Biomedical subjects

Ernst Wagner

Publications and source records attributed to Ernst Wagner.

At least 37 records · Page 2Linked to original sources

PubMed2005

Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo.

Nonviral vectors should undergo "virus-like" changes compatible with the steps of gene delivery. Poly(ethylene) glycol (PEG) shielding of DNA/polycation polyplexes protects from nonspecific interactions with the extracellular environment. pH-triggered removal of the shield within the endosome may be advantageous. Polycation and PEG were linked via acylhydrazides or pyridylhydrazines. The pyridylhydrazone prepared from polylysine and propionaldehyde-PEG showed the greatest acid-dependent hydrolysis; at pH 5, 37 degrees C for 10 min, 90% hydrolyzed, while at pH 7.4 the half-life was 1.5 h. Particle size and zeta potential measurements of the polyplexes showed complete deshielding within 1 h at pH 5, while at pH 7.4 the shield remained at 4 h, 37 degrees C. For gene transfection a targeting conjugate was also included in the polyplex, transferrin as ligand for K562 and Neuro2A cells and epidermal growth factor for HUH-7 and Renca-EGFR cells. Marker gene expression showed that the reversibly shielded polyplexes exhibited up to 2 log orders of magnitude higher gene expression in vitro and 1 log magnitude higher gene expression in an in vivo mouse model, compared to the stably shielded control polyplexes. Engineering of polyplexes with more dynamic domains is an encouraging new direction in nonviral vector design.

Animals↗

PubMed2005

Targeting of Polyplexes: Toward Synthetic Virus Vector Systems.

Dominating issues in gene vector optimization are specific in recognizing the target cells and exploiting the proper intracellular trafficking routes. Any progress in this area will result in improved specific gene transfer, reduce the required therapeutic vector doses and, in consequence, lower the overall toxicity to the host. To provide polyplexes with the ability to distinguish between non-target and target cells, cell-binding ligands have been incorporated which recognize target-specific cellular receptors. In addition, polyplex domains with unspecific binding capacity (such as surface charges) have to be shielded or removed. Cell-binding ligands can be small molecules, vitamins, carbohydrates, peptides or proteins such as growth factors or antibodies. Such ligands have been incorporated into polyplexes after chemical conjugation to cationic polymers. The choice of the ligand and physical properties of the DNA formulation strongly influence extracellular routing (circulation in blood, tissue distribution), uptake and intracellular delivery of polyplexes. Recent efforts are discussed that aim at the development of polyplexes into virus-like supramolecular complexes; such particles should undergo structural changes compatible with extracellular and intracellular targeting.

Journal Article↗

PubMed2005

Opening of size-selective pores in endosomes during human rhinovirus serotype 2 in vivo uncoating monitored by single-organelle flow analysis.

The effect of virus uncoating on endosome integrity during the early steps in viral infection was investigated. Using fluid-phase uptake of 10- and 70-kDa dextrans labeled with a pH-dependent fluorophore (fluorescein isothiocyanate [FITC]) and a pH-independent fluorophore (cyanine 5 [Cy5]), we determined the pHs of labeled compartments in intact HeLa cells by fluorescence-activated cell sorting analysis. Subsequently, the number and pH of fluorescent endosomes in cell homogenates were determined by single-organelle flow analysis. Cointernalization of adenovirus and 70-kDa FITC- and Cy5-labeled dextran (FITC/Cy5-dextran) led to virus-induced endosomal rupture, resulting in the release of the marker from the low-pH environment into the neutral cytosol. Consequently, in the presence of adenovirus, the number of fluorescent endosomes was reduced by 40% compared to that in the control. When human rhinovirus serotype 2 (HRV2) was cointernalized with 10-and 70-kDa FITC/Cy5-dextrans, the 10-kDa dextran was released, whereas the 70-kDa dextran remained within the endosomes, which also maintained their low pH. These data demonstrate that pores are generated in the membrane during HRV2 uncoating and RNA penetration into the cytosol without gross damage of the endosomes; 10-kDa dextran can access the cytosol through these pores. Whereas rhinovirus-mediated pore formation was prevented by the vacuolar ATPase inhibitor bafilomycin A1, adenovirus-mediated endosomal rupture also occurred in the presence of the inhibitor. This finding is in keeping with the low-pH requirement of HRV2 infection; for adenovirus, no pH dependence for endosomal escape was found with this drug.

Adenoviridae↗

PubMed2005

Specific targets in tumor tissue for the delivery of therapeutic genes.

