(2-ethyl-2-oxazoline)-PLA-g-PEI (PEOz-PLA-g-PEI) micelles for the delivery of minicircle DNA (mcDNA) vectors. In this copolymer the generally used PEG moieties are replaced by the biocompatible PEOz polymer backbone that assembles the hydrophilic shell. The obtained results show that amphiphilic micelles have low critical micellar concentration, are hemocompatible and exhibit stability upon incubation in serum. The uptake in MCF-7 cells was efficient and the nanocarriers achieved 2.7 fold higher expression than control particles. Moreover, mcDNA-loaded micelleplexes penetrated into 3D multicellular spheroids and promoted widespread gene expression. Additionally, to prove the concept of co-delivery, mcDNA and doxorubicin (Dox) were simultaneously encapsulated in PEOz-PLA-g-PEI carriers, with high efficiency. Dox-mcDNA micelleplexes exhibited extensive cellular uptake and demonstrated anti-tumoral activity. These findings led us to conclude that this system has a potential not only for the delivery of novel mcDNA vectors, but also for the co-delivery of drug-mcDNA combinations without PEG functionalization.

Mahindhoratep, S., Bouda, H. A., El Shafey, N., Scherman, D., Kichler, A., Pichon, C., Midoux, P., Mignet, N. and Bureau, M. F.  (2014)

NF-kB related transgene expression in mouse tibial cranial muscle after pDNA injection followed or not by electrotransfer

Biochim Biophys Acta 1840 (11) 3257-3263 - doi : 10.1016/j.bbagen.2014.06.013
BACKGROUND : When activated, NF-kappaB can promote the nuclear import and transcription of DNA possessing NF-kappaB consensus sequences. Here, we investigated whether NF-kappaB is involved in the plasmid electrotransfer process. METHODS : Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-kappaB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBalpha were studied as a marker of NF-kappaB activation. RESULTS : Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-kappaB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IkappaBalpha was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IkappaBalpha and thus of free NF-kappaB in the absence of any stimulation. GENERAL SIGNIFICANCE : pDNA electrotransfer can increase transgene expression independently of NF-kappaB. The insertion of NF-kappaB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-kappaB in muscle might be sufficient to enhance the activity of pDNA bearing NF-kappaB consensus sequences.

BACKGROUND : When activated, NF-kappaB can promote the nuclear import and transcription of DNA possessing NF-kappaB consensus sequences. Here, we investigated whether NF-kappaB is involved in the plasmid electrotransfer process. METHODS : Mouse tibial cranial muscles were transfected with plasmids encoding luciferase bearing or not NF-kappaB consensus sequences. Luciferase transgene expression was evaluated noninvasively by luminescence imaging and the number of pDNA copies in the same muscles by qPCR. RT-PCR of heat shock protein HsP70 mRNA evidenced cell stress. Western blots of phosphorylated IkBalpha were studied as a marker of NF-kappaB activation. RESULTS : Intra-muscular injection of a plasmid bearing a weak TATA-like promoter results in a very low muscle transfection level. Electrotransfer significantly increased both the number of pDNA copy and the transgene expression of this plasmid per DNA copy. Insertion of NF-kappaB consensus sequences into pDNA significantly increased the level of gene expression both with and without electrotransfer. Electrotransfer-induced cellular stress was evidenced by increased HsP70 mRNA. Phosphorylated IkappaBalpha was slightly increased by simple pDNA injection and a little more by electrotransfer. We also observed a basal level of phosphorylated IkappaBalpha and thus of free NF-kappaB in the absence of any stimulation. GENERAL SIGNIFICANCE : pDNA electrotransfer can increase transgene expression independently of NF-kappaB. The insertion of NF-kappaB consensus sequences into pDNA bearing a weak TATA-like promoter leads to enhanced transgene expression in muscle with or without gene electrotransfer. Finally, our results suggest that the basal amount of free NF-kappaB in muscle might be sufficient to enhance the activity of pDNA bearing NF-kappaB consensus sequences.

Xia, M., Gonzalez, P., Li, C.Y., Meng, G., Jiang, A.Q., Wang, H.W., Gao, Q., Debatin, K.M., Beltinger, C. and Wei, J.W.  (2014)

Mitophagy Enhances Oncolytic Measles Virus Replication by Mitigating DDX58/RIG-I-Like Receptor Signaling

Journal of Virology (2014) 88 (9) 5152-5164 - doi : 10.1128/jvi.03851-13
The success of future clinical trials with oncolytic viruses depends on the identification and the control of mechanisms that modulate their therapeutic efficacy. In particular, little is known about the role of autophagy in infection by attenuated measles virus of the Edmonston strain (MV-Edm). We investigated the interaction between autophagy, innate immune response, and oncolytic activity of MV-Edm, since the antiviral immune response is a known factor limiting virotherapies. We report that MV-Edm exploits selective autophagy to mitigate the innate immune response mediated by DDX58/RIG-I like receptors (RLRs) in non-small cell lung cancer (NSCLC) cells. Both RNA interference (RNAi) and overexpression approaches demonstrate that autophagy enhances viral replication and inhibits the production of type I interferons regulated by RLRs. We show that MV-Edm unexpectedly triggers SQSTM1/p62-mediated mitophagy, resulting in decreased mitochondrion-tethered mitochondrial antiviral signaling protein (MAVS) and subsequently weakening the innate immune response. These results unveil a novel infectious strategy based on the usurpation of mitophagy leading to mitigation of the innate immune response. This finding provides a rationale to modulate autophagy in oncolytic virotherapy.

The success of future clinical trials with oncolytic viruses depends on the identification and the control of mechanisms that modulate their therapeutic efficacy. In particular, little is known about the role of autophagy in infection by attenuated measles virus of the Edmonston strain (MV-Edm). We investigated the interaction between autophagy, innate immune response, and oncolytic activity of MV-Edm, since the antiviral immune response is a known factor limiting virotherapies. We report that MV-Edm exploits selective autophagy to mitigate the innate immune response mediated by DDX58/RIG-I like receptors (RLRs) in non-small cell lung cancer (NSCLC) cells. Both RNA interference (RNAi) and overexpression approaches demonstrate that autophagy enhances viral replication and inhibits the production of type I interferons regulated by RLRs. We show that MV-Edm unexpectedly triggers SQSTM1/p62-mediated mitophagy, resulting in decreased mitochondrion-tethered mitochondrial antiviral signaling protein (MAVS) and subsequently weakening the innate immune response. These results unveil a novel infectious strategy based on the usurpation of mitophagy leading to mitigation of the innate immune response. This finding provides a rationale to modulate autophagy in oncolytic virotherapy.

Tertil, M., Skrzypek, K., Florczyk, U., Weglarczyk, K., Was, H., Collet, G., Guichard, A., Gil, T., Kuzdzal, J., Jozkowicz, A., Kieda, C., Pichon, C. and Dulak, J.  (2014)

Regulation and Novel Action of Thymidine Phosphorylase in Non-Small Cell Lung Cancer : Crosstalk with Nrf2 and HO-1

PloS One (2014) 9 (5) e97070 - doi : 10.1371/journal.pone.0097070
Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell prolifera\tion and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1 beta and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1 beta and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC.

Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell prolifera\tion and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1 beta and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1 beta and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC.

Maury, B., Goncalves, C., Tresset, G., Zeghal, M., Cheradame, H., Guegan, P., Pichon, C. and Midoux, P.  (2014)

Influence of pDNA availability on transfection efficiency of polyplexes in non-proliferative cells

Biomaterials (2014) 35 (22) 5977-5985 - doi : 10.1016/j.biomaterials.2014.04.007
We succeeded in visualizing plasmid DNA (pDNA) in the nucleus and cytosol of non-proliferative cells after transfection with linear polyethylenemine (IPEI) and histidinylated IPEI (His(16)-IPEI). This was possible with confocal microscope by using pDNA labelled with quantum dots. Indeed pDNA labelled with Cy3 leads to false positive nuclear localization because the saturation of the fluorescence signal overestimated the volume occupied by Cy3-pDNA. Moreover, Cy3 brightness was too weak to detect low amount of pDNA. About 20 to 40 pDNA copies were detected in the nucleus after the transfection of pDNA labelled with quantum dots. Transfection efficiency and cellular imaging data suggested that the cytosolic availability of pDNA, including endosome escape and/or polyplexes dissociation, is crucial for its nuclear delivery. In vitro transcription assay and transfection of cells allowing cytosolic gene expression concluded to better cytosolic availability of pDNA within His(16)-IPEI polyplexes. Cryo-TEM analyses revealed that His(16)-IPEI polyplexes exhibited a spherical shape and an amorphous internal structure which differed from the high degree of order of IPEI polyplexes. Altogether, this comparative study indicated that the high transfection efficiency of non-proliferative cells with His(16)-IPEI polyplexes was related to the amorphous structure and the facilitated dissociation of the assemblies.

We succeeded in visualizing plasmid DNA (pDNA) in the nucleus and cytosol of non-proliferative cells after transfection with linear polyethylenemine (IPEI) and histidinylated IPEI (His(16)-IPEI). This was possible with confocal microscope by using pDNA labelled with quantum dots. Indeed pDNA labelled with Cy3 leads to false positive nuclear localization because the saturation of the fluorescence signal overestimated the volume occupied by Cy3-pDNA. Moreover, Cy3 brightness was too weak to detect low amount of pDNA. About 20 to 40 pDNA copies were detected in the nucleus after the transfection of pDNA labelled with quantum dots. Transfection efficiency and cellular imaging data suggested that the cytosolic availability of pDNA, including endosome escape and/or polyplexes dissociation, is crucial for its nuclear delivery. In vitro transcription assay and transfection of cells allowing cytosolic gene expression concluded to better cytosolic availability of pDNA within His(16)-IPEI polyplexes. Cryo-TEM analyses revealed that His(16)-IPEI polyplexes exhibited a spherical shape and an amorphous internal structure which differed from the high degree of order of IPEI polyplexes. Altogether, this comparative study indicated that the high transfection efficiency of non-proliferative cells with His(16)-IPEI polyplexes was related to the amorphous structure and the facilitated dissociation of the assemblies.

Kasparkova, J. Thibault, T.Kostrhunova, H., Stepankova, J., Vojtiskova, M., Muchova, T., Midoux, P., Malinge, J.M. and Brabec, V.  (2014)

Different affinity of nuclear factor-kappa B proteins to DNA modified by antitumor cisplatin and its clinically ineffective trans isomer

Febs Journal (2014) 281 (5) 1393-1408 - doi : 10.1111/febs.12711
Nuclear factor-kappa B (NF-kB) comprises a family of protein transcription factors that have a regulatory function in numerous cellular processes and are implicated in the cancer cell response to antineoplastic drugs, including cisplatin. We characterized the effects of DNA adducts of cisplatin and ineffective transplatin on the affinity of NF-kB proteins to their consensus DNA sequence (kB site). Although the kB site-NF-B protein interaction was significantly perturbed by DNA adducts of cisplatin, transplatin adducts were markedly less effective both in cell-free media and in cellulo using a decoy strategy derivatized-approach. Moreover, NF-B inhibitor JSH-23 [4-methyl-N-1-(3-phenylpropyl)benzene-1,2-diamine] augmented cisplatin cytotoxicity in ovarian cancer cells and the data showed strong synergy with JSH-23 for cisplatin. The distinctive structural features of DNA adducts of the two platinum complexes suggest a unique role for conformational distortions induced in DNA by the adducts of cisplatin with respect to inhibition of the binding of NF-kB to the platinated kB sites. Because thousands of B sites are present in the DNA, the mechanisms underlying the antitumor efficiency of cisplatin in some tumor cells may involve downstream processes after inhibition of the binding of NF-B to B site(s) by DNA adducts of cisplatin, including enhanced programmed cell death in response to drug treatment.

Nuclear factor-kappa B (NF-kB) comprises a family of protein transcription factors that have a regulatory function in numerous cellular processes and are implicated in the cancer cell response to antineoplastic drugs, including cisplatin. We characterized the effects of DNA adducts of cisplatin and ineffective transplatin on the affinity of NF-kB proteins to their consensus DNA sequence (kB site). Although the kB site-NF-B protein interaction was significantly perturbed by DNA adducts of cisplatin, transplatin adducts were markedly less effective both in cell-free media and in cellulo using a decoy strategy derivatized-approach. Moreover, NF-B inhibitor JSH-23 [4-methyl-N-1-(3-phenylpropyl)benzene-1,2-diamine] augmented cisplatin cytotoxicity in ovarian cancer cells and the data showed strong synergy with JSH-23 for cisplatin. The distinctive structural features of DNA adducts of the two platinum complexes suggest a unique role for conformational distortions induced in DNA by the adducts of cisplatin with respect to inhibition of the binding of NF-kB to the platinated kB sites. Because thousands of B sites are present in the DNA, the mechanisms underlying the antitumor efficiency of cisplatin in some tumor cells may involve downstream processes after inhibition of the binding of NF-B to B site(s) by DNA adducts of cisplatin, including enhanced programmed cell death in response to drug treatment.

Gaspar, V. M. Maia, C. J. Queiroz, J. A. Pichon, C. Correia, I. J. Sousa, F.  (2014)

Improved minicircle DNA biosynthesis for gene therapy applications

Human Gene Therapy Methods (2014) 25 (2) 93-105 - doi : 10.1089/hgtb.2013.020
Minicircular DNA (mcDNA) biopharmaceuticals have recently risen as a valuable alternative for the development of a next generation of bioactive therapeutics because they are more efficient and safer than standard plasmid DNA (pDNA). To date, the relatively insufficient knowledge regarding mcDNA biosynthesis is currently hindering its manufacture in suitable amounts for clinical trial evaluations. Addressing this limitation is therefore mandatory to bring forth the full therapeutic potential of this cutting-edge technology. Herein, we describe for the first time new processing parameters that improve the overall yield of mcDNA obtained from bacterial fermentations. We provide details for further in-line monitoring and optimization in view of the current good manufacturing guidelines. Our results show that by rising growth temperature to 42 degrees C, an increase in the overall minicircle producer plasmid yield is attained, while biomass amounts are reduced. Moreover, by monitoring in real time the dynamic recombination of parental plasmids to mcDNA, we found that this event is more efficient at specific time points, regardless of the growth temperature and inductor concentration used. These are important findings since mcDNA can be recovered with higher yields at these determined key stages. Indeed, the manipulation of these parameters resulted in a 2.21-fold increase in mcDNA production compared with the established growth temperatures for this technology. Overall, our findings highlight that to achieve maximum productivity while attaining pharmaceutical-grade mcDNA preparations, process design and biosynthesis optimization must take into account key parameters such as temperature, inductor concentration, and recovery time.

