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Daniel Scherman

Publications and source records attributed to Daniel Scherman.

At least 37 records · Page 2Linked to original sources
PubMed2003

Anionic polyethyleneglycol lipids added to cationic lipoplexes increase their plasmatic circulation time.

Cationic liposomes have been widely sensed as good DNA compacting delivery agents. Although their use generally met with encouraging results in vitro, the results in vivo were rather disappointing, as they strongly interact with the blood components before they can reach the therapeutic target. Polyethyleneglycol (PEG) shielding has been proposed as a way to alleviate this effect, but was still found unsatisfactory in most instances for systemic administration. We demonstrate here that the insertion of anionic functions between the lipid part and the PEG, at a correct distance to favor electrostatic interactions with the outer cationic layer of the lipoplexes, provides not only a decrease in the mean peripheral charge of the lipoplex (zeta potential), but also a greater colloidal stability of the particles in the presence of serum. Transfection in the lung is also decreased with negatively charged PEG shielding, although no significant changes are observed in the tumor. This encouraging new approach should consequently be combined with active extra-cellular receptor targeting to achieve the desired delivery of the therapeutic DNA to tumor tissues.

Animals↗
PubMed2003

NLS bioconjugates for targeting therapeutic genes to the nucleus.

One of the major steps limiting non-viral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of transfected cells. Trafficking of nuclear proteins from the cytoplasm into the nucleus through nuclear pore complexes is mediated by the presence of nuclear localization sequences (NLS) on proteins. Viral DNA and RNA also require interaction with cellular machinery for efficient nuclear import. In this article, we review the various strategies used to provide plasmid DNA with nuclear localization sequences, and discuss the possibility of developing efficient gene delivery systems based on these strategies.

Active Transport, Cell Nucleus↗
PubMed2003

Efficacy of interleukin-10 gene electrotransfer into skeletal muscle in mice with collagen-induced arthritis.

BACKGROUND: Gene therapy is very promising in the treatment of rheumatoid arthritis (RA). Electrotransfer is a recent method reported to enhance in vivo intramuscular DNA transfection. Interleukin-10 (IL-10) has antiinflammatory effects in RA and in collagen-induced arthritis (CIA), a murine model of RA. In order to improve our strategy of gene therapy, we used electrotransfer to enhance penetration into skeletal muscle with CIA of plasmids encoding IL-10. METHODS: CIA was induced in DBA/1 mice by immunization with bovine type II collagen. Injection into the tibial cranial muscle of low-dose (200 ng) pCOR plasmid encoding murine IL-10 (pCOR-CMV-mIL-10) was immediately followed by application of square-wave electric pulses (8 pulses of 200V/cm, 20 ms duration at 2 Hz). Control groups received empty plasmid or saline before electrotransfer. RESULTS: When electrotransfer was performed twice on days 10 and 25 postimmunization, CIA was significantly delayed (P < 0.05) and attenuated (P < 0.001) in groups treated by electrotransfer or pCOR-CMV-mIL-10 plasmid vs. control groups. When electrotransfer of pCOR-CMV-mIL-10 plasmid was performed on days 25 and 40 postimmunization, at disease onset, the clinical severity of CIA was reduced (P < 0.05). All groups which had been electrotransferred early or late by pCOR-CMV-mIL-10 plasmid showed suppression of histological signs of arthritis. CONCLUSIONS: Taken together, these data indicate that administration of an antiinflammatory plasmid-born gene by electrotransfer of naked DNA is effective in vivo in an arthritis model.

Animals↗
PubMed2003

Transcriptional activation of the metallothionein I gene by electric pulses in vivo: basis for the development of a new gene switch system.