Gene therapy is part of a growing field in molecular medicine, which will gain importance in the treatment of human diseases. Until now, almost two thirds of all clinical trials performed in gene therapy are directed against Cancer As solid tumors exceeding a certain size rely on blood supply, the administration of particulate gene delivery vectors via the bloodstream is a promising concept. Tumor cells and the tumor vasculature both offer specific molecular targets, which can be utilized for the site directed delivery of therapeutic genes. Passive targeting of macromolecular drugs including gene delivery vectors to tumors can be achieved by the so called enhanced permeability and retention (EPR) effect. The specificity can be markedly enhanced when tumor targeting ligands are used. Viral vectors, which usually do not have a natural tropism for tumor tissue, were generated to carry tumor targeting molecules on their surface. Synthetic gene delivery vectors, based on cationic lipids or cationic polymers were biochemically modified to incorporate ligands specific for tumor cells or tumor vasculature. For systemic application, these delivery systems have to fulfill certain conditions. The delivery vector should not induce any immunogenic and inflammatory responses. Several studies were conducted to reduce the immunogenicity of viral vectors; surface modification of non-viral gene delivery systems reduced their non-specific interaction with blood components. On the genetic level, tumor specific promoters add additional layers of specificity restricting the transgene expression to the tumor tissue. This review will cover the systemic application of particulate gene transfer vectors targeted to tumors and will give an overview of therapeutic concepts for cancer gene therapy.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

Targeting of polyplexes: toward synthetic virus vector systems.

Endocytosis↗

PubMed2004

Functional analysis of genomic DNA, cDNA, and nucleotide sequence of the mature C-type natriuretic peptide gene in vascular cells.

OBJECTIVE: The aim of this study was to investigate the effect of various C-type natriuretic peptide (CNP) sequences (genomic DNA [CNPDNA], cDNA derived from mRNA [CNPcDNA], and sequence coding for 22 amino acids of the mature CNP [CNP22aa]) on the growth of primary porcine vascular cells. METHODS AND RESULTS: Gene transfer was performed with cationic lipid DOCSPER or linear polyethylenimine. All 3 CNP sequences led to significant inhibition of smooth muscle cell (SMC) proliferation. In contrast, significant stimulation of cell growth was observed in endothelial cells (ECs) using CNPDNA or CNPcDNA but not CNP22aa. In a porcine restenosis model, a significant reduction in neointima hyperplasia was found 3 months after application of the CNPcDNA vector compared with the control transfection. CONCLUSIONS: The results demonstrate that the first intron in the CNP sequence does not contain any additional enhancer-binding sites. However, the signal sequence is indispensable for secretion of CNP and its appropriate physiological function. Furthermore, the results show for the first time the therapeutic effect of CNP using liposome-mediated gene transfer and local adventitial delivery. Advantages of the CNP gene are its dual effects with inhibition of SMC proliferation and simultaneous promotion of EC growth. Functional analysis of various C-type natriuretic peptide (CNP) sequences on growth of vascular cells. For the first time, dual therapeutic effects of CNP with inhibition of smooth muscle cell proliferation and stimulation of re-endothelialization were demonstrated in a pig restenosis model using liposome-mediated gene transfer and local adventitial delivery.

Angioplasty, Balloon↗

PubMed2004

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer.

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.

Animals↗

PubMed2004

Targeted nucleic acid delivery into tumors: new avenues for cancer therapy.

Unique properties of tumors, such as abnormalities in the cell cycle and apoptosis, migration and metastasis, neoangiogenesis or unique antigen profiles are targets for therapeutic anti-cancer strategies. Beyond the selection of such strategies, additional specificity for the targeted tumor tissue can be accomplished in cancer gene therapy in several ways. Upon systemic administration, appropriately packaged therapeutic nucleic acid may be preferentially transported into the tumor tissue (targeted delivery); formulation can mediate the intracellular uptake of the nucleic acid into the nucleus of target cells only (transductional targeting); and/or the use of specific promotor/enhancer elements can restrict transcription of therapeutic genes to the target cells only (transcriptional targeting). Options for physical and biological targeting of nucleic acid formulations into tumors and therapeutic approaches are reviewed.

Animals↗

PubMed2004

Strategies to improve DNA polyplexes for in vivo gene transfer: will "artificial viruses" be the answer?

For the purpose of introducing nucleic acids into cells, cationic polymers have been steadily improved as gene carriers. This has resulted in improved polymer-based gene transfer formulations, termed polyplexes, which efficiently transfect cell cultures and also have shown encouraging gene transfer potential in in vivo administration. Targeted delivery to liver, lung, tumor, or other tissues has been achieved in experimental animals by localized or systemic application. Therapeutic effect has been demonstrated, although efficiencies are still too low to justify clinical use. The limitations of first-generation polymeric carriers (modest activity and significant toxicity) have been addressed by developments of new biodegradable polycations, incorporation of targeting and intracellular transport functions, and polyplex formulations that avoid unspecific adverse interactions with the host. A key future step will be the development of polyplexes into artificial viruses, with virus-like entry functions presented by smart polymers and polymer conjugates. These polymers have to sense their biologic microenvironment, respond in a more dynamic manner to alterations in pH, ionic or redox environment, undergoing programmed structural changes compatible with the different gene delivery steps.

Animals↗

PubMed2004

Stabilized nonviral formulations for the delivery of MCP-1 gene into cells of the vasculoendothelial system.

