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Cryo-electron microscopy (cryo-EM) has recently provided invaluable experimental data about the full-length cystic fibrosis transmembrane conductance regulator (CFTR) 3D structure. However, this experimental information deals with inactive states of the channel, either in an apo, quiescent conformation, in which nucleotide-binding domains (NBDs) are widely separated or in an ATP-bound, yet closed conformation. Here, we show that 3D structure models of the open and closed forms of the channel, now further supported by metadynamics simulations and by comparison with the cryo-EM data, could be used to gain some insights into critical features of the conformational transition toward active CFTR forms. These critical elements lie within membrane-spanning domains but also within NBD1 and the N-terminal extension, in which conformational plasticity is predicted to occur to help the interaction with filamin, one of the CFTR cellular partners.

BACKGROUND To optimize synthetic gene delivery systems, there is a need to develop more efficient lipid formulations. Most cationic lipid formulations contain 'helper' neutral lipids because of their ability to increase DNA delivery, in particular by improving endosomal escape of DNA molecules via the pH-buffering effect of protonatable groups and/or fusion with the lipid bilayer of endosomes. METHODS We evaluated the influence of the linker structure between the two oleyl chains in the helper lipid on transfection efficiency in cell lines, as well as in primary cells (hepatocytes/cardiomyocytes). We reported the synthesis of two new pH-buffering imidazole helper lipids characterized by a polar headgroup containing one (compound 6) or two (compound 5) imidazole groups and two oleyl chains linked by an amide group. We studied their association with the aminoglycoside lipidic derivative dioleylsuccinylparomomycin (DOSP), which contains two oleyl chains linked to the aminoglycoside polar headgroup via an amide function. We compared the morphology and transfection properties of such binary liposomes of DOSP/5 and DOSP/6 with those of liposomes combining DOSP with another imidazole-based dioleyl helper lipid (MM27) in which a phosphoramido group acts as a linker between the two oleyl chains and imidazole function. RESULTS The phosphoramido linker in the helper lipid induces a major difference in terms of morphology and resistance to decomplexation at physical pH for DOSP/helper lipid complexes. CONCLUSIONS This hybrid dioleyl linker composition of DOSP/MM27 led to higher transfection efficiency in cell lines and in primary cells compared to complexes with homogeneous dioleyl linker.

International audience; 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

International audience; In this work that aims to synthesize and evaluate new cationic lipids as vectors for gene delivery, we report the synthesis of a series of cationic lipids in which a phosphate functional group acts as a linker to assemble on a molecular scale, two lipid chains and one cationic polar head. The mono or dicationic moiety is connected to the phosphate group by an aryl spacer. In this work, two synthesis strategies were evaluated. The first used the Atherton–Todd coupling reaction to introduce a phenolic derivative to dioleylphosphite. The second strategy used a sequential addition of lipid alcohol and a phenolic derivative on POCl3. The two methods are efficient, but the latter allows larger yields. Different polar head groups were introduced, thus producing amphiphilic compounds possessing either one permanent (N-methyl-imidazolium, pyridinium, trimethylammonium) or two permanent cationic charges. All these cationic lipids were formulated as liposomal solutions and characterized (size and zeta potential). They formed stable liposomal solutions both in water (at pH 7.0) and in a weakly acidic medium (at pH 5.5). Finally, this new generation of cationic lipids was used to deliver DNA into various human-derived epithelial cells cultured in vitro. Compared with Lipofectamine used as a reference commercial lipofection reagent, some cationic dialkylarylphosphates were able to demonstrate potent gene transfer abilities, and noteworthily, monocationic derivatives were much more efficient than dicationic analogues.