BACKGROUND: In vivo gene transfer to skeletal muscle is a promising strategy for the treatment of muscular disorders and for the systemic delivery of therapeutic proteins. Nevertheless, for a safe and effective protein production, the spatial and temporal control of gene expression is critical. The existing regulating systems rely on the use of an exogenously regulatory protein and/or an inducer drug whose pharmacological properties are of major concerns for therapeutic applications in humans. Therefore, new strategies based on endogenous regulatable elements have been explored. METHODS: Gene expression profiles of skeletal muscle submitted or not to electrical pulses and harvested at different times were compared using the Affymetrix GeneChip technology. The endogenous metallothionein promoter was studied by Northern blot and semiquantitative and quantitative RT-PCR. The inducibility of the metallothionein I promoter placed in a plasmid exogenous context was studied using the murine SEAP reporter gene. RESULTS: The expression of metallothionein I mRNA is significantly increased 6 h after electric pulses delivery. This induction is transient. Identical MT-I expression level is observed after several sequential series of pulses delivery. We demonstrated as well that the MT-II promoter was sensitive to electric pulses delivery. Moreover, the metallothionein I promoter, placed in a plasmid context in front of a reporter gene, was also activated by the application of transient electric field. CONCLUSIONS: We identified a promoter highly inducible by the controlled electric stimuli applied for electrotransfer experiments. The use of the metallothionein promoter is promising for the time-control by physical stimuli of the expression of a therapeutic gene.

Animals↗
PubMed2003

Enhanced antitumor activity of a combination of MBD2-antisense electrotransfer gene therapy and bleomycin electrochemotherapy.

BACKGROUND: MBD2 is a methylated DNA-binding protein that has been previously suggested to have transcriptional silencing as well as DNA demethylase activities. We have previously shown that electrotransfer of an MBD2-antisense encoding plasmid inhibits tumor growth in vivo. In this study we tested whether a combination of MBD2-antisense gene therapy and bleomycin chemotherapy has an augmented antitumor effect in comparison with either monotherapy. METHODS: Mice bearing human non-small-cell lung carcinoma line H1299 xenoplants were treated with electrotransfer of either bleomycin or MBD2-antisense expression plasmid or a combination of both therapies and tumor growth following treatment was monitored. RESULTS: A combination of electrotransfer of MBD2-antisense and bleomycin electrochemotherapy has an additive inhibitory effect on the rate of tumor growth and a synergistic effect on the number of tumor-free animals when compared with either monotherapy. CONCLUSIONS: Our results suggest that a combination of MBD2-antisense electrotransfer gene therapy and chemotherapy with bleomycin is a candidate new approach to anticancer therapy.

Animals↗
PubMed2003

Cationic lipids for transfection.

Among other strategies, the use of cationic lipids as autoassembling vehicles for non viral DNA transfection has received considerable attention. An exponentially growing litterature has been published on this topic (over 700 hits for the past decade, including 400 in the last two years). The present review focuses on the main present strategies aiming at improving cationic lipids induced transfection, and on some of the frequently encountered problems that should be solved to apply these non-viral vectors for human health. The review contains several sections dealing with the chemistry, physico-chemistry, cell biology, in vivo biology, and targeting of cationic-lipid DNA complexes.

Animals↗
PubMed2002

Interleukin-10 expression after intramuscular DNA electrotransfer: kinetic studies.

Transfected muscle can be used as a secreting tissue for therapeutic proteins. Skeletal muscle transfection is increased by suitable electric pulse application (electrotransfer). We and others had shown that electrotransfer of interleukin-10 encoding plasmid is an effective strategy in animal models of chronic diseases such as myocarditis, atherosclerosis, or rheumatoid arthritis. In the present work, we have studied murine interleukin-10 production and secretion after i.m. electrotransfer. In immunocompetent mice, serum and muscle mIL-10 levels were enhanced by electrotransfer. Serum mIL-10 concentration reached rapidly a peak level 2 days after electrotransfer. It then decreased to background at day 14. Muscle mIL-10 mRNA and protein remained more stable, being detectable up to 84 days after electrotransfer. A boost reinjection led to similar high level of circulating mIL-10. The fast decrease of serum mIL-10 was not observed in SCID mice.

Animals↗
PubMed2002

Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis.

Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 micros) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of (51)Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.

Animals↗
PubMed2002

Folate-targeted gene transfer in vivo.