Minicircular DNA (mcDNA) biopharmaceuticals have recently risen as a valuable alternative for the development of a next generation of bioactive therapeutics because they are more efficient and safer than standard plasmid DNA (pDNA). To date, the relatively insufficient knowledge regarding mcDNA biosynthesis is currently hindering its manufacture in suitable amounts for clinical trial evaluations. Addressing this limitation is therefore mandatory to bring forth the full therapeutic potential of this cutting-edge technology. Herein, we describe for the first time new processing parameters that improve the overall yield of mcDNA obtained from bacterial fermentations. We provide details for further in-line monitoring and optimization in view of the current good manufacturing guidelines. Our results show that by rising growth temperature to 42 degrees C, an increase in the overall minicircle producer plasmid yield is attained, while biomass amounts are reduced. Moreover, by monitoring in real time the dynamic recombination of parental plasmids to mcDNA, we found that this event is more efficient at specific time points, regardless of the growth temperature and inductor concentration used. These are important findings since mcDNA can be recovered with higher yields at these determined key stages. Indeed, the manipulation of these parameters resulted in a 2.21-fold increase in mcDNA production compared with the established growth temperatures for this technology. Overall, our findings highlight that to achieve maximum productivity while attaining pharmaceutical-grade mcDNA preparations, process design and biosynthesis optimization must take into account key parameters such as temperature, inductor concentration, and recovery time.

Brevet, D., Hocine, O., Delalande, A., Raehm, L., Charnay, C., Midoux, P., Durand, J.O. and Pichon, C.  (2014)

Improved gene transfer with histidine-functionalized mesoporous silica nanoparticles

International Journal of Pharmaceutics (2014) 471 (1-2) 197-205 - doi : 10.1016/j.ijpharm.2014.05.020
Mesoporous silica nanoparticles (MSN) were functionalized with aminopropyltriethoxysilane (MSN-NH2) then l-histidine (MSN-His) for pDNA delivery in cells and in vivo. The complexation of pDNA with MSN-NH2 and MSN-His was first studied with gel shift assay. pDNA complexed with MSN-His was better protected from DNase degradation than with MSN-NH2. An improvement of the transfection efficiency in cells was observed with MSN-His/pDNA compared to MSN-NH2/pDNA, which could be explained by a better internalization of MSN-His. The improvement of the transfection efficiency with MSN-His was also observed for gene transfer in Achilles tendons in vivo.

Mesoporous silica nanoparticles (MSN) were functionalized with aminopropyltriethoxysilane (MSN-NH2) then l-histidine (MSN-His) for pDNA delivery in cells and in vivo. The complexation of pDNA with MSN-NH2 and MSN-His was first studied with gel shift assay. pDNA complexed with MSN-His was better protected from DNase degradation than with MSN-NH2. An improvement of the transfection efficiency in cells was observed with MSN-His/pDNA compared to MSN-NH2/pDNA, which could be explained by a better internalization of MSN-His. The improvement of the transfection efficiency with MSN-His was also observed for gene transfer in Achilles tendons in vivo.

Majhen D., Stojanovic N., Vukic D., Pichon C., Leduc C., Osmak M. and Ambriovic-Ristov A.  (2014)

Increased Adenovirus Type 5 Mediated Transgene Expression Due to RhoB Down-Regulation

PLoS One 9 (1) e86698 - doi : 10.1371/journal.pone.0086698
Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.

Maurel, D. B. Benaitreau, D. Jaffre, C. Toumi, H. Portier, H. Uzbekov, R. Pichon, C. Benhamou, C. L. Lespessailles, E. Pallu, S.  (2014)

Effect of the alcohol consumption on osteocyte cell processes : a molecular imaging study

Journal of cellular and molecular medicine (2014) 969 247-274 - doi : 10.1007/978-1-62703-260-5_16
We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups : Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.

We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups : Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.

Goncalves C., Berchel M., Gosselin M.P., Malard V., Cheradame H., Jaffres P.A., Guegan P., Pichon C. and Midoux P.  (2014)

Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection

International journal of pharmaceutics (2014) 460 (1-2) 264-272 - doi : 10.1016/j.ijpharm.2013.11.005
Lipopolyplexes formulations resulting from association of nucleic acid, cationic liposomes and a cationic polymer are attracting formulations for siRNA delivery. Herein, imidazole- and imidazolium-based liposomes in association with histidinylated polymers are studied to produce siRNA lipopoplyplexes (LPRi) subsequently used for gene silencing. Several kinds of imidazole/histidine liposomes and cationic polymers are tested. The gene silencing effect is evaluated with synthetic siRNA directed against EGFP or luciferase mRNA, in HeLa cells stably expressing EGFP or B16F10 melanoma cells stably expressing luciferase, respectively. SiRNA formulations are compared with those prepared using some commercial transfection reagents. One formulation called His-lPEI LPRi100 comprising siRNA, histidinylated lPEI (His-lPEI) and liposomes 100 made with O,O-dioleyl-N-[3N-(N-methylimidazolium iodide)propylene] phosphoramidate and O,O-dioleyl-N-histamine phosphoramidate appears to give the best specific inhibition of gene expression at 10nM siRNA in a dose-dependent manner with low cytotoxicity. This formulation exhibits a size and a zeta potential of 60nm and +84mV, respectively. According to our previous works, histidinylated lipopolyplexes appears as a versatile formulation for DNA, mRNA and siRNA transfection.

Lipopolyplexes formulations resulting from association of nucleic acid, cationic liposomes and a cationic polymer are attracting formulations for siRNA delivery. Herein, imidazole- and imidazolium-based liposomes in association with histidinylated polymers are studied to produce siRNA lipopoplyplexes (LPRi) subsequently used for gene silencing. Several kinds of imidazole/histidine liposomes and cationic polymers are tested. The gene silencing effect is evaluated with synthetic siRNA directed against EGFP or luciferase mRNA, in HeLa cells stably expressing EGFP or B16F10 melanoma cells stably expressing luciferase, respectively. SiRNA formulations are compared with those prepared using some commercial transfection reagents. One formulation called His-lPEI LPRi100 comprising siRNA, histidinylated lPEI (His-lPEI) and liposomes 100 made with O,O-dioleyl-N-[3N-(N-methylimidazolium iodide)propylene] phosphoramidate and O,O-dioleyl-N-histamine phosphoramidate appears to give the best specific inhibition of gene expression at 10nM siRNA in a dose-dependent manner with low cytotoxicity. This formulation exhibits a size and a zeta potential of 60nm and +84mV, respectively. According to our previous works, histidinylated lipopolyplexes appears as a versatile formulation for DNA, mRNA and siRNA transfection.


2013   Références trouvées : 15

Maurel D. B., Benaitreau D., Jaffre C., Toumi H., Portier H., Uzbekov R., Pichon C., Benhamou C. L., Lespessailles E. and Pallu S.  (2013)

Effect of the alcohol consumption on osteocyte cell processes : a molecular imaging study

Journal of Cellular and Molecular Medicine (2013) 18 (8) 1680-1693 - doi : 10.1111/jcmm.12113
We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups : Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.

We have previously shown microarchitectural tissue changes with cellular modifications in osteocytes following high chronic alcohol dose. The aim of this study was to assess the dose effect of alcohol consumption on the cytoskeleton activity, the cellular lipid content and modulation of differentiation and apoptosis in osteocyte. Male Wistar rats were divided into three groups : Control (C), Alcohol 25% v/v (A25) or Alcohol 35% v/v (A35) for 17 weeks. Bone mineral density (BMD) was assessed by DXA, osteocyte empty lacunae, lacunae surface, bone marrow fat with bright field microscopy. Osteocyte lipid content was analysed with transmission electron microscopy (TEM) and epifluorescence microscopy. Osteocyte apoptosis was analysed with immunolabelling and TEM. Osteocyte differentiation and cytoskeleton activity were analysed with immunolabelling and real time quantitative PCR. At the end of the protocol, BMD was lower in A25 and A35 compared with C, while the bone marrow lipid content was increased in these groups. More empty osteocyte lacunae and osteocyte containing lipid droplets in A35 were found compared with C and A25. Cleaved caspase-3 staining and chromatin condensation were increased in A25 and A35 versus C. Cleaved caspase-3 was increased in A35 versus A25. CD44 and phosphopaxillin stainings were higher in A35 compared with C and A25. Paxillin mRNA expression was higher in A35 versus A25 and C and sclerostin mRNA expression was higher in A35 versus C. We only observed a dose effect of alcohol consumption on cleaved caspase-3 osteocyte immunostaining levels and on the number of lipid droplets in the bone marrow.

Félix R., Crottès D., Delalande A., Fauconnier J., Lebranchu Y., Le Guennec J.-Y., Velge-Roussel F.  (2013)

The Orai-1 and STIM-1 Complex Controls Human Dendritic Cell Maturation

PLoS One (2013) 8 (5) - doi : 10.1371/journal.pone.0061595
Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca(2+) signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca(2+) stores that results in a store-operated Ca(2+) entry (SOCE). This Ca(2+) influx was inhibited by 2-APB and exhibited a Ca(2+)permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca(2+) entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins.

Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the professional antigen presenting cells. Here, we described the role of Calcium released activated (CRAC) channels in the maturation and cytokine secretion of human DC. Recent works identified STIM1 and Orai1 in human T lymphocytes as essential for CRAC channel activation. We investigated Ca(2+) signaling in human DC maturation by imaging intracellular calcium signaling and pharmalogical inhibitors. The DC response to inflammatory mediators or PAMPs (Pathogen-associated molecular patterns) is due to a depletion of intracellular Ca(2+) stores that results in a store-operated Ca(2+) entry (SOCE). This Ca(2+) influx was inhibited by 2-APB and exhibited a Ca(2+)permeability similar to the CRAC (Calcium-Released Activated Calcium), found in T lymphocytes. Depending on the PAMPs used, SOCE profiles and amplitudes appeared different, suggesting the involvement of different CRAC channels. Using siRNAi, we identified the STIM1 and Orai1 protein complex as one of the main pathways for Ca(2+) entry for LPS- and TNF-α-induced maturation in DC. Cytokine secretions also seemed to be SOCE-dependent with profile differences depending on the maturating agents since IL-12 and IL10 secretions appeared highly sensitive to 2-APB whereas IFN-γ was less affected. Altogether, these results clearly demonstrate that human DC maturation and cytokine secretions depend on SOCE signaling involving STIM1 and Orai1 proteins.

Pichon C. and Midoux P.  (2013)

Mannosylated and histidylated LPR technology for vaccination with tumor antigen mRNA

Methods in molecular biology (Clifton, N.J.) (2013) 969 247-274 - doi : 10.1007/978-1-62703-260-5_16
mRNA-based vaccines are currently being developed for treating various diseases including cancers. For this purpose, synthetic or in vitro transcribed (IVT) mRNA encoding tumor antigen offers several advantages over plasmid DNA encoding tumor antigen including better delivery and security. In this chapter, we report the preparation of mannosylated mRNA nanoparticles termed mannosylated lipopolyplexes or Man-LPR loaded with mRNA encoding a melanoma antigen. This formulation enhances the transfection of dendritic cells (DCs) in vivo and the anti-B16F10 melanoma vaccination in mice. The mRNA is formulated with histidylated liposomes and a histidylated polymer. Those pH-sensitive vectors promote membrane destabilization in endosomes upon the protonation of their histidine groups, allowing nucleic acid delivery in the cytosol. To favor DCs targeting via the mannose receptor, a mannose lipid is incorporated in the liposomes. Here, we provide protocols for the preparation of mannosylated liposomes, the synthesis of mRNA, mice immunization based on systemic injection, measurement of the cellular immune response and determination of the number of transfected splenic DC.

mRNA-based vaccines are currently being developed for treating various diseases including cancers. For this purpose, synthetic or in vitro transcribed (IVT) mRNA encoding tumor antigen offers several advantages over plasmid DNA encoding tumor antigen including better delivery and security. In this chapter, we report the preparation of mannosylated mRNA nanoparticles termed mannosylated lipopolyplexes or Man-LPR loaded with mRNA encoding a melanoma antigen. This formulation enhances the transfection of dendritic cells (DCs) in vivo and the anti-B16F10 melanoma vaccination in mice. The mRNA is formulated with histidylated liposomes and a histidylated polymer. Those pH-sensitive vectors promote membrane destabilization in endosomes upon the protonation of their histidine groups, allowing nucleic acid delivery in the cytosol. To favor DCs targeting via the mannose receptor, a mannose lipid is incorporated in the liposomes. Here, we provide protocols for the preparation of mannosylated liposomes, the synthesis of mRNA, mice immunization based on systemic injection, measurement of the cellular immune response and determination of the number of transfected splenic DC.

