D. Scherman - Academia.edu (original) (raw)
Papers by D. Scherman
13th International Ceramics Congress - Part D, 2014
ZnGa2O4 (ZGO) is a normal spinel. When doped with Cr3+ ions, ZGO:Cr becomes a high brightness per... more ZnGa2O4 (ZGO) is a normal spinel. When doped with Cr3+ ions, ZGO:Cr becomes a high brightness persistent luminescence material with an emission spectrum perfectly matching the transparency window of living tissues. It allows in vivo mouse imaging with a better signal to background ratio than classical quantum dots. The most interesting characteristic of ZGO:Cr lies in the fact that its LLP can be excited with red light, well below its band gap energy and in the transparency window of living tissues. A mechanism based on the trapping of carriers localized around a special type of Cr3+ ions namely CrN2 can explain this singularity. The antisite defects of the structure are the main responsible traps in the persistent luminescence mechanism. When located around Cr3+ ions, they allow, via Cr3+ absorption, the storage of not only UV light but also all visible light from the excitation source.
Molecular Therapy, 2000
Numerous diseases are linked to the absence or insufficient concentration of a specific plasma pr... more Numerous diseases are linked to the absence or insufficient concentration of a specific plasma protein. Gene transfer is an appealing strategy for correction of such diseases. We report high and sustained plasma secretion of human secreted alkaline phosphatase and of human Factor IX by skeletal muscle of mice. This was obtained by delivering square-wave unipolar electric pulses of low field strength (200 V/cm) and long duration (20 ms) to skeletal muscle previously injected with plasmid DNA encoding for the secreted protein. This intramuscular electrotransfer method allows 30- to 150-fold increase in reporter protein secretion, compared to simple plasmid DNA injection. This increase allows one to obtain values of up to 2200 ng/ml of a reporter circulating protein. Moreover, this high level of secretion remains stable for several months.
![Research paper thumbnail of Characterization of the monoamine carrier of chromaffin granule membrane by binding of [2-3H]dihydrotetrabenazine](https://attachments.academia-assets.com/109578520/thumbnails/1.jpg)
](Proceedings of the National Academy of Sciences, 1983
Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-llb-H-benzo[a]qu... more Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-llb-H-benzo[a]quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane ofpurified bovine chromaffin granules. Specific binding was characterized by Kd and B.., values of 3.1 nM and 62 pmol/mg ofmembrane protein, respectively. It was reversible, with association and dissociation rate constants of 0.22 x 106 M'1 s-1 and 1.8 x 10-3 s-1, respectively. Binding sites were present
Proceedings of the National Academy of Sciences, 1999
We have recently discovered that cationic cholesterol derivatives characterized by guanidinium po... more We have recently discovered that cationic cholesterol derivatives characterized by guanidinium polar headgroups are very efficient for gene transfection in vitro and in vivo . In spite of being based on some rationale at the molecular level, the development of these new synthetic vectors was nevertheless empirical. Indeed, the factors and processes underlying cationic lipid-mediated gene transfer are still poorly understood. Thus, to get a better insight into the mechanisms involved, we have examined the supramolecular structure of lipid/DNA aggregates obtained when using reagent bis(guanidinium)-tren-cholesterol (BGTC), either alone or as a liposomal formulation with the neutral phospholipid dioleoyl phosphatidylethanolamine (DOPE). We here report the results of cryotransmission electron microscopy studies and small-angle x-ray scattering experiments, indicating the presence of multilamellar domains with a regular spacing of 70 Å and 68 Å in BGTC/DOPE–DNA and BGTC–DNA aggregates, r...
Nucleic Acids Research, 1999
Clinical applications of gene therapy mainly depend on the development of efficient gene transfer... more Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.
Journal of Medicinal Chemistry, 1998
We have designed and synthesized original cationic lipids for gene delivery. A synthetic method o... more We have designed and synthesized original cationic lipids for gene delivery. A synthetic method on solid support allowed easy access to unsymmetrically monofunctionalized polyamine building blocks of variable geometries. These polyamine building blocks were introduced into cationic lipids. To optimize the transfection efficiency in the novel series, we have carried out structure-activity relationship studies by introduction of variable-length lipids, of variable-length linkers between lipid and cationic moiety, and of substituted linkers. We introduce the concept of using the linkers within cationic lipids molecules as carriers of side groups harboring various functionalities (side chain entity), as assessed by the introduction of a library composed of cationic entities, additional lipid chains, targeting groups, and finally the molecular probes rhodamine and biotin for cellular traffic studies. The transfection activity of the products was assayed in vitro on Hela carcinoma, on NIH3T3, and on CV1 fibroblasts and in vivo on the Lewis Lung carcinoma model. Products from the series displayed high transfection activities. Results indicated that the introduction of a targeting side chain moiety into the cationic lipid is permitted. A primary physicochemical characterization of the DNA/lipid complexes was demonstrated with this leading compound. Selected products from the series are currently being developed for preclinical studies, and the labeled lipopolyamines can be used to study the intracellular traffic of DNA/cationic lipid complexes.
