Combining theoretical and experimental data to decipher CFTR 3D structures and functions.

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.

Polynucleotide transport through lipid membrane in the presence of starburst cyclodextrin-based poly(ethylene glycol)s.

Symmetrical cyclodextrin-based 14-arm star polymers with poly(ethylene glycol) PEG branches were synthesized and characterized. Interactions of the star polymers with lipid bilayers were studied by the "black lipid membrane" technique in order to demonstrate the formation of monomolecular artificial channels. The conditions for the insertion are mainly based on dimensions and amphiphilic properties of the star polymers, in particular the molar mass of the water-soluble polymer branches. Translocation of single-strand DNA (ssDNA) through those synthetic nanopores was investigated, and the close dimension between the cross-section of ssDNA and the cyclodextrin cavity led to an energy barrier that slowed down the translocation process.

Molecular modelling and molecular dynamics of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ATP-binding cassette (ABC) transporter superfamily that functions as an ATP-gated channel. Considerable progress has been made over the last years in the understanding of the molecular basis of the CFTR functions, as well as dysfunctions causing the common genetic disease cystic fibrosis (CF). This review provides a global overview of the theoretical studies that have been performed so far, especially molecular modelling and molecular dynamics (MD) simulations. A special emphasis is placed on the CFTR-specific evolution of an ABC transporter framework towards a channel function, as well as on the understanding of the effects of disease-causing mutations and their specific modulation. This in silico work should help structure-based drug discovery and design, with a view to develop CFTR-specific pharmacotherapeutic approaches for the treatment of CF in the context of precision medicine.

Important role of phosphoramido linkage in imidazole-based dioleyl helper lipids for liposome stability and primary cell transfection.

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.

Full-open and closed CFTR channels, with lateral tunnels from the cytoplasm and an alternative position of the F508 region, as revealed by molecular dynamics.

In absence of experimental 3D structures, several homology models, based on ABC exporter 3D structures, have provided significant insights into the molecular mechanisms underlying the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel whose defects are associated with cystic fibrosis (CF). Until now, these models, however, did not furnished much insights into the continuous way that ions could follow from the cytosol to the extracellular milieu in the open form of the channel. Here, we have built a refined model of CFTR, based on the outward-facing Sav1866 experimental 3D structure and integrating the evolutionary and structural information available today. Molecular dynamics simulations revealed significant conformational changes, resulting in a full-open channel, accessible from the cytosol through lateral tunnels displayed in the long intracellular loops (ICLs). At the same time, the region of nucleotide-binding domain 1 in contact with one of the ICLs and carrying amino acid F508, the deletion of which is the most common CF-causing mutation, was found to adopt an alternative but stable position. Then, in a second step, this first stable full-open conformation evolved toward another stable state, in which only a limited displacement of the upper part of the transmembrane helices leads to a closure of the channel, in a conformation very close to that adopted by the Atm1 ABC exporter, in an inward-facing conformation. These models, supported by experimental data, provide significant new insights into the CFTR structure-function relationships and into the possible impact of CF-causing mutations.

Cationic lipophosphoramidates containing a hydroxylated polar headgroup for improving gene delivery.

The structure of the cationic moiety of amphiphiles is a key factor which directly influences their transfection efficacy. Accordingly, in the present work, we have synthesized three new lipophosphoramide-based amphiphilic compounds incorporating a methoxy 5, hydroxyl 6, or dihydroxyl 7 functional group in their cationic part. Gene delivery efficacies of these novel vectors were compared to our benchmark compound, the arsenolipophosphoramidate KLN47, and to its trimethylammonium (TMA) analogue 4. We next studied the characteristics (size, ζ potential) of the nanometric assemblies formed (liposomes and lipid/DNA complexes), and the DNA binding ability of the cationic liposomes was characterized at the physicochemical level. In vitro, all of the cationic lipids evaluated were efficient not only to condense plasmids but also to transfect two types of human airway epithelial cells. Interestingly, in vivo administration to mice (via simple tail vein injection) showed that compound 6 was the most efficient in transfecting the lungs when compared to that of the other cationic lipids studied, including compound KLN47. All of these results suggest that a hydroxyethyldimethylammonium (HE-DMA) polar head could be a valuable alternative to a trimethylarsonium (TMAs) polar head and that they also invite further evaluation of the in vivo potential of compound 6 using more clinically relevant delivery procedures.

Poly(2-methyl-2-oxazoline)-b-poly(tetrahydrofuran)-b-poly(2-methyl-2-oxazoline) amphiphilic triblock copolymers: synthesis, physicochemical characterizations, and hydrosolubilizing properties.

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.

Cationic dialkylarylphosphates: a new family of bio-inspired cationic lipids for gene delivery.

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.

Effects of a novel archaeal tetraether-based colipid on the in vivo gene transfer activity of two cationic amphiphiles.