Orio J., Bellard E., Baaziz H., Pichon C., Mouritzen P., Rols M.P., Teissie J., Golzio M. and Chabot S.  (2013)

Sub-cellular temporal and spatial distribution of electrotransferred LNA/DNA oligomer

Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research (2013) 9 479-485
Low biological activity and inefficient targeted delivery in vivo have hindered RNA interference (RNAi)-based therapy from realising its full clinical potential. To overcome these hurdles, progresses have been made to develop new technologies optimizing oligonucleotides chemistry on one hand and achieving its effective delivery on the other hand. In this report, we achieved, by using the electropulsation technique (EP), efficient cellular delivery of chemically-modified oligonucleotide : The locked nucleic acid (LNA)/DNA oligomer. We used single cell level confocal fluorescence microscopy to follow the spatial and temporal distribution of electrotransferred cyanine 5 (Cy5)-labeled LNA/DNA oligomer. We observed that EP allowed LNA/DNA oligomer cellular uptake providing the oligomer a rapid access to the cytoplasm of HeLa cells. Within a few minutes after electrotransfer, Cy5-LNA/DNA oligomers shuttle from cytoplasm to nucleus whereas in absence of pulses application, Cy5-LNA/DNA oligomers were not detected. We then observed a redistribution of the Cy5 fluorescence that accumulated over time into cytoplasmic organelles. To go further and to identify these compartments, we used the HeLa GFP-Rab7 cell line to visualise late endosomes, and lysosomal or mitochondrial specific markers. Our results showed that the EP technique allowed direct entry into the cytoplasm of the Cy5-LNA/DNA oligomer bypassing the endocytosic pathway. However, in absence of pulses application, Cy5-LNA/DNA oligomer were able to enter cells through the endocytosic pathway. We demonstrated that EP is an efficient technique for LNA-based oligonucleotides delivery offering strong advantages by avoiding the endolysosomal compartmentalization, giving a rapid and free access to the cytoplasm and the nucleus where they can find their targets.

Low biological activity and inefficient targeted delivery in vivo have hindered RNA interference (RNAi)-based therapy from realising its full clinical potential. To overcome these hurdles, progresses have been made to develop new technologies optimizing oligonucleotides chemistry on one hand and achieving its effective delivery on the other hand. In this report, we achieved, by using the electropulsation technique (EP), efficient cellular delivery of chemically-modified oligonucleotide : The locked nucleic acid (LNA)/DNA oligomer. We used single cell level confocal fluorescence microscopy to follow the spatial and temporal distribution of electrotransferred cyanine 5 (Cy5)-labeled LNA/DNA oligomer. We observed that EP allowed LNA/DNA oligomer cellular uptake providing the oligomer a rapid access to the cytoplasm of HeLa cells. Within a few minutes after electrotransfer, Cy5-LNA/DNA oligomers shuttle from cytoplasm to nucleus whereas in absence of pulses application, Cy5-LNA/DNA oligomers were not detected. We then observed a redistribution of the Cy5 fluorescence that accumulated over time into cytoplasmic organelles. To go further and to identify these compartments, we used the HeLa GFP-Rab7 cell line to visualise late endosomes, and lysosomal or mitochondrial specific markers. Our results showed that the EP technique allowed direct entry into the cytoplasm of the Cy5-LNA/DNA oligomer bypassing the endocytosic pathway. However, in absence of pulses application, Cy5-LNA/DNA oligomer were able to enter cells through the endocytosic pathway. We demonstrated that EP is an efficient technique for LNA-based oligonucleotides delivery offering strong advantages by avoiding the endolysosomal compartmentalization, giving a rapid and free access to the cytoplasm and the nucleus where they can find their targets.

Krolikiewicz-Renimel, I. Michel, T. Destandau, E. Reddy, M. Andre, P. Elfakir, C. Pichon, C.  (2013)

Protective effect of a Butea monosperma (Lam.) Taub. flowers extract against skin inflammation : antioxidant, anti-inflammatory and matrix metalloproteinases inhibitory activities

Journal of ethnopharmacology (2013) 148 (2) 537-543 - doi : 10.1016/j.jep.2013.05.001
ETHNOPHARMACOLOGICAL RELEVANCE : Butea monosperma (Lam.) Taubert (Syn. Butea frondosa ; family Fabaceae) is a common plant of the Indian continent (Das et al., 2011 ; Sharma and Deshwal, 2011). The brightly orange flowers of this plant are widely used in traditional medicine and more particularly for inflammatory disease. AIM OF THE STUDY : In vitro anti-inflammatory mechanism of a hydroethanolic extract of B. monosperma flowers (BME) and more specifically of an enriched fraction in butrin and isobutrin (BI) was studied using cell culture of Normal Human Keratinocyte, cells involved in the skin inflammatory. MATERIALS AND METHODS : Dried and crushed B. monosperma flowers were extracted with Ethanol/H2O (70/30 v/v). The butrin/isobutrin fraction was obtained by centrifugal Partition Chromatography (CPC). Experiments were conducted on UV-B treated normal human epidermis keratinocytes, cells involved in the skin inflammatory response. To evaluate extract anti-inflammatory activity, cytokines IL-1beta, IL-6, IL-8, prostaglandin E2 and metalloproteinases MMP-1, -2, -9 and -10 were measured in the cells supernatant. RESULTS : Our data clearly showed that hydroalcoholic B. monosperma flower extract was able to decrease the secretion of IL-1beta, IL-6 and IL-8 pro-inflammatory cytokines of -32, -33 and -18% respectively. Interestingly, Prostaglandin E2 production and the secretion of MMP-1, -2, -9 and -10 were also inhibited. Same results were observed in presence of enriched fraction in butrin and isobutrin and confirmed the participation of these molecules in the anti-inflammatory activity. CONCLUSION : These results explain the anti-inflammatory activity of B. monosperma and confirm the interest to use it in traditional Indian medicine. Moreover, its metalloproteinases inhibitory activities coupled with its anti-inflammatory action also give anti-aging property to this plant.

ETHNOPHARMACOLOGICAL RELEVANCE : Butea monosperma (Lam.) Taubert (Syn. Butea frondosa ; family Fabaceae) is a common plant of the Indian continent (Das et al., 2011 ; Sharma and Deshwal, 2011). The brightly orange flowers of this plant are widely used in traditional medicine and more particularly for inflammatory disease. AIM OF THE STUDY : In vitro anti-inflammatory mechanism of a hydroethanolic extract of B. monosperma flowers (BME) and more specifically of an enriched fraction in butrin and isobutrin (BI) was studied using cell culture of Normal Human Keratinocyte, cells involved in the skin inflammatory. MATERIALS AND METHODS : Dried and crushed B. monosperma flowers were extracted with Ethanol/H2O (70/30 v/v). The butrin/isobutrin fraction was obtained by centrifugal Partition Chromatography (CPC). Experiments were conducted on UV-B treated normal human epidermis keratinocytes, cells involved in the skin inflammatory response. To evaluate extract anti-inflammatory activity, cytokines IL-1beta, IL-6, IL-8, prostaglandin E2 and metalloproteinases MMP-1, -2, -9 and -10 were measured in the cells supernatant. RESULTS : Our data clearly showed that hydroalcoholic B. monosperma flower extract was able to decrease the secretion of IL-1beta, IL-6 and IL-8 pro-inflammatory cytokines of -32, -33 and -18% respectively. Interestingly, Prostaglandin E2 production and the secretion of MMP-1, -2, -9 and -10 were also inhibited. Same results were observed in presence of enriched fraction in butrin and isobutrin and confirmed the participation of these molecules in the anti-inflammatory activity. CONCLUSION : These results explain the anti-inflammatory activity of B. monosperma and confirm the interest to use it in traditional Indian medicine. Moreover, its metalloproteinases inhibitory activities coupled with its anti-inflammatory action also give anti-aging property to this plant.

Gaspar V.M., Cruz C., Queiroz J.A., Pichon C., Correia I.J. and Sousa F.  (2013)

Sensitive detection of peptide-minicircle DNA interactions by surface plasmon resonance

Analytical chemistry (2013) 85 (4) 2304-2311 - doi : 10.1021/ac303288x
Minicircle DNA (mcDNA) is recently becoming an exciting source of genetic material for therapeutic purposes due to its exceptional biocompatibility and efficiency over typical DNA. However, its widespread use is yet restrained because of the absence of an efficient technology that allows its purification. Here, the precise conditions of mcDNA interaction with novel arginine-arginine dipeptide ligands were explored to promote binding and recovery of these biopharmaceuticals. Such interactions were investigated by taking advantage of a highly sensitive method based on surface plasmon resonance (SPR) to screen, in real-time, for ligand-coupled biomolecules, while preserving mcDNA integrity. Through this analytic approach, we detected dynamic binding responses that are dependent on buffer type, mcDNA electrokinetic potential, and temperature conditions. Remarkably, the results obtained revealed that the ligands possess high affinity to mcDNA molecules under low salt buffers, and low affinity in the presence of salt, suggesting that electrostatic interactions mainly govern ligand-analyte coupling. These findings provide important insights for an active manipulation of parameters that promote mcDNA recovery and purification. Above all, this study showed the crucial importance of SPR for future screening of other ligands that, like the one described herein, can be used to design mcDNA recovery platforms which will have significant impact in biopharmaceutical-based therapeutics.

Minicircle DNA (mcDNA) is recently becoming an exciting source of genetic material for therapeutic purposes due to its exceptional biocompatibility and efficiency over typical DNA. However, its widespread use is yet restrained because of the absence of an efficient technology that allows its purification. Here, the precise conditions of mcDNA interaction with novel arginine-arginine dipeptide ligands were explored to promote binding and recovery of these biopharmaceuticals. Such interactions were investigated by taking advantage of a highly sensitive method based on surface plasmon resonance (SPR) to screen, in real-time, for ligand-coupled biomolecules, while preserving mcDNA integrity. Through this analytic approach, we detected dynamic binding responses that are dependent on buffer type, mcDNA electrokinetic potential, and temperature conditions. Remarkably, the results obtained revealed that the ligands possess high affinity to mcDNA molecules under low salt buffers, and low affinity in the presence of salt, suggesting that electrostatic interactions mainly govern ligand-analyte coupling. These findings provide important insights for an active manipulation of parameters that promote mcDNA recovery and purification. Above all, this study showed the crucial importance of SPR for future screening of other ligands that, like the one described herein, can be used to design mcDNA recovery platforms which will have significant impact in biopharmaceutical-based therapeutics.

Dubois A.V., Midoux P., Gras D., Si-Tahar M., Brea D., Attucci S., Khelloufi M.K., Ramphal R., Diot P., Gauthier F. and Herve V.  (2013)

Poly-L-Lysine compacts DNA, kills bacteria, and improves protease inhibition in cystic fibrosis sputum

American journal of respiratory and critical care medicine (2013) 188 (6) 703-709 - doi : 10.1164/rccm.201305-0912OC
RATIONALE : Neutrophil serine proteases in cystic fibrosis (CF) lung secretions partially resist inhibition by natural and exogenous inhibitors, mostly because DNA impairs their control. Cationic polypeptides display the property of condensing DNA and retain antimicrobial properties. We hypothesized that DNA condensation by cationic polypeptides in CF sputum would result in a better control of CF inflammation and infection. OBJECTIVES : We examined whether poly-L-lysine would compact DNA in CF lung secretions and liquefy CF sputum, improve the control of extracellular proteases by exogenous inhibitors, and whether it displays antibacterial properties toward CF-associated bacteria. METHODS : We used fluorogenic methods to measure proteolytic activities and inhibition by protease inhibitors in whole sputum homogenates from patients with CF before and after treatment with poly-L-lysine. Antibacterial properties of poly-L-lysine were measured in bacterial cultures and in whole CF sputum. Poly-L-lysine toxicity was evaluated after aerosolization by histologic analysis, flow cytometry, and quantification of proinflammatory cytokines. MEASUREMENTS AND MAIN RESULTS : Poly-L-lysine compacts CF sputum DNA, generating a liquid phase that improves ciliary beating frequency at the lung epithelial surface, and allows the control of neutrophil elastase and cathepsin G by their natural inhibitors. It retains antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at doses that induce no inflammation in the mouse lung after aerosol administration. CONCLUSIONS : Poly-L-lysine may be an alternative to dornase-alpha to liquefy sputum with added benefits because it helps natural inhibitors to better control the deleterious effects of extracellularly released neutrophil serine proteases and has the ability to kill bacteria in CF sputum.

RATIONALE : Neutrophil serine proteases in cystic fibrosis (CF) lung secretions partially resist inhibition by natural and exogenous inhibitors, mostly because DNA impairs their control. Cationic polypeptides display the property of condensing DNA and retain antimicrobial properties. We hypothesized that DNA condensation by cationic polypeptides in CF sputum would result in a better control of CF inflammation and infection. OBJECTIVES : We examined whether poly-L-lysine would compact DNA in CF lung secretions and liquefy CF sputum, improve the control of extracellular proteases by exogenous inhibitors, and whether it displays antibacterial properties toward CF-associated bacteria. METHODS : We used fluorogenic methods to measure proteolytic activities and inhibition by protease inhibitors in whole sputum homogenates from patients with CF before and after treatment with poly-L-lysine. Antibacterial properties of poly-L-lysine were measured in bacterial cultures and in whole CF sputum. Poly-L-lysine toxicity was evaluated after aerosolization by histologic analysis, flow cytometry, and quantification of proinflammatory cytokines. MEASUREMENTS AND MAIN RESULTS : Poly-L-lysine compacts CF sputum DNA, generating a liquid phase that improves ciliary beating frequency at the lung epithelial surface, and allows the control of neutrophil elastase and cathepsin G by their natural inhibitors. It retains antimicrobial properties against Pseudomonas aeruginosa and Staphylococcus aureus at doses that induce no inflammation in the mouse lung after aerosol administration. CONCLUSIONS : Poly-L-lysine may be an alternative to dornase-alpha to liquefy sputum with added benefits because it helps natural inhibitors to better control the deleterious effects of extracellularly released neutrophil serine proteases and has the ability to kill bacteria in CF sputum.

Delalande A., Kotopoulis S., Postema M., Midoux P. and Pichon C.  (2013)

Sonoporation : mechanistic insights and ongoing challenges for gene transfer

Gene (2013) 525 (2) 191-199 - doi : 10.1016/j.gene.2013.03.095
Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble-cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Microbubbles first developed as ultrasound contrast agents have been used to assist ultrasound for cellular drug and gene delivery. Their oscillation behavior during ultrasound exposure leads to transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. In this review, we summarize current state of the art concerning microbubble-cell interactions and cellular effects leading to sonoporation and its application for gene delivery. Optimization of sonoporation protocol and composition of microbubbles for gene delivery are discussed.