Gene Therapy, 1997
Plasmid DNA used for nonviral therapeutic gene transfer After extensive washing of the column, pu... more Plasmid DNA used for nonviral therapeutic gene transfer After extensive washing of the column, purified plasmid or nucleic acid vaccination has to be highly purified, devoid DNA is eluted using an alkaline buffer. The binding conof contaminating components such as bacterial proteins, ditions of the plasmid DNA on to the column have been endotoxins, or bacterial chromosomal DNA. We have optimized, as well as the hybridization sequence and the developed a new affinity chromatography technique for linker group between the matrix and the third strand oligoplasmid DNA purification: triple-helix affinity chromato-nucleotide. The THAC technique makes it possible to purify graphy (THAC). This technique is based on the sequence-in one step supercoiled plasmid DNA, and to significantly specific interaction of an oligonucleotide forming a triple-reduce the level of contaminating RNA, endotoxins and helix with plasmid DNA. The oligonucleotide was covalently chromosomal DNA. In particular, a 100-fold reduction of linked to a chromatographic matrix, thus providing a re-chromosomal DNA contamination over that obtained with usable affinity support. By inserting a suitable homopurine conventional techniques can be achieved through a single sequence in the plasmid DNA, it is possible to obtain a additional THAC step. Further improvements of THAC triple-helix interaction that will only be stable at mild acidic technology are possible, and we anticipate that this tech-pH and that will dissociate in alkaline conditions. A crude nique can be scaled up for integration into a full commerlysate from a recombinant E. coli, or a pre-purified plasmid cial-scale DNA production process. DNA, is thus applied at acidic pH on to a THAC column.
Gene Therapy, 1997
Plasmids currently used for nonviral gene transfer have the excised in vivo after thermoinduction... more Plasmids currently used for nonviral gene transfer have the excised in vivo after thermoinduction of the integrase gene disadvantage of carrying a bacterial origin of replication leading to the formation of two supercoiled molecules: the and an antibiotic resistance gene. There is, therefore, a minicircle and the starting plasmid lacking the expression risk of uncontrolled dissemination of the therapeutic gene cassette. In various cell lines, purified minicircles exhibited and the antibiotic resistance gene. Minicircles are new a two-to 10-fold higher luciferase reporter gene activity DNA delivery vehicles which do not have such elements than the unrecombined plasmid. This could be due to either and are consequently safer as they exhibit a high level of the removal of unnecessary plasmid sequences, which biological containment. They are obtained in E. coli by att could affect gene expression, or the smaller size of minisite-specific recombination mediated by the phage inte-circle which may confer better extracellular and intracellular grase. The desired eukaryotic expression cassette bioavailability and result in improved gene delivery bounded by the attP and attB sites was cloned on a properties. recombinant plasmid. The expression cassette was
FEBS Letters, 1999
The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear ent... more The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear entry of proteins. We have developed a strategy for covalent coupling of one or a controlled number of NLS peptides to plasmid DNA at a specific site by triple helix formation. A psoralen-oligonucleotide-NLS peptide conjugate was synthesized and characterized by proteolysis with trypsin. This conjugate was used to covalently associate one NLS peptide to plasmid DNA by triple helix formation and photoactivation. The oligonucleotide-NLS peptide conjugate interacted with the NLS-receptor importin K K. The reporter gene was expressed after transfection of the modified plasmid in NIH 3T3 cells, indicating no loss of the gene expression functionality of the plasmid. On the other hand, no increase in expression was observed as a result of the NLS peptide. This site-specific coupling technology can be used to couple to a plasmid other ligands targeting to a specific receptor.