Nonviral systemic delivery is one of the most attractive approaches for cancer gene therapy. To achieve this goal, various laboratories have developed cationic liposomes. However, when injected intravenously, cationic lipid-DNA complexes accumulate mostly into and transfect lung tissue. Here, we describe a method by which these complexes can be targeted to tumors using folic acid. Adding polyethylene glycol (PEG)-lipids to the complexes dramatically reduced both lung accumulation and gene transfer to lungs and tumors after intravenous administration. The presence of folic acid at the distal end of the PEG-lipid did not modify tumor accumulation of the complexes. However, with folate-targeted complexes, gene transfer activity was restored in tumors while the activity in lungs was reduced by 50- to 100-fold compared with nontargeted lipid-DNA complexes. This approach provides a first in vivo proof of concept to achieve targeted tumor gene delivery.

Animals↗
PubMed2002

In vivo plasmid DNA electrotransfer.

In vivo electrotransfer is a physical technique for gene delivery in various mammalian tissues, which involves the injection of plasmid DNA into a target tissue and administration of an electric field. Its ease of performance, as well as recent understanding of its mechanism and applications to different mammalian tissues such as skeletal muscle, liver, brain and tumors, makes it a powerful technique. It could be used in gene therapy and as a laboratory tool to study gene functions.

Animals↗
PubMed2002

Sphingosine-based liposome as DNA vector for intramuscular gene delivery.

PURPOSE: The aim of this study was to develop a labile sphingosine-based liposome for intramuscular gene delivery. METHODS: Sphingosine-based liposomes were formulated in a range of solutions with phosphatidylcholine, then were associated to DNA. The physico-chemical characteristics of the sphingosine/EPC liposomes and sphingosine/EPC/DNA lipoplexes were determined. DNA stability within sphingosine-based liposomes was evaluated in the presence of a nuclease and mouse serum. In vivo gene transfer was studied by intramuscular injection with and without the electrotransfer technique. RESULTS: By increasing the charge ratios, colloidally stable sphingosine/DNA particles with a 170 nm average diameter and a positive zeta potential were obtained. Ethidium bromide was still able to insert into plasmid DNA within the lipoplexes, even though plasmid DNA was demonstrated to be complexed to the lipid by gel electrophoresis. Additionally, DNA was shown to be accessible to DNase I, but significantly resistant to serum enzymatic digestion. Upon intramuscular injection, lipoplexes induced an inhibition of gene expression as compared with naked DNA. CONCLUSIONS: The cationic sphingosine/EPC/DNA complexes form weakly compacted structure, potentially labile in vivo, which might be useful for in vivo gene transfer.

Animals↗
PubMed2002

Applications of plasmid electrotransfer.

The use of electric pulses to transfect cells has recently been extended to show the utility of this procedure in vivo. Electrotransfer has been performed in vivo on several tissue types including skin, blood vessels, liver, tumor, muscle, cornea, brain and spleen. The most widely targeted tissue has been skeletal muscle. In addition to its potential use in gene therapy, in vivo DNA electrotransfer is also, because of its simplicity, a powerful laboratory tool to study in vivo gene expression and function in a given tissue. Many published studies have now shown that plasmid electrotransfer can lead to a long-lasting therapeutic effect in various pathologies, such as cancer, blood disease, or muscle ischemia. The future potential for this gene therapy approach will include delivery for both local action or distal effect by secretion of the transgenic proteins in the circulation.

Animals↗
PubMedDate not supplied

Antisense MBD2 gene therapy inhibits tumorigenesis.

BACKGROUND: Aberration in the pattern of DNA methylation is one of the hallmarks of cancer. We present data suggesting that dysregulation of MBD2, a recently characterized member of a novel family of methylated DNA binding proteins, is involved in tumorigenesis. Two functions were ascribed to MBD2, DNA demethylase activity and repression of methylated genes. METHODS: Multiple antisense expression and delivery systems, transfection, electrotransfer and adenoviral were employed to demonstrate that MBD2 is essential in tumorigenesis, both ex vivo and in vivo. RESULTS: Inhibition of MBD2 by antisense expression resulted in inhibition of anchorage-independent growth of antisense transfected cancer cells or cells infected with an adenoviral vector expressing MBD2 antisense. Xenograft tumors treated with an adenoviral vector expressing MBD2 antisense or xenografts treated with electrotransferred plasmids expressing MBD2 antisense showed reduced growth. CONCLUSIONS: These results support the hypothesis that one or both of the functions described for MBD2 are critical in tumorigenesis and that MBD2 is a potential anticancer target.