Berchel M., Le Corre S., Le Gall T., Couthon-Gourvès H., Haelters J.P., Midoux P., Montier T., Lehn P. and Jaffrès P.A.  (2013)

Trimethylarsonium-Based Cationic Phospholipids for Gene Delivery

Phosphorus, Sulfur, and Silicon and the Related Elements (2013) 188 (1-2) 91-94 - doi : 10.1080/10426507.2012.741161
Lipophosphoramide-based cationic lipids are a class of synthetic vectors used for gene delivery that can be produced in multigram scale. The use of trimethylarsonium moiety as a cationic polar head was beneficial to produce efficient gene delivery vectors for in vivo applications. Moreover, this type of cationic lipid can also exhibit some bactericidal effects.

Lipophosphoramide-based cationic lipids are a class of synthetic vectors used for gene delivery that can be produced in multigram scale. The use of trimethylarsonium moiety as a cationic polar head was beneficial to produce efficient gene delivery vectors for in vivo applications. Moreover, this type of cationic lipid can also exhibit some bactericidal effects.

Bire S., Gosset D., Jegot G., Midoux P., Pichon C. and Rouleux-Bonnin F.  (2013)

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition

BMC Biotechnology (2013) 13 (1) 75 - doi : 10.1186/1472-6750-13-75
Background

Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage : stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components : a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. 

Results

The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. 

Conclusion

Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

Background
Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage : stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components : a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability.

Results
The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window.

Conclusion
Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking.

Gomez J.P., Pichon C. and Midoux P.  (2013)

Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers

Gene (2013) 525 (2) 182-190 - doi : 10.1016/j.gene.2013.03.055
DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 μg/cm(2).h and 0.385 μg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.

DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 μg/cm(2).h and 0.385 μg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.

Sedletska, Y. Culard, F. Midoux, P.and Malinge, J. M.  (2013)

Interaction studies of muts and mutl with DNA containing the major cisplatin lesion and its mismatched counterpart under equilibrium and nonequilibrium conditions

Biopolymers 99 (9) 636-647
The DNA mismatch repair (MMR) system participates in cis-diamminedichloroplatinum (II) (cisplatin) cytotoxicity through signaling of cisplatin DNA lesions by yet unknown molecular mechanisms. It is thus of great interest to determine whether specialized function of MMR proteins could be associated with cisplatin DNA damage. The major cisplatin 1,2-d(GpG) intrastrand crosslink and compound lesions arising from misincorporation of a mispaired base opposite either platinated guanine of the 1,2-d(GpG) adduct are thought to be critical lesions for MMR signaling. Previously, we have shown that cisplatin compound lesion with a mispaired thymine opposite the 3 platinated guanine triggers new Escherichia coli MutS ATP-dependent biochemical activities distinguishable from those encountered with DNA mismatch consistent with a role of this lesion in MMR-dependent signaling mechanism. In this report, we show that the major cisplatin 1,2-d(GpG) intrastrand crosslink does not confer novel MutS postrecognition biochemical activity as studied by surface plasmon resonance spectroscopy. A fast rate of MutS ATP-dependent dissociation prevents MutL recruitment to the major cisplatin lesion in contrast to cisplatin compound lesion which authorized MutS-dependent recruitment of MutL with a dynamic of ternary complex formation distinguishable from that encountered with DNA mismatch substrate. We conclude that the mode of cisplatin DNA damage recognition by MutS and the nature of MMR post-recognition events are lesion-dependent and suggest that MMR signaling through the major cisplatin lesion is unlikely to occur.

The DNA mismatch repair (MMR) system participates in cis-diamminedichloroplatinum (II) (cisplatin) cytotoxicity through signaling of cisplatin DNA lesions by yet unknown molecular mechanisms. It is thus of great interest to determine whether specialized function of MMR proteins could be associated with cisplatin DNA damage. The major cisplatin 1,2-d(GpG) intrastrand crosslink and compound lesions arising from misincorporation of a mispaired base opposite either platinated guanine of the 1,2-d(GpG) adduct are thought to be critical lesions for MMR signaling. Previously, we have shown that cisplatin compound lesion with a mispaired thymine opposite the 3 platinated guanine triggers new Escherichia coli MutS ATP-dependent biochemical activities distinguishable from those encountered with DNA mismatch consistent with a role of this lesion in MMR-dependent signaling mechanism. In this report, we show that the major cisplatin 1,2-d(GpG) intrastrand crosslink does not confer novel MutS postrecognition biochemical activity as studied by surface plasmon resonance spectroscopy. A fast rate of MutS ATP-dependent dissociation prevents MutL recruitment to the major cisplatin lesion in contrast to cisplatin compound lesion which authorized MutS-dependent recruitment of MutL with a dynamic of ternary complex formation distinguishable from that encountered with DNA mismatch substrate. We conclude that the mode of cisplatin DNA damage recognition by MutS and the nature of MMR post-recognition events are lesion-dependent and suggest that MMR signaling through the major cisplatin lesion is unlikely to occur.

Ezzine, S., Vassaux, G., Pitard, B., Barteau, B., Malinge, J. M., Midoux, P., Pichon, C. and Baril, P.  (2013)

RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression

Nucleic Acids Res - 1-14 - doi : 10.1093/nar/gkt797
Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.

Pigeon L., Gonçalves C., Gosset D., Pichon C., Midoux P.  (2013)

An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA increases polyplexes transfection efficiency

Small 2013 - doi : 10.1002/smll.201300217
Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

Lacerda, S., Bonnet, C.S., Pallier, A., Villette, S., Foucher, F., Westall, F., Buron, F., Suzenet, F., Pichon, C., Petoud, S. and Tóth, E.  (2013)

Lanthanide-Based, Near-Infrared Luminescent and Magnetic Lipoparticles : Monitoring Particle Integrity

Small (2013) 9 (16) 2662-2666 - doi : 10.1002/smll.201201923
Near-infrared emitting, magnetic particles for combined optical and MR detection based on liposomes or artificial lipoproteins are presented. They provide a novel strategy for the luminescence sensitization of lanthanide cations (Yb3+, Nd3+) without covalent bonds between the chromophore and the lanthanide, and provide an unambiguous tool for monitoring the integrity of the liponanoparticles, via emission in the NIR region.

Near-infrared emitting, magnetic particles for combined optical and MR detection based on liposomes or artificial lipoproteins are presented. They provide a novel strategy for the luminescence sensitization of lanthanide cations (Yb3+, Nd3+) without covalent bonds between the chromophore and the lanthanide, and provide an unambiguous tool for monitoring the integrity of the liponanoparticles, via emission in the NIR region.


2012   Références trouvées : 7

Rudd, L. Pichon, C. and Delalande, A.  (2012)

Ultrasound and microbubbles assisted-gene transfer : what is next ?

Ultraschall in der Medizin (2014) 22 (4) 391-393
Having first been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble—cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

Having first been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble—cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

Berchel, M., Le Gall, T., Couthon-Gourvès, H., Haelters, J.P., Montier, T., Midoux, P., Lehn, P. and Jaffrès, PA.   (2012)

Lipophosphonate/lipophosphoramidates : A family of synthetic vectors efficient for gene delivery

Biochimie 94 (1) 33-41
Lipophophoramidates constitute a class of synthetic vectors which were especially designed for gene delivery. In this family of compounds, the phosphorus functional group links two lipid chains to a spacer ended by a polar headgroup. Such vectors, which can readily be obtained, offer an alternative to the numerous examples of glycerolipid-based vectors that have been more exhaustively studied. Since the pioneering work describing this series of synthetic vectors, several chemical modifications have been proposed with the aim of correlating the molecular structure with the gene transfection efficacy. It has indeed been observed that some modifications which may be considered as minor at first glance, actually have important consequences on both the transfection efficacy and cytotoxic side effects. We herein discuss the modification of the structure of lipophosphoramidates, in particular of their lipidic part and of the nature of the cationic polar head which may be constituted by a trimethylammonium, trimethylphosphonium or trimethylarsonium motif. We also report that, as well as the in vitro transfection efficacy which governs the selection of the most promising vectors for in vivo studies, other aspects related to the synthetic pathway must be also considered for the development of new synthetic vectors (such as modularity of the synthesis, scaling-up).

Lipophophoramidates constitute a class of synthetic vectors which were especially designed for gene delivery. In this family of compounds, the phosphorus functional group links two lipid chains to a spacer ended by a polar headgroup. Such vectors, which can readily be obtained, offer an alternative to the numerous examples of glycerolipid-based vectors that have been more exhaustively studied. Since the pioneering work describing this series of synthetic vectors, several chemical modifications have been proposed with the aim of correlating the molecular structure with the gene transfection efficacy. It has indeed been observed that some modifications which may be considered as minor at first glance, actually have important consequences on both the transfection efficacy and cytotoxic side effects. We herein discuss the modification of the structure of lipophosphoramidates, in particular of their lipidic part and of the nature of the cationic polar head which may be constituted by a trimethylammonium, trimethylphosphonium or trimethylarsonium motif. We also report that, as well as the in vitro transfection efficacy which governs the selection of the most promising vectors for in vivo studies, other aspects related to the synthetic pathway must be also considered for the development of new synthetic vectors (such as modularity of the synthesis, scaling-up).

Delalande, A., Postema, M., Mignet, N., Midoux, P., and Pichon, C.  (2012)

Ultrasound and microbubble-assisted gene delivery : recent advances and ongoing challenges

Ther Deliv. 3 (10) 1199-1215
Having first been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble—cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

Having first been developed for ultrasound imaging, nowadays, microbubbles are proposed as tools for ultrasound-assisted gene delivery, too. Their behavior during ultrasound exposure causes transient membrane permeability of surrounding cells, facilitating targeted local delivery. The increased cell uptake of extracellular compounds by ultrasound in the presence of microbubbles is attributed to a phenomenon called sonoporation. Sonoporation has been successfully applied to deliver nucleic acids in vitro and in vivo in a variety of therapeutic applications. However, the biological and physical mechanisms of sonoporation are still not fully understood. In this review, we discuss recent data concerning microbubble—cell interactions leading to sonoporation and we report on the progress in ultrasound-assisted therapeutic gene delivery in different organs. In addition, we outline ongoing challenges of this novel delivery method for its clinical use.

Godin, F., Villette, S., Vallée, B., Doudeau, M., Morisset-Lopez, S., Ardourel, M., Hevor, T., Pichon, C. and Bénédetti, B.  (2012)

A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line

Biochemical and Biophysical Research Communications 418 (4) 689-694
Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line : CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using ‘‘in situ’’ proximity ligation assay (PLA).

Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line : CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using ‘‘in situ’’ proximity ligation assay (PLA).

Billiet L., Gomez J.P., Berchel M., Jaffrès P.A., Le Gall T., Montier T., Bertrand E., Cheradame H., Guégan P., Mével M., Pitard B., Benvegnu T., Lehn P., Pichon C. and Midoux P.   (2012)

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

Biomaterials 33 (10) 2980-2990
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.

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.

Bonnet, CS, Buron, F, Caillé, F, Shade, CM, Drahoš, B, Pellegatti, L, Zhang, J, Villette, S, Helm, L, Pichon, C, Suzenet, F, Petoud, S. & Tóth E.  (2012)

Pyridine-Based Lanthanide Complexes Combining MRI and NIR Luminescence Activities

Chemistry - a european journal 18 (5) 1419-1431
A series of novel triazole derivative pyridine-based polyamino-polycarboxylate ligands has been synthesized for lanthanide complexation. This versatile platform of chelating agents combines advantageous properties for both magnetic resonance (MR) and optical imaging applications of the corresponding Gd(3+) and near-infrared luminescent lanthanide complexes. The thermodynamic stability constants of the Ln(3+) complexes, as assessed by pH potentiometric measurements, are in the range log K(LnL) =17-19, with a high selectivity for lanthanides over Ca(2+) , Cu(2+) , and Zn(2+) . The complexes are bishydrated, an important advantage to obtain high relaxivities for the Gd(3+) chelates. The water exchange of the Gd(3+) complexes (k(ex) (298) =7.7-9.3×10(6) s(-1) ) is faster than that of clinically used magnetic resonance imaging (MRI) contrast agents and proceeds through a dissociatively activated mechanism, as evidenced by the positive activation volumes (ΔV(≠) =7.2-8.8 cm(3) mol(-1) ). The new triazole ligands allow a considerable shift towards lower excitation energies of the luminescent lanthanide complexes as compared to the parent pyridinic complex, which is a significant advantage in the perspective of biological applications. In addition, they provide increased epsilon values resulting in a larger number of emitted photons and better detection sensitivity. The most conjugated system PheTPy, bearing a phenyl-triazole pendant on the pyridine ring, is particularly promising as it displays the lowest excitation and triplet-state energies associated with good quantum yields for both Nd(3+) and Yb(3+) complexes. Cellular and in vivo toxicity studies in mice evidenced the non-toxicity and the safe use of such bishydrated complexes in animal experiments. Overall, these pyridinic ligands constitute a highly versatile platform for the simultaneous optimization of both MRI and optical properties of the Gd(3+) and the luminescent lanthanide complexes, respectively.