Expert Opinion on Therapeutic Patents, 1998
ABSTRACT The ability to produce and express foreign genes in humans might cure or even prevent ma... more ABSTRACT The ability to produce and express foreign genes in humans might cure or even prevent many important human diseases that are either treated poorly or are untreatable by present therapies. However, insufficient transgene expression in vivo has so far impaired the development of effective gene therapy. Thus, it is of interest to develop compositions and delivery methods for gene therapy that lead to high level transgene expression in a variety of cell and tissue types. Synthetic DNA delivery agents are of crucial interest for gene therapy as an alternative to viral vectors, since they potentially display fewer risks in terms of immunogenicity and propagation and are easier to produce under GMP conditions. In the last decade, a significant number of industrial and academic groups have emerged with a number of important patents claiming a variety of synthetic DNA delivery agents. Some of these agents were launched into clinical trials at very early stages of development leading to rather modest results. The lack of physicochemical characterisation of the self-assembling complexes in those early clinical trials prevented interpretation, correlation and comparison of transgene expression and biodistribution with the supramolecular state of these self-assemblies. On the other hand, in the last two years, a significant body of information has emerged from academic and academic-industrial groups regarding the exhaustive physicochemical characterisation of some complexes and the impact of these characteristics on transgene biodistribution and expression both in vitro and in vivo. Additionally, the intracellular fate of different self-assemblies has been studied by different groups. Together, these studies provide a rational basis for designing novel DNA delivery agents. Synthetic agents for gene delivery are classified into different chemical families. In this manuscript, we have focused on the cationic lipids family. We choose to show the approaches that introduce original elements into the backbone of the synthetic delivery agent. Data on the physicochemical characterisation of different synthetic agents for gene delivery are given when available. Clinical data (reported in other reviews) are beyond the scope of this article. Finally, a discussion on how to improve the results obtained so far in order to advance towards new human trials is presented.
Developmental Dynamics, 2006
We report here a method that allows fast, efficient, and low-cost screening for gene function in ... more We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.
Current Opinion in Biotechnology, 1998
Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major cha... more Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major challenges for the improvement of in vivo cationic lipid-mediated gene delivery reside in the design of more biocompatible lipoplexes mimicking viral-mediated gene delivery and in understanding the fate of the lipoplexes within the cells.
Cell Biology and Toxicology, 2004
In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle diso... more In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic delivery of therapeutic proteins. Electrotransfer is a powerful method for DNA transfer into skeletal muscle. In view of the broad potential gene therapy clinical application of electrotransfer o¡ers, it is important to perform toxicology studies on electrotransfered muscle tissue. We have investigated if the delivery of square wave electric pulses of low ¢eld strength and long duration to mouse tibial cranial muscle induced the expression of stress related genes. We have pro¢led gene expression patterns in muscles at di¡erent times after delivery of electric pulses using Stress/Toxicology microarrays. No signi¢cant variation in the expression of stress related-genes was detected between treated and non-treated muscles. This suggests that application of adequate, ¢netuned, electric pulses to the skeletal muscle is a non-toxic technique for gene therapy.
Blood, 2001
High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic ... more High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic anemia. Thus, it is clear that therapy for chronic anemia would greatly benefit from an erythropoietin derivative with increased erythropoietic activity rather than the native endogenous hormone. In this report, the activity of a human Epo-Epo dimer protein, obtained by recombinant technology, is described and compared with its Epo monomer counterpart produced under identical conditions. Although monomer Epo and dimer Epo-Epo had similar pharmacokinetics in normal mice, the increase in hematocrit value was greater with the dimer than with the monomer. Moreover, in clonogenic assays using CD34+ human hematopoietic cells, the human dimer induced a 3- to 4-fold-greater proliferation of erythroid cells than the monomer. Controlled secretion of dimeric erythropoietin was achieved in β-thalassemic mice by in vivo intramuscular electrotransfer of a mouse Epo-Epo plasmid containing the tetO elem...
Biochimie, 1986
Chromaffin granule membranes were incubated in the presence of low ATP concentrations, at low tem... more Chromaffin granule membranes were incubated in the presence of low ATP concentrations, at low temperature. A phosphorylated compound was rapidly formed which was stable in 10% trichloroacetic acid at 0 degree C. The lability of this compound in the presence of hydroxylamine or hot trichloroacetic acid indicated an acylphosphate, i.e., an ATPase phosphointermediate. Vanadate but not N-ethylmaleimide inhibited the formation of this derivative. Since the ATP-dependent generation of a transmembrane potential in chromaffin granule vesicles by the H+-pump was inhibited by N-ethylmaleimide but not by vanadate, the acylphosphate should not be associated with the H+-pump, i.e. ATPase I. We suggest that it is associated with ATPase II, an ATPase of unknown function present in chromaffin granule membrane preparations. This hypothesis is supported by the fact that ATPase II is vanadate sensitive and has a molecular mass of 140 kDa, properties similar to those of the phosphorylated intermediate.
Biochemistry, 1982
The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose m... more The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.