We report herein the synthesis of a novel family of cationic lipids, characterized by a trimethylammonium headgroup linked through a phosphate function to either two identical or two different lipid chains. The novelty of this study arises from the use of a trialkyl phosphate group to associate the hydrophobic domain to the cationic polar head. The structure of these new cationic lipids, which differs from that of previously reported lipophosphoramidates, is closer to the phospholipids encountered in the plasma membrane, and possesses a phosphocholine polar head. Evaluation of the transfection activity allowed us to compare the efficacy of cationic lipophosphates with that of lipophosphoramidates. These results demonstrate that cationic lipophosphates and lipophosphoramidates having otherwise identical chemical structures exhibit similar transfection efficacies. The second conclusion is that the structure of the lipid domain is a much more important parameter in governing transfection efficacy than the composition of the linker moiety. The best results were obtained with cationic lipophosphates or lipophosphoramidates possessing two different lipid chains, for example one oleyl chain with one phytanyl chain. These nonsymmetric cationic lipids exhibited transfection efficacies that were 10- to 200-fold better than those obtained with Lipofectamine used as a commercial standard.

International audience; Natural giant extracellular hemoglobins (Hbs) from polychaete annelids are currently actively investigated as promising oxygen carriers. Their powerful oxygenating ability and their safety have been demonstrated in preclinical studies, motivating their development for therapeutic and industrial applications. HEMARINA-M101 (M101) is derived from the marine invertebrate Arenicola marina. It is formulated as a manufactured product designated HEMOXYCarrier(®) (HEMARINA SA, France). The aim of the present study was to unveil the fate of M101 after a single intravenous (i.v.) injection in mice. For this purpose, M101 was tagged with a far-red fluorescent dye. Repeated non-invasive fluorescent imaging revealed a rapid diffusion of M101 in the whole body of animals, reaching all the examined organs such as brain, liver, lungs and ovaries. Functional M101 was circulating in bloodstream for several hours, without inducing any obvious side-effects. Last, a single i.v. injection of M101 in mice bearing human-derived subcutaneous tumors demonstrated the ability of this Hb to reduce hypoxia in poorly vascularized tissues, thus supporting the biological relevance of M101 oxygen release to vertebrate tissues. Altogether, these results further encourage the development of M101 as an oxygen carrying therapeutic.

International audience; Cationic lipids constitute a family of synthetic vectors commonly used for nucleic acids delivery. We herein report the results of a systematic study that aimed to compare the transfection efficacies of cationic lipophosphoramidates possessing either two identical lipid chains (termed symmetric cationic lipids) or two different lipid chains (non-symmetric cationic lipids). In addition, we also compared the transfection results of such a ‘molecular approach’ (the two different lipid chains being included in the same molecule) with those of a ‘supramolecular approach’ in which two types of symmetrical cationic lipids were mixed in one liposomal formulation. Thus, the present work allowed us first to optimize the methods used to synthesize non-symmetric cationic lipophosphoramidates. In addition, we could also identify two non-symmetric cationic lipids exhibiting high transfection efficiencies with a series of mammalian cell lines, both vectors being characterized by a single phytanyl chain and either an oleyl or a lauryl lipid chain.

Background CFTR is the only ABC transporter functioning as a chloride (Cl − ) channel. We studied molecular determinants, which might distinguish CFTR from standard ABC transporters, and focused on the interface formed by the intracellular loops from the membrane spanning domains. Methods Residues from ICL2 and ICL4 in close proximity were targeted, and their involvement in the functioning of CFTR was studied by whole cell patch clamp recording. Results We identified 2 pairs of amino acids, at the extremity of the bundle formed by the four intracellular loops, whose mutation i) decreases the Cl − current of CFTR (couple E267–K1060) or ii) increases it with a change of the electrophysiological signature (couple S263–V1056). Conclusions These results highlight the critical role of these ICL residues in the assembly of the different domains and/or in the Cl − permeation pathway of CFTR.