A series of novel triazole derivative pyridine-based polyamino-polycarboxylate ligands has been synthesized for lanthanide complexation. This versatile platform of chelating agents combines advantageous properties for both magnetic resonance (MR) and optical imaging applications of the corresponding Gd(3+) and near-infrared luminescent lanthanide complexes. The thermodynamic stability constants of the Ln(3+) complexes, as assessed by pH potentiometric measurements, are in the range log K(LnL) =17-19, with a high selectivity for lanthanides over Ca(2+) , Cu(2+) , and Zn(2+) . The complexes are bishydrated, an important advantage to obtain high relaxivities for the Gd(3+) chelates. The water exchange of the Gd(3+) complexes (k(ex) (298) =7.7-9.3×10(6)  s(-1) ) is faster than that of clinically used magnetic resonance imaging (MRI) contrast agents and proceeds through a dissociatively activated mechanism, as evidenced by the positive activation volumes (ΔV(≠) =7.2-8.8 cm(3) mol(-1) ). The new triazole ligands allow a considerable shift towards lower excitation energies of the luminescent lanthanide complexes as compared to the parent pyridinic complex, which is a significant advantage in the perspective of biological applications. In addition, they provide increased epsilon values resulting in a larger number of emitted photons and better detection sensitivity. The most conjugated system PheTPy, bearing a phenyl-triazole pendant on the pyridine ring, is particularly promising as it displays the lowest excitation and triplet-state energies associated with good quantum yields for both Nd(3+) and Yb(3+) complexes. Cellular and in vivo toxicity studies in mice evidenced the non-toxicity and the safe use of such bishydrated complexes in animal experiments. Overall, these pyridinic ligands constitute a highly versatile platform for the simultaneous optimization of both MRI and optical properties of the Gd(3+) and the luminescent lanthanide complexes, respectively.

Perche, F. Lambert, O. Berchel, M. Jaffrès, P.A. Pichon, C. Midoux, P.  (2012)

Gene transfer by histidylated lipopolyplexes : A dehydration method allowing preservation of their physicochemical parameters and transfection efficiency

International Journal of Pharmaceutics 423 (1) 144-150
Lipid-Polycation-DNA complexes (LPD) is a promising non-viral system for nucleic acids delivery. Usually, LPD are prepared just before their use. In the present work, we have examined whether dehydration of a new type of LPD (named LPD100) might be a storage option. LPD100 comprises PEGylated histidylated polylysine/pDNA polyplexes and a liposomal formulation made with lipophosphoramidates containing N-methylimidazolium and histamine polar heads. LPD100 were dehydrated by evaporation, and the physicochemical parameters and transfection efficiency (TE) of reconstituted LPD100 were compared to that of fresh LPD100. LPD100 previously dehydrated in the presence of 20% saccharose, displayed comparable size and surface charge as freshly prepared LPD100 but gave a better TE. CryoTEM experiments showed that the reconstituted LPD100 exhibited a shape similar to fresh ones. Moreover, when LPD100 were prepared with dehydrated pDNA/polymer complexes and fresh liposomes, TE was as efficient as with fresh LPD100 while a small increase of their size were observed. These results demonstrate that evaporation of LPD100 in the presence of saccharose is a powerful method to store them for a long period of time.

Lipid-Polycation-DNA complexes (LPD) is a promising non-viral system for nucleic acids delivery. Usually, LPD are prepared just before their use. In the present work, we have examined whether dehydration of a new type of LPD (named LPD100) might be a storage option. LPD100 comprises PEGylated histidylated polylysine/pDNA polyplexes and a liposomal formulation made with lipophosphoramidates containing N-methylimidazolium and histamine polar heads. LPD100 were dehydrated by evaporation, and the physicochemical parameters and transfection efficiency (TE) of reconstituted LPD100 were compared to that of fresh LPD100. LPD100 previously dehydrated in the presence of 20% saccharose, displayed comparable size and surface charge as freshly prepared LPD100 but gave a better TE. CryoTEM experiments showed that the reconstituted LPD100 exhibited a shape similar to fresh ones. Moreover, when LPD100 were prepared with dehydrated pDNA/polymer complexes and fresh liposomes, TE was as efficient as with fresh LPD100 while a small increase of their size were observed. These results demonstrate that evaporation of LPD100 in the presence of saccharose is a powerful method to store them for a long period of time.


2011   Références trouvées : 12

Delalande, A. ; Bouakaz, A. ; Renault, G. ; Tabareau, F. ; Kotopoulis, S. ; Midoux, P. ; Arbeille, B. ; Uzbekov, R. ; S., C. ; Postema, M. and Pichon, C.  (2011)

Ultrasound and microbubble-assisted gene delivery in Achilles tendons : long lasting gene expression and restoration of fibromodulin KO phenotype

Journal of Controlled Release 2011, 156, (2), 223-230
The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

The aim of this study is to deliver genes in Achilles tendons using ultrasound and microbubbles. The rationale is to combine ultrasound-assisted delivery and the stimulation of protein expression induced by US. We found that mice tendons injected with 10 μg of plasmid encoding luciferase gene in the presence of 5×10⁵ BR14 microbubbles, exposed to US at 1 MHz, 200 kPa, 40% duty cycle for 10 min were efficiently transfected without toxicity. The rate of luciferase expression was 100-fold higher than that obtained when plasmid alone was injected. Remarkably, the luciferase transgene was stably expressed for up to 108 days. DNA extracted from these sonoporated tendons was efficient in transforming competent E. coli bacteria, indicating that persistent intact pDNA was responsible for this long lasting gene expression. We used this approach to restore expression of the fibromodulin gene in fibromodulin KO mice. A significant fibromodulin expression was detected by quantitative PCR one week post-injection. Interestingly, ultrastructural analysis of these tendons revealed that collagen fibrils diameter distribution and circularity were similar to that of wild type mice. Our results suggest that this gene delivery method is promising for clinical applications aimed at modulating healing or restoring a degenerative tendon while offering great promise for gene therapy due its safety compared to viral methods.

Breton, M. ; Berret, J. F. ; Bourgaux, C. ; Kral, T. ; Hof, M. ; Pichon, C. ; Bessodes, M. ; Scherman, D. andMignet, N.  (2011)

Protonation of Lipids Impacts the Supramolecular and Biological Properties of Their Self-Assembly

Langmuir 27 (20) 12336-12345
We assessed in this work how a chemical structure difference could influence a supramolecular organization and then its biological properties. In our case study, we considered two amphiphilic lipidic gene vectors. The chemical difference was situated on their hydrophilic part which was either a pure neutral thiourea head or a mixture of three thiourea function derivatives, thiourea, iminothiol, and charged iminothiol. This small difference was obtained thanks to the last chemical deprotection conditions of the polar head hydroxyl groups. Light, neutron, and X-ray scattering techniques have been used to investigate the spatial structure of the liposomes and lipoplexes formed by the lipids. The chemical structure difference impacts the supramolecular assemblies of the lipids and with DNA as shown by fluorescence correlation spectroscopy (FCS), X-ray, and neutron scattering. Hence the structures formed were found to be highly different in terms of liposomes to DNA ratio and size and polydispersity of the aggregates. Finally, the transfection and internalization results proved that the differences in the structure of the lipid aggregates fully affect the biological properties of the lipopolythiourea compounds. The lipid containing three functions is a better gene transfection agent than the lipid which only contains one thiourea moiety. As a conclusion, we showed that the conditions of the last chemical step can influence the lipidic supramolecular structure which in turn strongly impacts their biological properties.

We assessed in this work how a chemical structure difference could influence a supramolecular organization and then its biological properties. In our case study, we considered two amphiphilic lipidic gene vectors. The chemical difference was situated on their hydrophilic part which was either a pure neutral thiourea head or a mixture of three thiourea function derivatives, thiourea, iminothiol, and charged iminothiol. This small difference was obtained thanks to the last chemical deprotection conditions of the polar head hydroxyl groups. Light, neutron, and X-ray scattering techniques have been used to investigate the spatial structure of the liposomes and lipoplexes formed by the lipids. The chemical structure difference impacts the supramolecular assemblies of the lipids and with DNA as shown by fluorescence correlation spectroscopy (FCS), X-ray, and neutron scattering. Hence the structures formed were found to be highly different in terms of liposomes to DNA ratio and size and polydispersity of the aggregates. Finally, the transfection and internalization results proved that the differences in the structure of the lipid aggregates fully affect the biological properties of the lipopolythiourea compounds. The lipid containing three functions is a better gene transfection agent than the lipid which only contains one thiourea moiety. As a conclusion, we showed that the conditions of the last chemical step can influence the lipidic supramolecular structure which in turn strongly impacts their biological properties.

Bertrand, E. ; Goncalves, C. ; Billiet, L. ; Gomez, J. P. ; Pichon, C. ; Cheradame, H. ; Midoux, P. and Guégan, P  (2011)

Histidinylated linear PEI : a new efficient non-toxic polymer for gene transfer

Chem. Commun. 47 (46) 12547-12549
A series of linear polyethylenimine (lPEI) substituted with histidine residue (His-lPEI) was synthesized using the Michael reaction in order to provide new highly efficient vectors for gene therapy applications (up to 95% of transfected cells) with remarkable low cytotoxicity compared to lPEI-based polyplexes.

A series of linear polyethylenimine (lPEI) substituted with histidine residue (His-lPEI) was synthesized using the Michael reaction in order to provide new highly efficient vectors for gene therapy applications (up to 95% of transfected cells) with remarkable low cytotoxicity compared to lPEI-based polyplexes.

Berchel, M. ; Haelters, J. P. ; Couthon-Gourves, H. ; Deschamps, L. ; Midoux, P. ; Lehn, P. ; Jaffres, P. A.  (2011)

Modular Construction of Fluorescent Lipophosphoramidates by Click Chemistry

European Journal of Organic Chemistry 2011 (31) 6294-6303
The synthesis of fluorescent lipophosphoramidates is reported. Their modular construction allows several variations of the molecular structure including the separation of the fluorescent probe from the lipophosphoramidate moiety by a short methylene or by a tetraethylene glycol spacer. The last step of the convergent synthesis is a Huisgen click reaction, which assembles the fluorescent probe with the lipophosphoramidate part. Five fluorescent probes have been considered in this study including coumarin, NBD, fluorescein, naphthalimide and cyanine.

The synthesis of fluorescent lipophosphoramidates is reported. Their modular construction allows several variations of the molecular structure including the separation of the fluorescent probe from the lipophosphoramidate moiety by a short methylene or by a tetraethylene glycol spacer. The last step of the convergent synthesis is a Huisgen click reaction, which assembles the fluorescent probe with the lipophosphoramidate part. Five fluorescent probes have been considered in this study including coumarin, NBD, fluorescein, naphthalimide and cyanine.

Bennevault-Celton, V. ; Urbach, A. ; Martin, O. ; Pichon C. ; Guégan, P. and Midoux, P.  (2011)

Supramolecular Assemblies of Histidinylated α-Cyclodextrin in the Presence of DNA Scaffold during CDplexes Formation

Bioconjugate Chemistry 22 (12) 2404-2414
α-Cyclodextrin was transformed in a cationic unit after per substitution with histidine (His-α-CD) and lysine (Lys-α-CD) molecules on the primary face. His-α-CD and Lys-α-CD were used to form electrostatic complexes (CDplexes) with a plasmid DNA encoding luciferase gene, and the ability of CDplexes to transfect mammalian cells was examined using HEK293-T7 cells. The luciferase activity in cells transfected with His-α-CDplexes was 8-fold higher than that obtained Lys-α-CDplexes. When the transfection was carried out in the presence of chloroquine, the luciferase activity with His-α-CDplexes and Lys-α-CDplexes increased 6 and 25 times, respectively. The lower enhancement with His-α-CDplexes confirmed that histidine induced a proton sponge effect inside endosomes upon imidazole protonation, favoring DNA delivery in the cytosol. At the same time, we found that the condensation of DNA with His-α-CD was unexpectedly stronger than that obtained with the lysyl-α-CD counterpart. Moreover, it was as strong as that observed with high molecular weight polylysine. NMR (ROESY and DOSY) investigations in the absence of DNA showed that an inclusion complex is formed between the imidazole ring of histidine and the hydrophobic cavity of CD but no His-α-CD polymers can be formed by intermolecular interactions. These results suggest that intermolecular interactions between imidazole and His-α-CD cavity could be involved to form supramolecular assemblies in the presence of a DNA scaffold leading to DNA condensation into low diameter particles.

α-Cyclodextrin was transformed in a cationic unit after per substitution with histidine (His-α-CD) and lysine (Lys-α-CD) molecules on the primary face. His-α-CD and Lys-α-CD were used to form electrostatic complexes (CDplexes) with a plasmid DNA encoding luciferase gene, and the ability of CDplexes to transfect mammalian cells was examined using HEK293-T7 cells. The luciferase activity in cells transfected with His-α-CDplexes was 8-fold higher than that obtained Lys-α-CDplexes. When the transfection was carried out in the presence of chloroquine, the luciferase activity with His-α-CDplexes and Lys-α-CDplexes increased 6 and 25 times, respectively. The lower enhancement with His-α-CDplexes confirmed that histidine induced a proton sponge effect inside endosomes upon imidazole protonation, favoring DNA delivery in the cytosol. At the same time, we found that the condensation of DNA with His-α-CD was unexpectedly stronger than that obtained with the lysyl-α-CD counterpart. Moreover, it was as strong as that observed with high molecular weight polylysine. NMR (ROESY and DOSY) investigations in the absence of DNA showed that an inclusion complex is formed between the imidazole ring of histidine and the hydrophobic cavity of CD but no His-α-CD polymers can be formed by intermolecular interactions. These results suggest that intermolecular interactions between imidazole and His-α-CD cavity could be involved to form supramolecular assemblies in the presence of a DNA scaffold leading to DNA condensation into low diameter particles.