Biochemical Pharmacology, 1981
The carboxylic ionophore X-14547 A induces release of catecholamines from bovine chromaffin granu... more The carboxylic ionophore X-14547 A induces release of catecholamines from bovine chromaffin granules. This release is rapid and depends upon the ratio of ionophore to granule protein. When compared to X-537 A (lasolacid) which is known to mediate catecholamine release from the same preparation, X-14547 A is about 10 times more potent on a molar basis. Catecholamine release by X-14547 A is not accompanied by release of other components (ATP, protein or dopamine ~hydroxylase) of the granule matrix and therefore is not caused by lysis of the granule as previously proposed for X-537 A. X-14547 A also differs from X-537 A by its lack of specificity, both adrenaline and noradrenaline being equally released by this ionophore. These differences might reflect different release mechanisms. The absence of noradrenaline transport across a chloroform phase by X-14547 A suggests that its action might not involve direct translocation of the catecholamine cation across the granule membrane.
Biochemical Pharmacology, 1980
Reserpine, tetrabenazine and the neuroleptics chlorpromazine and haloperidol blocked the ATP-depe... more Reserpine, tetrabenazine and the neuroleptics chlorpromazine and haloperidol blocked the ATP-dependent uptake of noradrenaline and tyramine by ghosts derived from bovine chromaffin granules. The drugs did not affect chromaffin granules energization since they were without any effect on the membrane ATPase activity and on the transmembrane potential and pH-gradient generated by the ATP-dependent H+-translocase. Differences were observed in the inhibitory effect of the drugs on the monoamine uptake by ghosts acidic with respect to the external medium. These differences were accounted for by the existence under these conditions of two mechanisms of uptake, as shown by kinetic experiments. Noradrenaline was taken up by a carrier-mediated process which was blocked by all drugs, whereas tyramine transport involved non-specific diffusion of its unprotonated form, a process which was sensitive to neuroleptics and high doses of reserpine. From the kinetic and pharmacological studies of tyramine uptake, it is concluded that the ATP-dependent active transport of monoamines requires a carrier-mediated process even for amines which are rapidly transported by non-specific diffusion through the membrane.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1998
Most of the cationic lipids used for gene transfer experiments drastically lose their efficiency ... more Most of the cationic lipids used for gene transfer experiments drastically lose their efficiency in the presence of serum. We used a cationic lipid with a spermine head group and its fluorescent analog to study the cellular uptake and the intracellular fate of lipoplexes in the presence and absence of serum. We found that the amount of DNA and lipid taken up by the cells was not related to the efficacy of the gene transfer. When the lipofection was performed in the presence of serum, lipoplexes were contained within small intracellular vesicles. In the absence of serum, the vesicles were larger and heterogeneous in size and shape. By analysis of their size distribution, we showed that lipoplexes preformed in the absence of serum tended to aggregate. This aggregation was inhibited in the presence of serum. We used a carbonate formulation that led to the preformation of large particles: those large particles gave a high lipofection efficiency in the presence of serum and their intracellular distribution was identical to that observed in the absence of serum. q 1998 Elsevier Science B.V.
Molecular pharmacology, 1986
meta-Iodobenzylguanidine, an adrenal imaging agent used for the scintigraphic detection of human ... more meta-Iodobenzylguanidine, an adrenal imaging agent used for the scintigraphic detection of human pheochromocytoma, is a substrate for the monoamine uptake system of chromaffin granules. It is accumulated by bovine chromaffin granule membrane vesicles in the presence of ATP, and it can be released by an osmotic shock. The uptake is dependent upon the generation of an H+-electrochemical gradient by an ATP-dependent H+ pump since it is blocked by an H+ ionophore and since meta-iodobenzylguanidine uptake can be driven by imposing an artificial pH gradient (inside acidic) on the membrane vesicles. The transport is saturable and its Km value (2.0 microM at pH 8.0) is similar to that of noradrenaline (5.3 microM). Transport occurs through the monoamine transporter since it is blocked by the same inhibitors, tetrabenazine and reserpine, and also by the transporter substrates noradrenaline and serotonin. Noradrenaline inhibits meta-iodobenzylguanidine uptake competitively (Ki = 13 microM). I...