International audience; Gene therapy of diseases like cystic fibrosis (CF) would consist of delivering a gene medicine towards the lungs via the respiratory tract into the target epithelial cells. Accordingly, poly-functional nano-carriers are required in order to overcome the various successive barriers of such a complex environment, such as airway colonization with bacterial strains. In this work, the antibacterial effectiveness of a series of cationic lipids is investigated before evaluating its compatibility with gene transfer into human bronchial epithelial cells. Among the various compounds considered, some bearing a trimethyl-arsonium headgroup demonstrate very potent biocide effects towards clinically relevant bacterial strains. In contrast to cationic lipids exhibiting no or insufficient antibacterial potency, arsonium-containing lipophosphoramides can simultaneously inhibit bacteria while delivering DNA into eukaryotic cells, as efficiently and safely as in absence of bacteria. Moreover, such vectors can demonstrate antibacterial activity in vitro while retaining high gene transfection efficiency to the nasal epithelium as well as to the lungs in mice in vivo. Arsonium-containing amphiphiles are the first synthetic compounds shown to achieve efficient gene delivery in the presence of bacteria, a property particularly suitable for gene therapy strategies under infected conditions such as within the airways of CF patients.

Background Neutral amphiphilic triblock copolymers have been shown to be efficient for gene transfection in vivo, especially by direct injection into the muscle. To contribute to a better understanding of the underlying mechanisms, in the present study, we investigated the properties of a poly(ethylene oxide-b-4-vinylpyridine) diblock copolymer as vector for nucleic acid transfer, with the particular aim of shedding some light on a possible mechanism explaining the internalization of DNA by the transfected cells. Methods Complexation of plasmid DNA with the PEO-b-P4VP diblock copolymer was investigated by ethidium bromide exclusion and gel electrophoresis assays. Interaction of the copolymer with a lipid model membrane was evaluated by electrophysiological assays and quantification of plasmid DNA was performed by quantitative polymerase chain reaction. In vivo luciferase transfection assays were finally performed. Results The diblock copolymer was found to poorly interact with DNA up to a mass ratio (copolymer/DNA) as high as 150. At a concentration of 36 µg/ml, it induced the formation of mainly transient (but sometimes permanent) pores and the formation of those pores allowed the translocation of plasmid DNA across the model membrane. However, only low transgene expression was obtained; the luciferase levels observed with the diblock being of the same order of magnitude as those observed with the corresponding PEO and P4VP homopolymers. Conclusions These results strongly suggest that gene transfection by neutral block copolymers may involve the formation of cellular pores; in addition, they also highlight that in vivo gene transfection requires the use of adequately soluble block copolymers. Copyright © 2011 John Wiley & Sons, Ltd.

Background Genetic modification of human monocyte-derived dendritic cells (DC) with cDNA sequences encoding tumor-associated antigens (TAA) is a promising strategy for cancer immunotherapy. The present study aimed to develop a nonviral gene transfer method based on the use of the cationic lipophosphoramide reagent, KLN-5, as an alternative to the commonly used viral vectors. Methods First, the efficiency of KLN5 for gene transfection into DC was investigated using the green fluorescent protein (GFP) reporter gene. The highest transfection efficiency/cell viability ratio was determined by flow cytometry. Next, DC were transfected with a plasmid encoding NY-ESO-1, a TAA expressed in numerous cancers, according to the transfection protocol previously established with the GFP reporter. Transfected DC were then co-cultured with a CD8+ NY-ESO-1 specific HLA-A*02.01 T cell clone to control their ability to correctly process and present the corresponding epitope in the HLA-A*02.01 context. Finally, T cell activation was assessed via flow cytometry-based detection of interferon-γ production. Results An optimal KLN5/plasmid DNA ratio allowing both significant transgene expression and high viability of DC could be determined. Under the established experimental conditions, antigen processing and presentation of the immunodominant (SLLMWITQC157–165) epitope in the HLA-A*0201 context was demonstrated by activation of the NY-ESO-1-specific CD8+ T cell clone. Conclusions KLN5-based gene transfection into DC allows the efficient induction of TAA presentation and may thus represent a novel attractive nonviral approach for cancer vaccination. Copyright © 2008 John Wiley & Sons, Ltd.