Baril, P. ; Touchefeu, Y. ; Cany, J. ; Cherel, Y. ; Thorne, S. H. ; Tran, L. ; Conchon, S. and Vassaux, G.  (2011)

Differential biodistribution of oncolytic poxvirus administered systemically in an autochthonous model of hepatocellular carcinoma

The Journal of Gene Medicine (2011) 13 (12) 692-701
Background Preclinical studies have demonstrated that, unlike oncolytic adenoviruses, oncolytic vaccinia viruses can reach implanted tumors upon systemic injection. However, the biodistribution of this oncolytic agent in in situ autochthonous tumor models remains poorly characterized. In the present study, we assessed this biodistribution in a model of mouse hepatocellular carcinoma (HCC) obtained after injection of the carcinogen diethylnitrosamine (DEN). Methods Twelve months after DEN administration, histology, quantitative reverse transcription-polymerase chain reaction, in situ hybridization and viral titration were used to characterize tumors, as well as to assess the viral load of the livers upon either intravenous or intraperitoineal injection. Results The results obtained showed that the architecture of the liver was lost, with a noticeable absence of sinusoids, as well as the presence of steatosis and α-fetoprotein-positive HCC tumor nodules. Bioluminescence imaging and measures of the infective virus load demonstrated that intravenous injection of 108 plaque-forming units of the recombinant vaccinia virus led to a predominant transduction of the liver, whereas intraperitoneal injection resulted in a lower level of liver transduction accompanied by an increased infection of the lungs, spleen, kidneys and bowels. Immunohistochemical analysis of liver sections of animals injected intravenously with the virus revealed a preferential localization of vaccinia-specific immunoreactivity in the tumors. Conclusions The findings of the present study emphasize the importance of the route of administration of the vector and highlight the relevance of systemic injection of oncolytic vaccinia virus in the context of hepatocellular carcinoma.

Background Preclinical studies have demonstrated that, unlike oncolytic adenoviruses, oncolytic vaccinia viruses can reach implanted tumors upon systemic injection. However, the biodistribution of this oncolytic agent in in situ autochthonous tumor models remains poorly characterized. In the present study, we assessed this biodistribution in a model of mouse hepatocellular carcinoma (HCC) obtained after injection of the carcinogen diethylnitrosamine (DEN). Methods Twelve months after DEN administration, histology, quantitative reverse transcription-polymerase chain reaction, in situ hybridization and viral titration were used to characterize tumors, as well as to assess the viral load of the livers upon either intravenous or intraperitoineal injection. Results The results obtained showed that the architecture of the liver was lost, with a noticeable absence of sinusoids, as well as the presence of steatosis and α-fetoprotein-positive HCC tumor nodules. Bioluminescence imaging and measures of the infective virus load demonstrated that intravenous injection of 108 plaque-forming units of the recombinant vaccinia virus led to a predominant transduction of the liver, whereas intraperitoneal injection resulted in a lower level of liver transduction accompanied by an increased infection of the lungs, spleen, kidneys and bowels. Immunohistochemical analysis of liver sections of animals injected intravenously with the virus revealed a preferential localization of vaccinia-specific immunoreactivity in the tumors. Conclusions The findings of the present study emphasize the importance of the route of administration of the vector and highlight the relevance of systemic injection of oncolytic vaccinia virus in the context of hepatocellular carcinoma.

Maurel, D. B., Jaffre, C., Rochefort, G. Y., Aveline, P. C., Boisseau, N., Uzbekov, R., Gosset, D., Pichon, C., Fazzalari, N. L., Pallu, S. & Benhamou, C. L.  (2011)

Low bone accrual is associated with osteocyte apoptosis in alcohol-induced osteopenia.

Bone 49 (3) 543-552
Introduction : Alcohol is known to decrease bone mineral density (BMD) and to induce trabecular microarchitecture deterioration. However, little is known about the effects of chronic alcohol consumption on osteocytes in situ. The aim of this study was to assess the effects of a high alcohol dose on osteocytes in an alcohol-induced osteopenia model. Materials and methods : 24 male Wistar rats, 2-months old were separated in 2 groups : Control (C) or Alcohol (A35). The rats in the A35 group drank a beverage composed of 35% ethanol v/v mixed to water for 17 weeks. BMD was assessed by DXA, while the microarchitecture was analyzed using mu CT. Bone remodeling was studied measuring serum concentration of osteocalcin, NTx and TRAP. Bone marrow adiposity, osteoblastic lineage differentiation, osteocyte morphology and apoptosis were assessed using bright field, epifluorescence, transmission electron and confocal microscopy. Results : BMD, trabecular thickness, TRAP and NTx concentration were significantly decreased in A35, while cortical thickness was thinner. There were 10 fold more cells stained with cleaved caspase-3, and 35% more empty lacunae in A35, these data indicating a large increase in osteocyte apoptosis in the A35 group. The number of lipid droplets in the marrow was increased in A35 (7 fold). Both the osteocyte apoptosis and the fat bone marrow content strongly correlated with femur BMD (p = 0.0017, r = -0.72 and p = 0.002, r = 0.70) and whole body BMD. Conclusion : These data suggest that low BMD is associated with osteocyte apoptosis and bone marrow fat content in alcohol-induced osteopenia.

Introduction : Alcohol is known to decrease bone mineral density (BMD) and to induce trabecular microarchitecture deterioration. However, little is known about the effects of chronic alcohol consumption on osteocytes in situ. The aim of this study was to assess the effects of a high alcohol dose on osteocytes in an alcohol-induced osteopenia model. Materials and methods : 24 male Wistar rats, 2-months old were separated in 2 groups : Control (C) or Alcohol (A35). The rats in the A35 group drank a beverage composed of 35% ethanol v/v mixed to water for 17 weeks. BMD was assessed by DXA, while the microarchitecture was analyzed using mu CT. Bone remodeling was studied measuring serum concentration of osteocalcin, NTx and TRAP. Bone marrow adiposity, osteoblastic lineage differentiation, osteocyte morphology and apoptosis were assessed using bright field, epifluorescence, transmission electron and confocal microscopy. Results : BMD, trabecular thickness, TRAP and NTx concentration were significantly decreased in A35, while cortical thickness was thinner. There were 10 fold more cells stained with cleaved caspase-3, and 35% more empty lacunae in A35, these data indicating a large increase in osteocyte apoptosis in the A35 group. The number of lipid droplets in the marrow was increased in A35 (7 fold). Both the osteocyte apoptosis and the fat bone marrow content strongly correlated with femur BMD (p = 0.0017, r = -0.72 and p = 0.002, r = 0.70) and whole body BMD. Conclusion : These data suggest that low BMD is associated with osteocyte apoptosis and bone marrow fat content in alcohol-induced osteopenia.

Perche, F., Benvegnu, T., Berchel, M., Lebègue, L., Pichon, C., Jaffrès, P.-A. & Midoux, P.,  (2011)

Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA

Nanomedicine : Nanotechnology, Biology and Medicine 7 (4) 445-453

Jaffres, P. A., Fraix, A., Lorilleux, C., Berchel, M., Couthon-Gourves, H., Haelters, J. P., Yaouanc, J. J., Burel, L., Giamarchi, P., Midoux, P., Montier, T. & Lehn, P.  (2011)

New lipo-phospharamidates for gene delivery

Phosphorus Sulfur and Silicon and the Related Elements 186 (4) 918-920

Doucet, J. Briki, F. Gourrier, A. Pichon, C. Gumez, L. Bensamoun, S. & Sadoc, J. F.  (2011)

Modeling the lateral organization of collagen molecules in fibrils using the paracrystal concept

Journal of Structural Biology 173 (2) 197-201

Delalande, A. Kotopoulis, S. Rovers, T. Pichon, C. & Postema, M.  (2011)

Sonoporation at low mechanical index

Bubbles Science Engineering and Technology - 3 (1) 3-12

Perche, F., Gosset, D., Mével, M., Miramon, M.-L., Yaouanc, J.J., Pichon, C., Benvegnu, T., Jaffrès, P.A. & Midoux, P.  (2011)

Selective delivery in dendritic cells with Mannosylated and Histidylated Lipopolyplexes.

J. Drug Target. 19 (5) 315-325


2010   Références trouvées : 8

Gumez, L. Bensamoun, S. F. Doucet, J. Haddad, O. Hawse, J. R. Subramaniam, M. Spelsberg, T. C. & Pichon, C.  (2010)

Molecular structure of tail tendon fibers in TIEG1 knockout mice using synchrotron diffraction technology.

Journal of Applied Physiology - 108 (6) 1706-1710

Kaddur, K., Lebegue, L., Tranquart, F., Midoux, P., Pichon, C. and Bouakaz, A.  (2010)

Transient transmembrane release of green fluorescent proteins with sonoporation.

IEEE Trans Ultrason Ferroelectr. Freq. Control. 57, 1558-1567.

Pichon, C., Billiet, L. and Midoux, P.  (2010)

Chemical vectors for gene delivery : uptake and intracellular trafficking.

Curr. Opin. 21 (5) 640-645

Sacquin-Mora, S., Delalande, O. & Baaden, M.  (2010)

Functional modes and residue flexibility control the anisotropic response of Guanylate Kinase to mechanical stress

Biophysical Journal (2010) 99 (10) : 3412-3419 - doi : 0.1016/j.bpj.2010.09.026
The coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. In particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. We study such phenomena using a method for investigating local flexibility on the residue scale that combines a reduced protein representation with Brownian dynamics simulations. We performed calculations on the enzyme guanylate kinase to study its mechanical response when submitted to anisotropic deformations. The resulting modifications of the protein's rigidity profile can be related to the changes in substrate binding affinity observed experimentally. Further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme's catalytic rate. Eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechanical and dynamical points of view. Such enzyme-engineering approaches open the possibility to tune catalytic function by varying selected external forces.

The coupling between the mechanical properties of enzymes and their biological activity is a well-established feature that has been the object of numerous experimental and theoretical works. In particular, recent experiments show that enzymatic function can be modulated anisotropically by mechanical stress. We study such phenomena using a method for investigating local flexibility on the residue scale that combines a reduced protein representation with Brownian dynamics simulations. We performed calculations on the enzyme guanylate kinase to study its mechanical response when submitted to anisotropic deformations. The resulting modifications of the protein’s rigidity profile can be related to the changes in substrate binding affinity observed experimentally. Further analysis of the principal components of motion of the trajectories shows how the application of a mechanical constraint on the protein can disrupt its dynamics, thus leading to a decrease of the enzyme’s catalytic rate. Eventually, a systematic probe of the protein surface led to the prediction of potential hotspots where the application of an external constraint would produce a large functional response both from the mechanical and dynamical points of view. Such enzyme-engineering approaches open the possibility to tune catalytic function by varying selected external forces.

Suwalski, A., Dabboue, H., Delalande, A., Bensamoun, S., Canon, F., Midoux, P., Saillant, G., Klatzmann, D., Salvetat, J. & Pichon, C.  (2010)

Accelerated Achilles tendon healing by PDGF gene delivery with mesoporous silica nanoparticles.

Biomaterials 31, 5237-5245.

Fisichella, M., Dabboue, H., Bhattacharyya, S., Lelong, G., Warmont, F., Midoux, P., Pichon, C., Guérin, M., Hevor, T. and Salvetat, J.-P.  (2010)

Uptake of Functionalized Mesoporous Silica Nanoparticles by Human Cancer Cells.

J. Nanosci. Nanotechnol. 10 (4) : 2314-2324

Delalande, A., Bureau, M.F., Midoux, P., Bouakaz, A. & Pichon, C.  (2010)

Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy.

Ultrasonics 50, 269-272.

Breton, M., Leblond, J., Seguin, J., Midoux, P., Schermani, D., Herscovici, J., Pichon, C. & Mignet, N.  (2010)

Comparative gene transfer between cationic and thiourea lipoplexes.

J. Gene Med. 12, 45-54.


2009   Références trouvées : 3

Bure, C., Pichon, C. & Midoux, P.  (2009)

Involvement of histidines 11, 15 and 19 in the binding of zinc to the fusogenic H5WYG peptide

(2009) J Mass Spectrom 44, 1163-1170.

Midoux, P., Pichon, C., Yaouanc, J.J. & Jaffres, P.A.   (2009)

Chemical vectors for gene delivery : a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers.

Br. J. Pharmacol. 157, 166-178.

Goncalves, C., Ardourel, M.Y., Decoville, M., Breuzard, G., Midoux, P., Hartmann, B. & Pichon, C.  (2009)

An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression.

J. Gene Med. 11, 401-411.


2008   Références trouvées : 8

Pichon, C., Kaddur, K., Midoux, P., Tranquart, F. & Bouakaz, A.  (2008)

Recent advances in gene delivery with ultrasound and microbubbles.

J. Exp. Nanosci. 3, 17-40.

Midoux, P., Breuzard, G., Gomez, J.P. & Pichon, C.  (2008)

Polymer-Based Gene Delivery : A Current Review on the Uptake and Intracellular Trafficking of Polyplexes.

Curr. Gene Ther. 8, 335-352.

Mevel, M., Neveu, C., Goncalves, C., Yaouanc, J.J., Pichon, C., Jaffres, P.A. & Midoux, P.  (2008)

Novel neutral imidazole-lipophosphoramides for transfection assays.

Chem. Commun. 27, 3124-3126 (hot article le 08 juillet 2008).

Mevel, M., Breuzard, G., Yaouanc, J.J., Clement, J.C., Lehn, P., Pichon, C., Jaffres, P.A. & Midoux, P.  (2008)

Synthesis and transfection activity of new cationic phosphoramidate lipids : High efficiency of an imidazolium derivative.

ChemBioChem 9, 1462-1471.

Lopez-Crapez, E., Malinge, J.M., Gatchitch, F., Casano, L., Langlois, T., Pugniere, M., Roquet, F., Mathis, G. & Bazin, H.  (2008)

A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions.

Anal. Biochem. 383, 301-306.

Castellano-Castillo, M., Kostrhunova, H., Marini, V., Kasparkova, J., Sadler, P.J., Malinge, J.M. & Brabec, V.  (2008)

Binding of mismatch repair protein MutS to mispaired DNA adducts of intercalating ruthenium(II) arene complexes.

J. Biol. Inorg. Chem. 13, 993-999.

Breuzard, G., Tertil, M., Goncalves, C., Cheradame, H., Geguan, P., Pichon, C. & Midoux, P.  (2008)

Nuclear delivery of N kappa B-assisted DNA/polymer complexes : plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging.

Nucleic Acids Res. 36, 12, e71.

Bonnet, N., Benhamou, C.L., Malaval, L., Goncalves, C., Vico, L., Eder, V., Pichon, C. & Courteix, D.  (2008)

Low Dose Beta-Blocker Prevents Ovariectomy-induced Bone Loss in Rats Without Affecting Heart Functions.