13th International Ceramics Congress - Part D, 2014
ZnGa2O4 (ZGO) is a normal spinel. When doped with Cr3+ ions, ZGO:Cr becomes a high brightness per... more ZnGa2O4 (ZGO) is a normal spinel. When doped with Cr3+ ions, ZGO:Cr becomes a high brightness persistent luminescence material with an emission spectrum perfectly matching the transparency window of living tissues. It allows in vivo mouse imaging with a better signal to background ratio than classical quantum dots. The most interesting characteristic of ZGO:Cr lies in the fact that its LLP can be excited with red light, well below its band gap energy and in the transparency window of living tissues. A mechanism based on the trapping of carriers localized around a special type of Cr3+ ions namely CrN2 can explain this singularity. The antisite defects of the structure are the main responsible traps in the persistent luminescence mechanism. When located around Cr3+ ions, they allow, via Cr3+ absorption, the storage of not only UV light but also all visible light from the excitation source.
Molecular Therapy, 2000
Numerous diseases are linked to the absence or insufficient concentration of a specific plasma pr... more Numerous diseases are linked to the absence or insufficient concentration of a specific plasma protein. Gene transfer is an appealing strategy for correction of such diseases. We report high and sustained plasma secretion of human secreted alkaline phosphatase and of human Factor IX by skeletal muscle of mice. This was obtained by delivering square-wave unipolar electric pulses of low field strength (200 V/cm) and long duration (20 ms) to skeletal muscle previously injected with plasmid DNA encoding for the secreted protein. This intramuscular electrotransfer method allows 30- to 150-fold increase in reporter protein secretion, compared to simple plasmid DNA injection. This increase allows one to obtain values of up to 2200 ng/ml of a reporter circulating protein. Moreover, this high level of secretion remains stable for several months.
Proceedings of the National Academy of Sciences, 1983
Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-llb-H-benzo[a]qu... more Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9, 10-dimethoxy-1,2,3,4,6,7-hexahydro-llb-H-benzo[a]quinolizine), a derivative of the neuroleptic tetrabenazine, binds to the membrane ofpurified bovine chromaffin granules. Specific binding was characterized by Kd and B.., values of 3.1 nM and 62 pmol/mg ofmembrane protein, respectively. It was reversible, with association and dissociation rate constants of 0.22 x 106 M'1 s-1 and 1.8 x 10-3 s-1, respectively. Binding sites were present
Proceedings of the National Academy of Sciences, 1999
We have recently discovered that cationic cholesterol derivatives characterized by guanidinium po... more We have recently discovered that cationic cholesterol derivatives characterized by guanidinium polar headgroups are very efficient for gene transfection in vitro and in vivo . In spite of being based on some rationale at the molecular level, the development of these new synthetic vectors was nevertheless empirical. Indeed, the factors and processes underlying cationic lipid-mediated gene transfer are still poorly understood. Thus, to get a better insight into the mechanisms involved, we have examined the supramolecular structure of lipid/DNA aggregates obtained when using reagent bis(guanidinium)-tren-cholesterol (BGTC), either alone or as a liposomal formulation with the neutral phospholipid dioleoyl phosphatidylethanolamine (DOPE). We here report the results of cryotransmission electron microscopy studies and small-angle x-ray scattering experiments, indicating the presence of multilamellar domains with a regular spacing of 70 Å and 68 Å in BGTC/DOPE–DNA and BGTC–DNA aggregates, r...
Nucleic Acids Research, 1999
Clinical applications of gene therapy mainly depend on the development of efficient gene transfer... more Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.
Journal of Medicinal Chemistry, 1998
We have designed and synthesized original cationic lipids for gene delivery. A synthetic method o... more We have designed and synthesized original cationic lipids for gene delivery. A synthetic method on solid support allowed easy access to unsymmetrically monofunctionalized polyamine building blocks of variable geometries. These polyamine building blocks were introduced into cationic lipids. To optimize the transfection efficiency in the novel series, we have carried out structure-activity relationship studies by introduction of variable-length lipids, of variable-length linkers between lipid and cationic moiety, and of substituted linkers. We introduce the concept of using the linkers within cationic lipids molecules as carriers of side groups harboring various functionalities (side chain entity), as assessed by the introduction of a library composed of cationic entities, additional lipid chains, targeting groups, and finally the molecular probes rhodamine and biotin for cellular traffic studies. The transfection activity of the products was assayed in vitro on Hela carcinoma, on NIH3T3, and on CV1 fibroblasts and in vivo on the Lewis Lung carcinoma model. Products from the series displayed high transfection activities. Results indicated that the introduction of a targeting side chain moiety into the cationic lipid is permitted. A primary physicochemical characterization of the DNA/lipid complexes was demonstrated with this leading compound. Selected products from the series are currently being developed for preclinical studies, and the labeled lipopolyamines can be used to study the intracellular traffic of DNA/cationic lipid complexes.