Cationic lipids nowadays constitute a promising alternative to recombinant viruses for gene transfer. We have recently explored the transfection potential of a new class of lipids based upon the use of aminoglycosides as cationic polar headgroups. The encouraging results obtained with a first cholesterol derivative of kanamycin A prompted us to investigate this family of vectors further, by modulating the constituent structural units of the cationic lipid. For this study, we have investigated the transfection properties of a series of new derivatives based on a kanamycin A scaffold. The results primarily confirm that aminoglycoside-based lipids are efficient vectors for gene transfection both in vitro and in vivo (mouse airways). Furthermore, a combination of transfection and physicochemical data revealed that some modifications of the constitutive subunits of kanamycin A-based vectors were associated with substantial changes in their transfection properties.

Silver based Metal Organic Framework (MOF) materials were synthesized by the reaction of phospho-benzoic acid with silver salts under hydrothermal treatment and with/without urea. According to this procedure, either both functional groups (phosphonic acid and carboxylic acid) were engaged in iono-covalent bonds with silver atoms or, on the contrary, only the phosphonic acid function is engaged. Consequently, these materials exhibited different stability and their ability to release silver salts, when they were placed in water, was different. The antibacterial properties of these materials were attributed to the silver salt, released in solution.

Using bioinformatics methods, we have previously identified Glu235 and Glu340 as the putative acid/base catalyst and nucleophile, respectively, in the active site of human glucocerebrosidase. Thus, we undertook site-directed mutagenesis studies to obtain experimental evidence supporting these predictions. Recombinant retroviruses were used to express wild-type and E235A and E340A mutant proteins in glucocerebrosidase-deficient murine cells. In contrast to wild-type enzyme, the mutants were found to be catalytically inactive. We also report the results of various studies (Western blotting, glycosylation analysis, subcellular fractionation, and confocal microscopy) indicating that the wild-type and mutant enzymes are identically processed and sorted to the lysosomes. Thus, enzymatic inactivity of the mutant proteins is not the result of incorrect folding/processing. These findings indicate that Glu235 plays a key role in the catalytic machinery of human glucocerebrosidase and may indeed be the acid/base catalyst. As concerns Glu340, the results both support our computer-based predictions and confirm, at the biological level, previous identification of Glu340 as the nucleophile by use of active site labeling techniques. Finally, our findings may help to better understand the molecular basis of Gaucher disease, the human lysosomal disease resulting from deficiency in glucocerebrosidase.

The clan GH-A is a group of more than 200 proteins representing nine established families of glycosyl hydrolases that act on a large variety of substrates. This clan includes five enzymes implicated in lysosomal storage diseases: beta-glucuronidase (Sly disease), beta-glucocerebrosidase (Gaucher disease), beta-galactosidase (Landing disease and Morquito type B disease), beta-mannosidase (mannosidosis) and alpha-L-iduronidase (Hurler-Scheie disease). Examination of known 3D structures from some families of the clan allowed us to deduce structural and functional features shared by these proteins. We then used the hydrophobic cluster analysis method to study the protein sequences of the entire clan. Our results reveal that, despite low levels of sequence identity, all the proteins of the clan (including the aforementioned lysosomal enzymes) likely share a similar catalytic domain consisting of an (alpha/beta)8 barrel with conserved functional amino acids located at the C-terminal ends of six of the eight strands constituting the beta-barrel. Interestingly, several mutations reported to be responsible for lysosomal storage diseases are located within these conserved regions of the lysosomal enzyme catalytic domains.

Here we introduce the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines, produced by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), which are published in this issue of the journal with our endorsement, and will be incorporated into our Instructions to Authors.