J. Cell Physiol. 217, 819-827.


2007   Références trouvées : 4

Kaddur, K ; Palanchon, P ; Tranquart, F ; Pichon, C ; Bouakaz, A   (2007)

Sonopermeabilization : therapeutic alternative with ultrasound and microbubbles

Journal de Radiologie 88 (11) Part 2 1777-1786
Future applications of ultrasound and microbubbles extend to more than imaging applications. Over the last few years, it was reported that sonographic contrast agent effects under ultrasound, modulate transiently cell membrane permeability. This process, named sonoporation and classified as a new physical method to transfer genes or drugs, consists of using a physical energy source to modulate membrane integrity. The possibility to transfer therapeutic genes would be a new tool for gene therapy and could constitute an alternative method. After in vitro and in vivo studies presentation, the therapeutic potential of sonoporation will be investigated in this paper.

Future applications of ultrasound and microbubbles extend to more than imaging applications. Over the last few years, it was reported that sonographic contrast agent effects under ultrasound, modulate transiently cell membrane permeability. This process, named sonoporation and classified as a new physical method to transfer genes or drugs, consists of using a physical energy source to modulate membrane integrity. The possibility to transfer therapeutic genes would be a new tool for gene therapy and could constitute an alternative method. After in vitro and in vivo studies presentation, the therapeutic potential of sonoporation will be investigated in this paper.

Mockey, M ; Bourseau, E ; Chandrashekhar, V ; Chaudhuri, A ; Lafosse, S ; Le Cam, E ; Quesniaux, VFJ ; Ryffel, B ; Pichon, C ; Midoux, P  (2007)

mRNA-based cancer vaccine : prevention of B16 melanoma progression and metastasis by systemic injection of MART1 mRNA histidylated lipopolyplexes

Cancer Gene Therapy 14 (9) 802-814
Immunization with mRNA encoding tumor antigen is an emerging vaccine strategy for cancer. In this paper, we demonstrate that mice receiving systemic injections of MART1 mRNA histidylated lipopolyplexes were specifically and significantly protected against B16F10 melanoma tumor progression. The originality of this work concerns the use of a new tumor antigen mRNA formulation as vaccine, which allows an efficient protection against the growth of a highly aggressive tumor model after its delivery by intravenous route.

Immunization with mRNA encoding tumor antigen is an emerging vaccine strategy for cancer. In this paper, we demonstrate that mice receiving systemic injections of MART1 mRNA histidylated lipopolyplexes were specifically and significantly protected against B16F10 melanoma tumor progression. The originality of this work concerns the use of a new tumor antigen mRNA formulation as vaccine, which allows an efficient protection against the growth of a highly aggressive tumor model after its delivery by intravenous route.

Breuzard, G ; Tertil, M ; Pichon, C ; Midoux, P  (2007)

Intracellular trafficking and nuclear import of polyplexes by FRET coupled with cellular imaging

Human Gene Therapy 18 1048-1048

Sedletska, Y ; Fourrier, L ; Malinge, JM  (2007)

Modulation of MutS ATP-dependent functional activities by DNA containing a cisplatin compound lesion (base damage and mismatch)

Journal of Molecular Biology 369 (1) 27-40
DNA damage-dependent signaling by the DNA mismatch repair (MMR) system is thought to mediate cytotoxicity of the anti-tumor drug cisplatin through molecular mechanisms that could differ from those required for normal mismatch repair. The present study investigated whether ATP-dependent biochemical properties of Escherichia coli MutS protein differ when the protein interacts with a DNA oligonucleotide containing a GT mismatch versits a unique site specifically placed cisplatin compound lesion, a cisplatin 1,2-d(GpG) intrastrand cross-link with a mispaired thymine opposite the 3' platinated guanine.

DNA damage-dependent signaling by the DNA mismatch repair (MMR) system is thought to mediate cytotoxicity of the anti-tumor drug cisplatin through molecular mechanisms that could differ from those required for normal mismatch repair. The present study investigated whether ATP-dependent biochemical properties of Escherichia coli MutS protein differ when the protein interacts with a DNA oligonucleotide containing a GT mismatch versits a unique site specifically placed cisplatin compound lesion, a cisplatin 1,2-d(GpG) intrastrand cross-link with a mispaired thymine opposite the 3’ platinated guanine.


2006   Références trouvées : 2

Mockey, M ; Goncalves, C ; Dupuy, FP ; Lemoine, FM ; Pichon, C ; Midoux, P  (2006)

mRNA transfection of dendritic cells : Synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level

Biochemical and Biophysical Research Communications 340 (4) 1062-1068
The occurrence of translation mechanism in the cytosol offers advantages to mRNA transfer over DNA-based transfection in nondividing cells. Here, we sought to optimize mRNA constructs allowing a high level of protein upon lipofection. We found that luciferase into mouse dendritic cells (JAWSII cells) was similar to 20-fold higher when the luciferase mRNA was capped with 3'-O-methyl-m7(5')GpPP(5')G (anti-reverse cap analogue ; ARCA) than with m7(5')Gppp(5')G (CAP).

The occurrence of translation mechanism in the cytosol offers advantages to mRNA transfer over DNA-based transfection in nondividing cells. Here, we sought to optimize mRNA constructs allowing a high level of protein upon lipofection. We found that luciferase into mouse dendritic cells (JAWSII cells) was similar to 20-fold higher when the luciferase mRNA was capped with 3’-O-methyl-m7(5’)GpPP(5’)G (anti-reverse cap analogue ; ARCA) than with m7(5’)Gppp(5’)G (CAP).

Mennesson, E ; Erbacher, P ; Kuzak, M ; Kieda, C ; Midoux, P ; Pichon, C  (2006)

DNA/cationic polymer complex attachment on a human vascular endothelial cell monolayer exposed to a steady laminar flow

Journal of Controlled Release 114 (3) 389-397
This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe.

This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe.


2005   Références trouvées : 3

Mennesson, E ; Erbacher, P ; Piller, W ; Kieda, C ; Midoux, P ; Pichon C  (2005)

Transfection efficiency and uptake process of polyplexes in human lung endothelial cells : a comparative study in non-polarized and polarized cells

Journal of Gene Medicine 7 (6) 729-738
Background Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. Methods After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles.

Background Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. Methods After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles.

Mennesson, E ; Erbacher, P ; Piller, V ; Kieda, C ; Midoux, P ; Pichon, C  (2005)

Transfection efficiency and uptake process of polyplexes in human lung endothetial cells : A comparative study in non-polarized and polarized cells

Journal of Gene Medicine 7 (6) 729-738
Background : Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells.

Background : Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells.

Malinge, JM   (2005)

Cisplatin is a DNA-damaging antitumour compound triggering multifactorial biochemical responses in cancer cells : importance of apoptotic pathways

Current Medicinal Chemistry- Anti-Cancer Agents 5 (3) 251-265


2004   Références trouvées : 6

Kamal, A., Kumar, B.A., Arifuddin, M. and Midoux, P.   (2004)

An efficient and facile nitration of phenols with nitric acid/zinc chloride under ultrasonic conditions

Ultrasonics Sonochemistry 11, 455-457.
An efficient and facile nitration of phenols using nitric acid/zinc chloride under ultrasonic condition has exhibited significant chemo as well as regioselectivity. This study has been extended to other aromatic compounds like naphthalene and anthracene.

An efficient and facile nitration of phenols using nitric acid/zinc chloride under ultrasonic condition has exhibited significant chemo as well as regioselectivity. This study has been extended to other aromatic compounds like naphthalene and anthracene.

Elmadbouh, I ; Rossignol, P ; Meilhac, O ; Vranckx, R ; Pichon, C ; Pouzet, B ; Midoux, P ; Michel, JB  (2004)

Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications

Journal of Gene Medicine 6 (10) 1112-1124
Background : Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo.

Background : Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo.

Singh, RS ; Goncalves, C ; Sandrin, P ; Pichon, C ; Midoux, P ; Chaudhuri, A  (2004)

On the gene delivery efficacies of pH-sensitive cationic lipids via endosomal protonation : a chemical biology investigation

Chemistry & Biology 11 (6) 883-883

Montier, T ; Delepine, P ; Pichon, C ; Ferec, C ; Porteous, DJ ; Midoux, P  (2004)

Non-viral vectors in cystic fibrosis gene therapy : progress and challenges

Trends in Biotechnology 22 (11) 586-592
Although the viability of cystic fibrosis (CF) gene transfer to airway epithelium has been demonstrated in vitro and in animal models, so far none of the clinical investigations using adenovirus, adeno-associated virus, lentivirus, cationic lipids or polymers has shown a persistent correction of the ion transport defects that occur in CF. Despite disappointing results, these studies have shown that non-viral vectors could represent a viable alternative for gene therapy in CF airway epithelium. The transfer efficiency of non-viral vectors is currently low, however, and thus these systems are not clinically relevant as yet. Before clinical application, several limitations encountered by non-viral delivery systems must be addressed. Recent progress has been made towards overcoming these limitations and towards making non-viral gene therapy a more realistic option for CF.

Although the viability of cystic fibrosis (CF) gene transfer to airway epithelium has been demonstrated in vitro and in animal models, so far none of the clinical investigations using adenovirus, adeno-associated virus, lentivirus, cationic lipids or polymers has shown a persistent correction of the ion transport defects that occur in CF. Despite disappointing results, these studies have shown that non-viral vectors could represent a viable alternative for gene therapy in CF airway epithelium. The transfer efficiency of non-viral vectors is currently low, however, and thus these systems are not clinically relevant as yet. Before clinical application, several limitations encountered by non-viral delivery systems must be addressed. Recent progress has been made towards overcoming these limitations and towards making non-viral gene therapy a more realistic option for CF.

Kumar, VV ; Singh, RS ; Goncalves, C ; Sandrin, P ; Pichon, C ; Midoux, P ; Chaudhuri, A  (2004)

New histidylated cationic lipids for DNA- and mRNA-based lipofection

Molecular Therapy 9 S258-S259 Art No. 682 Suppl. 1

Goncalves, C ; Mennesson, E ; Fuchs, R ; Gorvel, JP ; Midoux, P ; Pichon, C  (2004)

Macropinocytosis of polyplexes and recycling of plasmid from clathrin-dependent pathway impair the transfection efficiency into human hepatocarcinoma cells

Molecular Therapy 9 S317-S317 Art No. 835 Suppl. 1


2003   Références trouvées : 4

Kumar, VV ; Pichon, C ; Refregiers, M ; Guerin, B ; Midoux, P ; Chaudhuri, A  (2003)

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids : evidence for histidine-mediated membrane fusion at acidic pH

Gene Therapy 10 (15) 1206-1215
Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles.

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles.

Midoux, P ; LeCam, E ; Coulaud, D ; Delain, E ; Pichon, C - Editor(s) : Luo, D ; Saltzman, WM ; Luo, D ; Saltzman, WM  (2003)

Histidine containing peptides and polypeptides as nucleic acid vectors

Synthetic Dna Delivery Systems 7 23-44

Madbouh, IEL ; Rossignol, P ; Midoux, P ; Michel, JB  (2003)

Optimization of primary cultured vascular cell transfection with non-viral vectors

Journal of Hypertension 21 (8) A6-A6

Fourrier, L ; Brooks, P ; Malinge, JM  (2003)

Binding discrimination of MutS to a set of lesions and compound lesions (base damage and mismatch) reveals its potential role as a cisplatin-damaged DNA sensing protein

Journal of Biological Chemistry 278 (23) 21267-21275
The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin. It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions. This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis. Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions. The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate.

The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin. It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions. This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis. Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions. The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate.


2002   Références trouvées : 4

Pichon, C ; Guerin, B ; Refregiers, M ; Goncalves, C ; Vigny, P ; Midoux, P  (2002)

Zinc improves gene transfer mediated by DNA/cationic polymer complexes

Journal of Gene Medicine 4 (5) 548-559
Background : The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn2+ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His-pLK) complexes.

Background : The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn2+ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His-pLK) complexes.

Pichon, C ; LeCam, E ; Guerin, B ; Coulaud, D ; Delain, E ; Midoux, P  (2002)

Poly[Lys-(AEDTP)] : A cationic polymer that allows dissociation of pDNA/cationic polymer complexes in a reductive medium and enhances polyfection

Bioconjugate Chemistry 13 (1) 76-82
Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polymer are widely used to develop polymer-based gene delivery systems. However, the plasmid must be released from its vector once inside the cells for an efficient expression of the exogenous gene in the cell nucleus. We have designed a disulfide-containing cationic polymer termed poly [Lys-(AEDTP)] which allowed for the formation of polyplexes and the release of the plasmid in a reductive medium. The amino groups of polylysine were substituted with 3-(2-aminoethyldithio-)propionyl residues in order to have each amino group of poly [Lys-(AEDTP)] interacting with a phosphate DNA linked to the polymer backbone via a disulfide bond.

Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polymer are widely used to develop polymer-based gene delivery systems. However, the plasmid must be released from its vector once inside the cells for an efficient expression of the exogenous gene in the cell nucleus. We have designed a disulfide-containing cationic polymer termed poly [Lys-(AEDTP)] which allowed for the formation of polyplexes and the release of the plasmid in a reductive medium. The amino groups of polylysine were substituted with 3-(2-aminoethyldithio-)propionyl residues in order to have each amino group of poly [Lys-(AEDTP)] interacting with a phosphate DNA linked to the polymer backbone via a disulfide bond.

Goncalves, C ; Pichon, C ; Guerin, B ; Midoux, P  (2002)

Intracellular processing and stability of DNA complexed with histidylated polylysine conjugates

Journal of Gene Medicine 4 (3) 271-281
Background : Glycosylated polylysines and histidylated polylysines complexed with plasmid DNA (pDNA) were proposed to develop polymer-based gene delivery systems. The present work has been undertaken in two steps to study the uptake and the intracellular processing of pDNA, which are still poorly understood in the polyfection pathway.