Gene Therapy, 1997
Plasmid DNA used for nonviral therapeutic gene transfer After extensive washing of the column, pu... more Plasmid DNA used for nonviral therapeutic gene transfer After extensive washing of the column, purified plasmid or nucleic acid vaccination has to be highly purified, devoid DNA is eluted using an alkaline buffer. The binding conof contaminating components such as bacterial proteins, ditions of the plasmid DNA on to the column have been endotoxins, or bacterial chromosomal DNA. We have optimized, as well as the hybridization sequence and the developed a new affinity chromatography technique for linker group between the matrix and the third strand oligoplasmid DNA purification: triple-helix affinity chromato-nucleotide. The THAC technique makes it possible to purify graphy (THAC). This technique is based on the sequence-in one step supercoiled plasmid DNA, and to significantly specific interaction of an oligonucleotide forming a triple-reduce the level of contaminating RNA, endotoxins and helix with plasmid DNA. The oligonucleotide was covalently chromosomal DNA. In particular, a 100-fold reduction of linked to a chromatographic matrix, thus providing a re-chromosomal DNA contamination over that obtained with usable affinity support. By inserting a suitable homopurine conventional techniques can be achieved through a single sequence in the plasmid DNA, it is possible to obtain a additional THAC step. Further improvements of THAC triple-helix interaction that will only be stable at mild acidic technology are possible, and we anticipate that this tech-pH and that will dissociate in alkaline conditions. A crude nique can be scaled up for integration into a full commerlysate from a recombinant E. coli, or a pre-purified plasmid cial-scale DNA production process. DNA, is thus applied at acidic pH on to a THAC column.
Gene Therapy, 1997
Plasmids currently used for nonviral gene transfer have the excised in vivo after thermoinduction... more Plasmids currently used for nonviral gene transfer have the excised in vivo after thermoinduction of the integrase gene disadvantage of carrying a bacterial origin of replication leading to the formation of two supercoiled molecules: the and an antibiotic resistance gene. There is, therefore, a minicircle and the starting plasmid lacking the expression risk of uncontrolled dissemination of the therapeutic gene cassette. In various cell lines, purified minicircles exhibited and the antibiotic resistance gene. Minicircles are new a two-to 10-fold higher luciferase reporter gene activity DNA delivery vehicles which do not have such elements than the unrecombined plasmid. This could be due to either and are consequently safer as they exhibit a high level of the removal of unnecessary plasmid sequences, which biological containment. They are obtained in E. coli by att could affect gene expression, or the smaller size of minisite-specific recombination mediated by the phage inte-circle which may confer better extracellular and intracellular grase. The desired eukaryotic expression cassette bioavailability and result in improved gene delivery bounded by the attP and attB sites was cloned on a properties. recombinant plasmid. The expression cassette was
FEBS Letters, 1999
The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear ent... more The nuclear localization signal (NLS) of the SV40 large T antigen efficiently induces nuclear entry of proteins. We have developed a strategy for covalent coupling of one or a controlled number of NLS peptides to plasmid DNA at a specific site by triple helix formation. A psoralen-oligonucleotide-NLS peptide conjugate was synthesized and characterized by proteolysis with trypsin. This conjugate was used to covalently associate one NLS peptide to plasmid DNA by triple helix formation and photoactivation. The oligonucleotide-NLS peptide conjugate interacted with the NLS-receptor importin K K. The reporter gene was expressed after transfection of the modified plasmid in NIH 3T3 cells, indicating no loss of the gene expression functionality of the plasmid. On the other hand, no increase in expression was observed as a result of the NLS peptide. This site-specific coupling technology can be used to couple to a plasmid other ligands targeting to a specific receptor.