Gene therapy for treating inherited diseases like cystic fibrosis might be achieved using multimodular nonviral lipid-based systems. To date, most optimizations have concerned cationic lipids rather than colipids. In this study, an original archaeal tetraether derivative was used as a colipid in combination with one or the other of two monocationic amphiphiles. The liposomes obtained, termed archaeosomes, were characterized regarding lipid self-assembling properties, macroscopic/microscopic structures, DNA condensation/neutralization/relaxation abilities, and colloidal stability in the presence of serum. In addition, gene transfer experiments were conducted in mice with lipid/DNA complexes being administered via systemic or local delivery routes. Altogether, the results showed that the tetraether colipid can provide complexes with different in vivo transfection abilities depending on the lipid combination, the lipid/colipid molar ratio, and the administration route. This original colipid appears thus as an innovative modular platform endowed with properties possibly beneficial for fine-tuning of in vivo lipofection and other biomedical applications.

Cationic lipophosphoramidates with two different lipid chains: synthesis and evaluation as gene carriers.

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.

Structure-function analysis of the human ferroportin iron exporter (SLC40A1): effect of hemochromatosis type 4 disease mutations and identification of critical residues.

Ferroportin (SLC40A1) is the only known iron exporter in mammals and is considered a key coordinator of the iron balance between intracellular and systemic iron homeostasis. However, the structural organization of ferroportin in the lipid bilayer remains controversial and very little is known about the mechanism underlying iron egress. In the present study, we have developed an approach based on comparative modeling, which has led to the construction of a model of the three-dimensional (3D) structure of ferroportin by homology to the crystal structure of a Major Facilitator Superfamily member (EmrD). This model predicts atomic details for the organization of ferroportin transmembrane helices and is in agreement with our current understanding of the ferroportin function and its interaction with hepcidin. Using in vitro experiments, we demonstrate that this model can be used to identify novel critical amino acids. In particular, we show that the tryptophan residue 42 (p.Trp42), which is localized within the extracellular end of the ferroportin pore, is likely involved in both the iron export function and in the mechanism of inhibition by hepcidin. Thus, our 3D model provides a new perspective for understanding the molecular basis of ferroportin functions and dysfunctions.

Cationic lipophosphoramidates with two disulfide motifs: synthesis, behaviour in reductive media and gene transfection activity.

Lipophosphoramidates have previously been identified as efficient vectors for gene delivery. The incorporation of functional groups that respond to a physiological stimulus is hypothesised to further improve the efficacy of this type of vector and eventually reduce its cytotoxicity. In the present work, we report the effects of the incorporation of two disulfide motifs into the hydrophobic domain, close to the phosphoramidate group. Three cationic vectors possessing such a red/ox sensitive function were synthesised. The capability of one of them (5b) to compact DNA is reported jointly with its ability to release that DNA in the presence of a reducing agent. Finally, compound 5b was tested as a vector for gene delivery into human cells in vitro and its cytotoxicity was also evaluated.

Folate-equipped nanolipoplexes mediated efficient gene transfer into human epithelial cells.

Since recombinant viral vectors have been associated with serious side effects, such as immunogenicity and oncogenicity, synthetic delivery systems represent a realistic alternative for achieving efficacy in gene therapy. A major challenge for non-viral nanocarriers is the optimization of transgene expression in the targeted cells. This goal can be achieved by fine-tuning the chemical carriers and the adding specific motifs to promote cellular penetration. Our study focuses on the development of novel folate-based complexes that contain varying quantities of folate motifs. After controlling for their physical properties, neutral folate-modified lipid formulations were compared in vitro to lipoplexes leading to comparable expression levels. In addition, no cytotoxicity was detected, unlike what was observed in the cationic controls. Mechanistically, the delivery of the transgene appeared to be, in part, due to endocytosis mediated by folate receptor targeting. This mechanism was further validated by the observation that adding free folate into the medium decreased luciferase expression by 50%. In vivo transfection with the folate-modified MM18 lipid, containing the highest amount of FA-PEG(570)-diether co-lipid (w:w; 90:10), at a neutral charge ratio, gave luciferase transgene expression. These studies indicate that modification of lipids with folate residues could enhance non-toxic, cell-specific gene delivery.

Lipothiophosphoramidates for gene delivery: critical role of the cationic polar headgroup.

When considering a family of cationic lipids designed for gene delivery, the nature of the cationic polar head probably has a great influence on both the transfection efficacy and toxicity. Starting from a cationic lipothiophosphoramidate bearing a trimethylammonium headgroup, we report herein the impact on gene transfection activity of the replacement of the trimethylammonium moiety by a trimethylphosphonium or a trimethylarsonium group. A series of three different human epithelial cell lines were used for the experimental transfection studies (HeLa, A549 and 16HBE14o(-)). The results basically showed that such structural modifications of the cationic headgroup can lead to a high transfection efficacy at low lipid/DNA charge ratios together with a low cytotoxicity. It thus appears that the use of a trimethylarsonium cationic headgroup for the design of efficient gene carriers, which was initially proposed in the lipophosphoramidate series, can be extended to other series of cationic lipids and might therefore have great potential for the development of novel non-viral vectors in general.