Background : Glycosylated polylysines and histidylated polylysines complexed with plasmid DNA (pDNA) were proposed to develop polymer-based gene delivery systems. The present work has been undertaken in two steps to study the uptake and the intracellular processing of pDNA, which are still poorly understood in the polyfection pathway.

Montero, EI ; Perez, JM ; Schwartz, A ; Fuertes, MA ; Malinge, JM ; Alonso, C ; Leng, M ; Navarro-Ranninger, C  (2002)

Apoptosis induction and DNA interstrand cross-link formation by cytotoxic trans-[PtCl2(NH(CH3)(2))(NHCH(CH3)(2))] : Cross-linking between d(G) and complementary d(C) within oligonucleotide duplexes

Chembiochem 3 (1) 61-67
We have investigated the cytotoxic activity, the induction of apoptosis, and the interstrand cross-linking efficiency in the A2780cisR ovarian tumor cell line, after replacement of the two NH3 nonleaving groups in trans-[PtCl2(NH3)(2)] (trans-DDP) by dimethylamine and isopropylamine. The data show that trans-[PtCl2(NH(CH3)(2))(NHCH(CH3)(2))] is able to circumvent resistance to cis-[PtCl2(NH3)(2)] (cis-DDP, cisplatin) in A2780cisR cells.

We have investigated the cytotoxic activity, the induction of apoptosis, and the interstrand cross-linking efficiency in the A2780cisR ovarian tumor cell line, after replacement of the two NH3 nonleaving groups in trans-[PtCl2(NH3)(2)] (trans-DDP) by dimethylamine and isopropylamine. The data show that trans-[PtCl2(NH(CH3)(2))(NHCH(CH3)(2))] is able to circumvent resistance to cis-[PtCl2(NH3)(2)] (cis-DDP, cisplatin) in A2780cisR cells.


2001   Références trouvées : 4

Pichon, C ; Goncalves, C ; Midoux, P  (2001)

Histidine-rich peptides and polymers for nucleic acids delivery

Advanced Drug Delivery Reviews 53 (1) 75-94
Nucleic acids transfer into mammalian cells requires devices to improve their escape from endocytic vesicles where they are mainly confined following cellular uptake. In this review, we describe histidine-rich molecules that enable the transfer of plasmid and oligonucleotides (ODN) in human and non-human cultured cells. An histidine-rich peptide which permeabilizes biological membrane at pH 6.4, favored the transfection mediated by lactosylated polylysine/pDNA complexes.

Nucleic acids transfer into mammalian cells requires devices to improve their escape from endocytic vesicles where they are mainly confined following cellular uptake. In this review, we describe histidine-rich molecules that enable the transfer of plasmid and oligonucleotides (ODN) in human and non-human cultured cells. An histidine-rich peptide which permeabilizes biological membrane at pH 6.4, favored the transfection mediated by lactosylated polylysine/pDNA complexes.

Ohan, J ; Gilbert, MA ; Leseche, G ; Panis, Y ; Midoux, P ; Drouet, L  (2001)

Nonviral gene transfer into primary cultures of human and porcine mesothelial cells

In Vitro Cellular & Developmental Biology-Animal 37 (7) 402-407
Due to their abundance and accessibility, mesothelial cells may be suitable toots for recombinant reagent expression by gene transfer. Genetically modified porcine mesothelial cells (PMCs) may have the potential for the treatment of vascular diseases in humans. We studied the effect of various transfection reagents on the primary culture of PMCs and human mesothelial cells (HMCs). The cells were transfected with a plasmid encoding a reporter gene (luciferase or green fluorescent protein [GFP]) under the control of the cytomegalovirus promoter.

Due to their abundance and accessibility, mesothelial cells may be suitable toots for recombinant reagent expression by gene transfer. Genetically modified porcine mesothelial cells (PMCs) may have the potential for the treatment of vascular diseases in humans. We studied the effect of various transfection reagents on the primary culture of PMCs and human mesothelial cells (HMCs). The cells were transfected with a plasmid encoding a reporter gene (luciferase or green fluorescent protein [GFP]) under the control of the cytomegalovirus promoter.

Bello Roufai, M. and Midoux, P.  (2001)

Histidylated polylysine as DNA vector : Elevation of the imidazole protonation and reduced cellular uptake without change in the polyfection efficiency of serum stabilized negative polyplexes

Bioconjugate Chemistry 12 (1) 92-99
We have reported that polylysine substituted with histidyl residues (His) was suited to make complexes with plasmid DNA (pDNA) and to transfect cells in vitro in the presence of serum. The present study was performed to determine whether the acetylation of the a -amino group of histidyl residues (AcHis) had an influence on the size and the charge of polyplexes and on their transfection efficiency. We found that the presence of free a -amino groups allowed the formation of smaller polyplexes but did not modify the zeta potential of +17 mV. At a physiological salt concentration, the adsorption of many serum proteins on His- and AcHis-polyplexes reduced their size below 100 nm, inhibited their aggregation, and reversed their zeta potential to -25 mV.

We have reported that polylysine substituted with histidyl residues (His) was suited to make complexes with plasmid DNA (pDNA) and to transfect cells in vitro in the presence of serum. The present study was performed to determine whether the acetylation of the a -amino group of histidyl residues (AcHis) had an influence on the size and the charge of polyplexes and on their transfection efficiency. We found that the presence of free a -amino groups allowed the formation of smaller polyplexes but did not modify the zeta potential of +17 mV. At a physiological salt concentration, the adsorption of many serum proteins on His- and AcHis-polyplexes reduced their size below 100 nm, inhibited their aggregation, and reversed their zeta potential to -25 mV.

Fourrier, L ; Malinge, JM ; Brooks, P  (2001)

Analysis of the binding of MutS to cisplatin-modified DNA

Journal of Inorganic Biochemistry 86 (1) 220-220


2000   Références trouvées : 10

Dorange, F ; El Mehdaoui, S ; Pichon, C ; Coursaget, P ; Vautherot, JF  (2000)

Marek’s disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes : high-level expression and characterization of MDV-1 VP22 and VP16

Journal of General Virology 81 2219-2230 Part 9
Genes UL49 and UL48 of Marek's disease virus 1 (MDV-1) strain RB1B, encoding the respective homologues of herpes simplex virus type 1 (HSV-1) genes VP22 and VP16, were cloned into a baculovirus vector. Seven anti-VP22 MAbs and one anti-VP16 MAb were generated and used to identify the tegument proteins in cells infected lytically with MDV-1.

Genes UL49 and UL48 of Marek’s disease virus 1 (MDV-1) strain RB1B, encoding the respective homologues of herpes simplex virus type 1 (HSV-1) genes VP22 and VP16, were cloned into a baculovirus vector. Seven anti-VP22 MAbs and one anti-VP16 MAb were generated and used to identify the tegument proteins in cells infected lytically with MDV-1.

Fajac, I ; Allo, JC ; Souil, E ; Merten, M ; Pichon, C ; Figarella, C ; Monsigny, M ; Briand, P ; Midoux, P  (2000)

Histidylated polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells

Journal of Gene Medicine 2 (5) 368-378
Background : We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes.

Background : We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes.

Pichon, C ; Roufai, MB ; Monsigny, M ; Midoux, P  (2000)

Histidylated oligolysines increase the transmembrane passage and the biological activity of antisense oligonucleotides

Nucleic Acids Research 28 (2) 504-512
We have designed histidylated oligolysines which increase the uptake, the cytosolic delivery and the nuclear accumulation of antisense oligonucleotides (ODN), Flow cytometry analysis showed a 10-fold enhancement of the ODN uptake in the presence of histidylated oligolysines. The intracellular localizations of fluorescein-labeled ODN and of rhodamine-labeled histidylated oligolysines were investigated by confocal microscopy. Histidylated oligolysines favor the cytosolic delivery of ODN from endosomes and increase their nuclear accumulation.

We have designed histidylated oligolysines which increase the uptake, the cytosolic delivery and the nuclear accumulation of antisense oligonucleotides (ODN), Flow cytometry analysis showed a 10-fold enhancement of the ODN uptake in the presence of histidylated oligolysines. The intracellular localizations of fluorescein-labeled ODN and of rhodamine-labeled histidylated oligolysines were investigated by confocal microscopy. Histidylated oligolysines favor the cytosolic delivery of ODN from endosomes and increase their nuclear accumulation.

Freulon, I ; Monsigny, M ; Midoux, P ; Mayer, R  (2000)

Spacer length dependence on the efficiency of dimeric anionic peptides in gene transfer by glycosylated polylysine/plasmid complexes

Bioscience Reports 20 (5) 383-398
Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol, It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-ß Ala-ß Ala residues. but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the calls to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.

Amphiphilic anionic peptides have been used to enhance the efficiency of transfection by helping plasmids to escape from endosomes to the cytosol, It has been shown that efficiency of an eicosamers containing five glutamyl residues (E5), can be considerably enhanced either by transforming it into a dimer or by adding a tripeptide WYG in a C-terminal position (E5WYG). The dimerization of the peptide E5WYG leads to a more efficient tool when the dimerization device includes the tripeptide WYG unit and a longer spacer arm made of Gly-ß Ala-ß Ala residues. but to a 10-fold less efficient tool when the dimerization device includes a shorter spacer, a glycyl residue. Both dimers are taken up by the calls to a similar extent. Both dimers seem to be surrounded similarly as far as the environmental pH is concerned. In contrast, we found a correlation between the propensity of the peptides to adopt a helical structure at neutral pH and the gene transfer efficiency.

Allo, JC ; Midoux, P ; Merten, M ; Souil, E ; Lipecka, J ; Figarella, C ; Monsigny, M ; Briand, P ; Fajac, I  (2000)

Efficient gene transfer into human normal and cystic fibrosis tracheal gland serous cells with synthetic vectors

American Journal of Respiratory Cell and Molecular Biology 22 (2) 166-175
Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of re porter gene transfer into immortalized normal (MM-39) and CF (CF-KM I) human airway epithelial gland serous cells using various synthetic vectors : glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using a-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF trans membrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using a-glycosylated poly lysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane.

Submucosal gland serous cells are believed to play a major role in the physiopathology of cystic fibrosis (CF) and may represent an important target for CF gene therapy. We have studied the efficiency of re porter gene transfer into immortalized normal (MM-39) and CF (CF-KM I) human airway epithelial gland serous cells using various synthetic vectors : glycosylated polylysines (glycofectins), polyethylenimine (PEI) (25 and 800 kD), lipofectin, and lipofectAMINE. In both cell lines, a high luciferase activity was achieved with various glycofectins, with PEI 25 kD, and with lipofectAMINE. After three transfections applied daily using a-glycosylated polylysine, 20% of the cells were transfected. At 24 h after CF trans membrane conductance regulator (CFTR) gene transfer into CF-KM4 cells using a-glycosylated poly lysine, the immunolocalization of CFTR was analyzed by laser scanning confocal microscopy and the transgenic CFTR was detected by an intense labeling of the plasma membrane.

Malinge, JM   (2000)

Obituary : Remembering Marc Leng

Metal-Based Drugs 7 (3) 165-166

Malinge, JM ; Giraud-Panis, MJ ; Leng, M  (2000)

Interstrand cross-links of cisplatin induce striking distortions in DNA (vol 77, pg 23, 1999)

Journal of Inorganic Biochemistry 81 (1-2) 119-119

Malinge, JM ; Schwartz, A ; Leng, M  (2000)

Gel electrophoresis and bending in cisplatin-modified linear DNA

Dna-Protein Interactions 25-35 - Editor(s) : Travers, A ; Buckle, M ; Travers, A ; Buckle, M

Gomez, JP ; Ghisdal, P ; Morel, N   (2000)

Changes of the potassium currents in rat aortic smooth muscle cells during postnatal development

Pflügers Archives 441 388-397
This study was designed to investigate the role and regulation of arterial K+ channels during postnatal development. Rat thoracic aortic segments were suspended for isometric tension and resting membrane potential (RMP) recording. Contraction in response to 4-aminopyridine (4-AP) was similar in 4-, 8- and 12-week-old rats but was higher in 1-day-old rats. Contraction in response to tetraethylammonium (TEA) increased after 4 weeks. TEA increased the contractions evoked by noradrenaline in the aorta from 8- and 12-week-old rats but not from 1-day- and 4-week-old rats. RMP did not change during development. Patch-clamp studies of freshly isolated smooth muscle cells from the same aortas bathed in Ca2+-free medium showed a voltage-dependent Kf current (IK) sensitive to 4-AP.

This study was designed to investigate the role and regulation of arterial K+ channels during postnatal development. Rat thoracic aortic segments were suspended for isometric tension and resting membrane potential (RMP) recording. Contraction in response to 4-aminopyridine (4-AP) was similar in 4-, 8- and 12-week-old rats but was higher in 1-day-old rats. Contraction in response to tetraethylammonium (TEA) increased after 4 weeks. TEA increased the contractions evoked by noradrenaline in the aorta from 8- and 12-week-old rats but not from 1-day- and 4-week-old rats. RMP did not change during development. Patch-clamp studies of freshly isolated smooth muscle cells from the same aortas bathed in Ca2+-free medium showed a voltage-dependent Kf current (IK) sensitive to 4-AP.

Ghisdal, P ; Gomez, JP ; Morel, N   (2000)

Action of a NO donor on the excitation-contraction pathway activated by noradrenaline in rat superior mesenteric artery

Journal of Physiology (London) 522 83-96.
1. The aim of the present study was to investigate the actions of NO donors in rat superior mesenteric artery stimulated with noradrenaline by studying their effects on isometric tension, membrane potential (V-m), cytosolic calcium concentration ([Ca2+](cyt)) and accumulation of inositol phosphates.

1. The aim of the present study was to investigate the actions of NO donors in rat superior mesenteric artery stimulated with noradrenaline by studying their effects on isometric tension, membrane potential (V-m), cytosolic calcium concentration ([Ca2+](cyt)) and accumulation of inositol phosphates.