Expert Opinion on Therapeutic Patents, 1998
ABSTRACT The ability to produce and express foreign genes in humans might cure or even prevent ma... more ABSTRACT The ability to produce and express foreign genes in humans might cure or even prevent many important human diseases that are either treated poorly or are untreatable by present therapies. However, insufficient transgene expression in vivo has so far impaired the development of effective gene therapy. Thus, it is of interest to develop compositions and delivery methods for gene therapy that lead to high level transgene expression in a variety of cell and tissue types. Synthetic DNA delivery agents are of crucial interest for gene therapy as an alternative to viral vectors, since they potentially display fewer risks in terms of immunogenicity and propagation and are easier to produce under GMP conditions. In the last decade, a significant number of industrial and academic groups have emerged with a number of important patents claiming a variety of synthetic DNA delivery agents. Some of these agents were launched into clinical trials at very early stages of development leading to rather modest results. The lack of physicochemical characterisation of the self-assembling complexes in those early clinical trials prevented interpretation, correlation and comparison of transgene expression and biodistribution with the supramolecular state of these self-assemblies. On the other hand, in the last two years, a significant body of information has emerged from academic and academic-industrial groups regarding the exhaustive physicochemical characterisation of some complexes and the impact of these characteristics on transgene biodistribution and expression both in vitro and in vivo. Additionally, the intracellular fate of different self-assemblies has been studied by different groups. Together, these studies provide a rational basis for designing novel DNA delivery agents. Synthetic agents for gene delivery are classified into different chemical families. In this manuscript, we have focused on the cationic lipids family. We choose to show the approaches that introduce original elements into the backbone of the synthetic delivery agent. Data on the physicochemical characterisation of different synthetic agents for gene delivery are given when available. Clinical data (reported in other reviews) are beyond the scope of this article. Finally, a discussion on how to improve the results obtained so far in order to advance towards new human trials is presented.
Developmental Dynamics, 2006
We report here a method that allows fast, efficient, and low-cost screening for gene function in ... more We report here a method that allows fast, efficient, and low-cost screening for gene function in the vascular system of the vertebrate embryo. Through intracardiac delivery of nucleic acids optimally compacted by a specific cationic lipid, we are able to induce in vivo endothelial cell-specific gain-of-function during development of the vascular network in the chick embryo. When the nucleic acids are delivered during the period of intraembryonic hematopoiesis, aortic hemangioblasts, the forerunners of the hematopoietic stem cells known to derive from the aortic endothelium, are also labeled. Similarly, we show that siRNA could be used to induce loss-of-function in vascular endothelial cells. This gene transfer technique was also applied to the mouse embryo with a high efficiency. The present method allows large-scale analysis and may represent a new and versatile tool for functional genomics.
Current Opinion in Biotechnology, 1998
Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major cha... more Cationic lipids are widely used for in vitro gene transfer due to their efficiency. The major challenges for the improvement of in vivo cationic lipid-mediated gene delivery reside in the design of more biocompatible lipoplexes mimicking viral-mediated gene delivery and in understanding the fate of the lipoplexes within the cells.
Cell Biology and Toxicology, 2004
In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle diso... more In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic delivery of therapeutic proteins. Electrotransfer is a powerful method for DNA transfer into skeletal muscle. In view of the broad potential gene therapy clinical application of electrotransfer o¡ers, it is important to perform toxicology studies on electrotransfered muscle tissue. We have investigated if the delivery of square wave electric pulses of low ¢eld strength and long duration to mouse tibial cranial muscle induced the expression of stress related genes. We have pro¢led gene expression patterns in muscles at di¡erent times after delivery of electric pulses using Stress/Toxicology microarrays. No signi¢cant variation in the expression of stress related-genes was detected between treated and non-treated muscles. This suggests that application of adequate, ¢netuned, electric pulses to the skeletal muscle is a non-toxic technique for gene therapy.
Blood, 2001
High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic ... more High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic anemia. Thus, it is clear that therapy for chronic anemia would greatly benefit from an erythropoietin derivative with increased erythropoietic activity rather than the native endogenous hormone. In this report, the activity of a human Epo-Epo dimer protein, obtained by recombinant technology, is described and compared with its Epo monomer counterpart produced under identical conditions. Although monomer Epo and dimer Epo-Epo had similar pharmacokinetics in normal mice, the increase in hematocrit value was greater with the dimer than with the monomer. Moreover, in clonogenic assays using CD34+ human hematopoietic cells, the human dimer induced a 3- to 4-fold-greater proliferation of erythroid cells than the monomer. Controlled secretion of dimeric erythropoietin was achieved in β-thalassemic mice by in vivo intramuscular electrotransfer of a mouse Epo-Epo plasmid containing the tetO elem...
Biochimie, 1986
Chromaffin granule membranes were incubated in the presence of low ATP concentrations, at low tem... more Chromaffin granule membranes were incubated in the presence of low ATP concentrations, at low temperature. A phosphorylated compound was rapidly formed which was stable in 10% trichloroacetic acid at 0 degree C. The lability of this compound in the presence of hydroxylamine or hot trichloroacetic acid indicated an acylphosphate, i.e., an ATPase phosphointermediate. Vanadate but not N-ethylmaleimide inhibited the formation of this derivative. Since the ATP-dependent generation of a transmembrane potential in chromaffin granule vesicles by the H+-pump was inhibited by N-ethylmaleimide but not by vanadate, the acylphosphate should not be associated with the H+-pump, i.e. ATPase I. We suggest that it is associated with ATPase II, an ATPase of unknown function present in chromaffin granule membrane preparations. This hypothesis is supported by the fact that ATPase II is vanadate sensitive and has a molecular mass of 140 kDa, properties similar to those of the phosphorylated intermediate.