Adenoviridae↗
PubMedDate not supplied

Design, synthesis, and evaluation of gadolinium cationic lipids as tools for biodistribution studies of gene delivery complexes.

Gadolinium-chelating cationic lipids have been synthesized to obtain lipoplexes with MRI contrast properties. These compounds were designed to follow the biodistribution of synthetic DNA for gene delivery by nuclear magnetic resonance imaging. The lipid MCO-I-68 was synthesized, and chelate complexes with gadolinium were formed and characterized in terms of physicochemical and DNA binding properties. The transfection activity of MCO-I-68-Gd/DNA complexes was assayed in vitro on NIH 3T3. Different formulations of the product were tested. When up to 5% of the gadolinium lipid complexes were co-formulated with the cationic lipid RPR120535 used as a reference, the transfection levels were maintained as compared to RPR120535 alone. To date, only a liposomal formulation of a gadolinium-cationic lipid chelate without DNA had been observed using magnetic resonance imaging. In vivo intratumoral administration of MCO-I-68-Gd/DNA lipoplexes to tumor model led to an important increase of the NMR signal. It was demonstrated that the new complexes also acted as transfection carriers when they were formulated from liposomes.

3T3 Cells↗
PubMedDate not supplied

DNA complexing lipopolythiourea.

We present a neutral lipopolythiourea (DTTU) as a potential DNA-binding agent. Light scattering experiments showed that mixing a lipopolythiourea with dipalmitoylphosphatidylcholine (DPPC/DTTU) led to small particles with sizes ranging from 100 to 150 nm at optimum conditions. Setting a fixed DNA amount, an increasing amount of DTTU/DPPC or DPPC lipids was added. Particle size increased only with DTTU/DPPC, indicating that interaction occurred between the DTTU/DPPC particles and DNA. In the same way, only DTTU/DPPC limited the ethidium bromide accessibility to plasmid DNA. These data suggest that DTTU/DPPC liposomes associate to DNA, which was confirmed by agarose gel experiments. To prove the active part of the DTTU lipid itself in DNA compaction, pegoylated-lipid was used. Cholesterol-PEG(2000) alone was not able to condense DNA. In contrast, DTTU/PEG-cholesterol was able to retain plasmid DNA on an agarose gel. In vivo injection of DTTU/DPPC/complexes was studied. Circulation time increase for noncationic particles as compared to cationic. More obvious was the lack of nonspecific accumulation in the lung, where a gain of 3 to 40 fold was measured.

1,2-Dipalmitoylphosphatidylcholine↗
PubMedDate not supplied

A new triantennary galactose-targeted PEGylated gene carrier, characterization of its complex with DNA, and transfection of hepatoma cells.

Nonviral gene vectors remain inefficient in vivo largely because of their rapid clearance from the circulation and also their nonspecific association with the extracellular matrix. To overcome such drawbacks, cationic lipoplexes are now frequently coated with hydrophilic polymers such as PEGs to reduce nonspecific interactions, and ligands are also linked to their surface to obtain cell-specific gene transfer. In view of the development of vectors for systemic gene delivery, we have designed and studied lipoplexes that carry a triantennary galactosyl ligand attached to the distal end of a (PEG)45-conjugated lipid. We incorporated this targeted PEGylated lipid into lipoplexes using two strategies of formulation, i.e., using either preformed micelles or liposomes. We demonstrated that the incorporation of PEG chains stabilized lipoplexes and masked, but only partially, the positive charges exposed on the surface of the particles. We have also shown that incorporation into lipoplexes of a lipidated PEG chain, bearing a ligand at its distal end, yielded particles that exhibited an accessible ligand throughout the whole range of cationic lipid to DNA ratios. We obtained a targeted transfection in HepG2 cells with one of the formulations. Our results strengthen the validity of using a ligand carried by a long PEG spacer arm for targeted gene transfer.

Carcinoma, Hepatocellular↗