Prostaglandin E1 (PGE1) was used to prevent veno-occlusive disease (VOD) of the liver after allogeneic bone marrow transplantation (BMT) for leukaemia. It was given in continuous i.v. infusion from day--8 to day 30 after BMT at a dose of 0.3 micrograms/kg/h. The patients were studied according to the risk factors for VOD: diagnosis, intensification of the conditioning and previous liver abnormalities. The diagnosis of VOD was made on at least two of the following factors: weight gain, hepatomegaly, jaundice, ascitis, pain of the right upper quadrant, increased platelet consumption. 109 patients were studied, 50 were treated by PGE1 and 59 did not receive it. The actuarial incidence of VOD was 12.2% in the PGE1 group and 25.5% in the non PGE1 group (P = 0.05). In acute leukaemia, the incidence was 39.1% in the non-treated group and 12.8% in the PGE1 treated group (P = 0.02). Patients with previous hepatitis had an incidence of 62.5% in the non treated group and 15.5% in the treated group (P = 0.05). A positive cytomegalovirus (CMV) serology seemed to increase the risk of VOD: the incidence of VOD was 31.4% in non-treated patients and 22% in PGE1 treated patients. The multivariate analysis of the risk factors for VOD shows that unfavourable factors were: recipient positive CMV serology (P = 0.06), hepatic disease prior to transplant (P = 0.02) and the absence of PGE1 treatment (P = 0.02). This study suggests that prophylactic PGE1 treatment may decrease the incidence of VOD in patients treated for leukaemia by allogeneic bone marrow transplantation.

Artificial self-assembling systems are currently widely investigated as an alternative approach to recombinant viruses for gene transfection in vitro and in vivo. Cationic lipids are particularly attractive, as a great variety of well-characterized reagents can be synthesized from there. Over the last few years, numerous cationic lipid systems have been developed and shown to be efficient for in vitro transfection. However, although some promising results have been reported in the in vivo setting (even in clinical gene therapy trials in man), the in vivo use of cationic lipid-based systems is still problematic, especially when considering the systemic route of administration. Herein, we summarize our own research on a particular class of cationic lipids, cholesterol derivatives characterized by polar headgroups with guanidinium functions, in order to illustrate the basic principles of and the positive results already obtained by cationic lipid-mediated gene delivery as well as the remaining problems that need to be urgently resolved, particularly as regards the systemic administration. In this forward-looking review, we also discuss the present efforts to develop modular systems for improved in vivo transfection. Indeed, lipid-based vectors offer the possibility to create sophisticated modular gene delivery systems capable of self-assembly via hydrophobic interaction between their components, the role of the different functional elements being to help in overcoming the distinct extracellular and cellular barriers to in vivo gene transfection into the various somatic target tissues.

B. Hoffmann1, J.-P. Mornon1, B. Boucherle2, A. Fortune2, R. Haudecoeur2, C. Boinot3, M. Jollivet3, J.-L. Decout2, F. Becq3, P. Lehn4, I. Callebaut1. 1Universite Pierre et Marie Curie, CNRS, MNHN, IRD, IMPMC, UMR 7590, Paris, France; 2Universite Joseph Fourier, UJF/CNRS, DPM, UMR 5063, Grenoble, France; 3Universite de Poitiers, CNRS, Laboratoire STIM, ERL 7368, Poitiers, France; 4Universite de Bretagne Occidentale, INSERM U 1078, Brest, France

It's widely believed that gene therapy will become an efficient way for the treatment of diseases such as acquired (cancers) or genetic diseases (cystic fibrosis) and for vaccination. The essential...

We used minisatellite probes to analyse by DNA fingerprints the long-term engraftment (median 4.3 years, range 1-2) of 21 bone marrow transplantation recipients for severe aplastic anaemia. Patients received their graft from histocompatible siblings. They were conditioned with cyclophosphamide (150 mg/kg) and a 6GY thoracoabdominal irradiation and did not have ex-vivo T cell depletion of marrow donor. DNA was extracted peripheral mononuclear cells and analysed by Southern blotting with 32P-labelled single-stranded RNA probes. Seven out of 21 donor-recipient pairs were sex-mismatched and additionally studied with a probe detecting a male specific repeated sequence on the Y chromosome. Red cell surface phenotype was also used as marker of engraftment in most cases. Long-term engraftment appeared complete for all patients studied with respect to the three methods.