Biochemistry, 1982
The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose m... more The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.
Biochemical Pharmacology, 1981
The carboxylic ionophore X-14547 A induces release of catecholamines from bovine chromaffin granu... more The carboxylic ionophore X-14547 A induces release of catecholamines from bovine chromaffin granules. This release is rapid and depends upon the ratio of ionophore to granule protein. When compared to X-537 A (lasolacid) which is known to mediate catecholamine release from the same preparation, X-14547 A is about 10 times more potent on a molar basis. Catecholamine release by X-14547 A is not accompanied by release of other components (ATP, protein or dopamine ~hydroxylase) of the granule matrix and therefore is not caused by lysis of the granule as previously proposed for X-537 A. X-14547 A also differs from X-537 A by its lack of specificity, both adrenaline and noradrenaline being equally released by this ionophore. These differences might reflect different release mechanisms. The absence of noradrenaline transport across a chloroform phase by X-14547 A suggests that its action might not involve direct translocation of the catecholamine cation across the granule membrane.
Biochemical Pharmacology, 1980
Reserpine, tetrabenazine and the neuroleptics chlorpromazine and haloperidol blocked the ATP-depe... more Reserpine, tetrabenazine and the neuroleptics chlorpromazine and haloperidol blocked the ATP-dependent uptake of noradrenaline and tyramine by ghosts derived from bovine chromaffin granules. The drugs did not affect chromaffin granules energization since they were without any effect on the membrane ATPase activity and on the transmembrane potential and pH-gradient generated by the ATP-dependent H+-translocase. Differences were observed in the inhibitory effect of the drugs on the monoamine uptake by ghosts acidic with respect to the external medium. These differences were accounted for by the existence under these conditions of two mechanisms of uptake, as shown by kinetic experiments. Noradrenaline was taken up by a carrier-mediated process which was blocked by all drugs, whereas tyramine transport involved non-specific diffusion of its unprotonated form, a process which was sensitive to neuroleptics and high doses of reserpine. From the kinetic and pharmacological studies of tyramine uptake, it is concluded that the ATP-dependent active transport of monoamines requires a carrier-mediated process even for amines which are rapidly transported by non-specific diffusion through the membrane.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1998
Most of the cationic lipids used for gene transfer experiments drastically lose their efficiency ... more Most of the cationic lipids used for gene transfer experiments drastically lose their efficiency in the presence of serum. We used a cationic lipid with a spermine head group and its fluorescent analog to study the cellular uptake and the intracellular fate of lipoplexes in the presence and absence of serum. We found that the amount of DNA and lipid taken up by the cells was not related to the efficacy of the gene transfer. When the lipofection was performed in the presence of serum, lipoplexes were contained within small intracellular vesicles. In the absence of serum, the vesicles were larger and heterogeneous in size and shape. By analysis of their size distribution, we showed that lipoplexes preformed in the absence of serum tended to aggregate. This aggregation was inhibited in the presence of serum. We used a carbonate formulation that led to the preformation of large particles: those large particles gave a high lipofection efficiency in the presence of serum and their intracellular distribution was identical to that observed in the absence of serum. q 1998 Elsevier Science B.V.
Molecular pharmacology, 1986
meta-Iodobenzylguanidine, an adrenal imaging agent used for the scintigraphic detection of human ... more meta-Iodobenzylguanidine, an adrenal imaging agent used for the scintigraphic detection of human pheochromocytoma, is a substrate for the monoamine uptake system of chromaffin granules. It is accumulated by bovine chromaffin granule membrane vesicles in the presence of ATP, and it can be released by an osmotic shock. The uptake is dependent upon the generation of an H+-electrochemical gradient by an ATP-dependent H+ pump since it is blocked by an H+ ionophore and since meta-iodobenzylguanidine uptake can be driven by imposing an artificial pH gradient (inside acidic) on the membrane vesicles. The transport is saturable and its Km value (2.0 microM at pH 8.0) is similar to that of noradrenaline (5.3 microM). Transport occurs through the monoamine transporter since it is blocked by the same inhibitors, tetrabenazine and reserpine, and also by the transporter substrates noradrenaline and serotonin. Noradrenaline inhibits meta-iodobenzylguanidine uptake competitively (Ki = 13 microM). I...