Evidence of DNA transfer across a model membrane by a neutral amphiphilic block copolymer.

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.

Cationic lipo-thiophosphoramidates for gene delivery: synthesis, physico-chemical characterization and gene transfection activity--comparison with lipo-phosphoramidates.

The synthesis of cationic lipo-thiophosphoramidates, a new family of cationic lipids designed for gene delivery, is reported herein. This new class of lipids is less polar than its oxygenated equivalent the lipo-phosphoramidates. Fluorescence anisotropy and FRET were used to determine the fluidity and fusogenicity of the lipo-phosphoramidates 3a-b and lipo-thiophosphoramidates 7a-b. The determination of both the size and the zeta potential of the nano-objects (liposomes and lipoplexes) and the determination of the DNA binding ability of the liposomes have completed the physico-chemical characterizations of the cationic lipids studied. Finally, the cationic lipids 3a-b and 7a-c have been evaluated as synthetic vectors for gene transfection into a variety of mammalian cell lines. The lipo-thiophosphoramidate 7a proved to be an efficient and low toxicity synthetic vector even when used at low lipid to DNA charge ratios.

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

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

The ARRIVE guidelines, a welcome improvement to standards for reporting animal research.

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.

Combining keratinocyte growth factor transfection into the airways and tracheal occlusion in a fetal sheep model of congenital diaphragmatic hernia.

BACKGROUND: In utero tracheal occlusion (TO) has been developed to improve the lung hypoplasia associated with congenital diaphragmatic hernia (CDH). However, although TO stimulates fetal lung growth, it results in a decrease of alveolar type II cells (ATII) and surfactant production. Because keratinocyte growth factor (KGF) is a potent stimulus of ATII proliferation and maturation, we evaluated, in a fetal lamb model of CDH, a gene therapy strategy combining TO and ovine KGF transfection into the fetal airways using bisguanidinium-tren-cholesterol/dioleoyl-phosphatidylethanolamine (BGTC/DOPE) cationic liposomes. METHODS: Three groups of sheep fetuses with CDH and a group of normal fetuses were studied. The fetuses of the three groups with CDH (KGF, Medium and Hernia groups) underwent surgery at 85 days of gestation to create a diaphragmatic hernia. The KGF and medium group fetuses underwent a second surgery step at day 125 to perform TO associated with injection of the KGF transfection mixture (KGF group) or control medium (Medium group), whereas the fetuses of the Hernia group were left untreated. Normal fetuses were used as a control (Normal group). All fetuses were euthanized at 132 days of gestation and various analytical studies [lung weight, radial alveolar count (RAC), KGF and surfactant protein B (SPB) expression, number of ATII cells] were performed to assess the efficiency of KGF transfection and its effects on fetal lung development. RESULTS: TO was associated with lung hyperplasia and increased RAC in the Medium and KGF groups versus the Hernia group. Expression of KGF was increased in the KGF group compared to all other groups and was associated with an increased synthesis of SPB by alveolar cells and an ectopic synthesis of SPB by bronchiolar cells compared to TO treatment alone. CONCLUSIONS: Thus, BGTC/DOPE liposomes can mediate efficient KGF transfection into the airways in a fetal sheep model of CDH. Furthermore, combining KGF transfection and TO resulted not only (as did TO alone) in the correction of the CDH-associated lung hypoplasia and decreased RAC, but also in increased SPB synthesis, suggesting a better maturation of the re-growing lung (compared to TO alone). Additional studies are required to further explore the therapeutic potential of such a combined strategy; in particular, studies evaluating the lung function of in utero-treated CDH lamb newborns.

Molecular models of the open and closed states of the whole human CFTR protein.

Cystic fibrosis transmembrane conductance regulator (CFTR), involved in cystic fibrosis (CF), is a chloride channel belonging to the ATP-binding cassette (ABC) superfamily. Using the experimental structure of Sav1866 as template, we previously modeled the human CFTR structure, including membrane-spanning domains (MSD) and nucleotide-binding domains (NBD), in an outward-facing conformation (open channel state). Here, we constructed a model of the CFTR inward-facing conformation (closed channel) on the basis of the recent corrected structures of MsbA and compared the structural features of those two states of the channel. Interestingly, the MSD:NBD coupling interfaces including F508 (DeltaF508 being the most common CF mutation) are mainly left unchanged. This prediction, completed by the modeling of the regulatory R domain, is supported by experimental data and provides a molecular basis for a better understanding of the functioning of CFTR, especially of the structural features that make CFTR the unique channel among the ABC transporters.