PURPOSE: The purpose of this study was to develop a stabilized non-viral gene transfer system for the efficient delivery and expression of monocyte chemoattractant protein 1 (MCP-1) gene in cells of the vasculoendothelial system. METHODS: Plasmid DNA was condensed with polyethylenimine (PEI), conjugates of PEI with polyethylene glycol (PEG), and PEI conjugates with the membrane-active peptide melittin. Surface charge and particle size of the resulting gene transfer particles were analyzed by laser light scattering. Reporter gene studies and toxicity assays were conducted on smooth muscle cells and endothelial cells of human, porcine, or rat origin. RESULTS: Nonviral gene carriers containing PEI and PEG were developed that could be produced in batches of several milligrams and conveniently stored as frozen samples. Incorporation of PEG into the transfection complex significantly reduced cellular toxicity. The cryoconserved gene transfer particles mediated high expression of luciferase, enhanced green fluorescent protein (EGFP), or secreted alkaline phosphatase reporter genes. Highest reporter gene expression was achieved with PEI polyplexes containing PEG and melittin. The gene for MCP-1 was efficiently delivered into target cells and resulted in expression of up to 125 ng/ml secreted bioactive MCP-1 protein per 50,000 cells. CONCLUSIONS: Gene carriers based on PEI and PEG display reduced toxicity, can be stored in frozen form without loss of biological activity, and can efficiently transfect cells of the vasculoendothelial system. Such gene carriers hold a potential for use in arterial gene transfer and local secretion of MCP-1 as trigger of therapeutic arteriogenesis in arterial occlusion diseases.

Animals↗

PubMed2004

Photochemically enhanced gene delivery of EGF receptor-targeted DNA polyplexes.

Epidermal growth factor receptor (EGFR) targeted DNA polyplexes, containing polyethylenimine (PEI) conjugated with EGF protein as cell-binding ligand for endocytosis and polyethylene glycol (PEG) for masking the polyplex surface charge, mediated a 3- to 30-fold higher luciferase gene expression in HUH7, HepG2 and A431 cell transfections than analogous untargeted PEG-PEI polyplexes. Transfection levels can be further enhanced by treatment of cells with amphiphilic photosensitizers followed by illumination. In this process photosensitizers localized in membranes of endocytic vesicles are activated by light, resulting in the destruction of endocytic membrane structures and releasing co-endocytosed polyplexes into the cell cytosol. Photochemical enhanced gene expression was observed in all cell lines, with the magnitude of enhancement depending on the particular PEI polyplex formulation and cell line, ranging between 2- and 600-fold. Importantly, improved gene transfer retained EGF receptor specificity, as demonstrated by comparison with ligand-free polyplexes and by receptor antibody or ligand competition experiments. These results suggest that this combined procedure enables a dual mode of targeting polyplexes: biological targeting via EGFR interaction, combined with physical targeting with light to direct a photochemical delivery of therapeutic genes to a desired location.

Animals↗

PubMed2003

Tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes.

Surface-shielded DNA delivery systems have been synthesized with virus-like characteristics that target gene expression into distant tumor tissues. Polyethylenimine (PEI)/DNA complexes ('polyplexes') conjugated with the cell-binding ligand transferrin (Tf) or epidermal growth factor (EGF) were used to achieve receptor-mediated endocytosis. The surface charge of the complexes was masked by covalently linking PEI to polyethylene glycol (PEG). Three alternatives for generating these surface-shielded formulations were utilized, attaching ligand and PEG molecules to PEI either before or after DNA complex formation. The stabilized formulations could be ultra-concentrated, stored frozen, and applied systemically after thawing. Intravenous injection of Tf-PEG-coated polyplexes resulted in gene transfer to subcutaneous Neuro2a neuroblastoma tumors of syngeneic A/J mice; EGF-PEG-coated polyplexes were intravenously applied for targeting human hepatocellular carcinoma xenografts in SCID mice. In these models, luciferase marker gene expression levels in tumor tissues were 10- to 100-fold higher than in other organ tissues. Repeated systemic application of Tf-PEG-PEI/DNA complexes encoding tumor necrosis factor alpha (TNF-alpha) into tumor-bearing mice induced tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origin (Neuro2a, M-3 or B16 melanoma).

Animals↗

PubMed2003

Nanoparticles bearing polyethyleneglycol-coupled transferrin as gene carriers: preparation and in vitro evaluation.

The aims of this work were to determine the stability of pDNA against various conditions during microencapsulation, prepare transferrin (TF)-conjugated PEGylated polycyanoacrylate nanoparticles (TF-PEG-nanoparticles), and assess its physicochemical characteristics and in vitro targeting cells association. The open circular forms of pDNA obviously increased when pDNA was emulsified into organic solution under sonification. When pDNA solution (pH 7.0) contained 1, 3 or 5% (w/v) PVA, after sonification, average 48.2, 59.4 and 62.1% of double-supercoiled DNA (dsDNA) were preserved, respectively. When medium of pDNA was 0.9% NaCl (pH 7.0), 0.1M NaHCO(3) (pH 8.0) or phosphate buffer (pH 8.0), average 53.1, 69.3 and 56.9% of dsDNA remained after sonification, respectively. Poly(aminopoly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate) (poly(H(2)NPEGCA-co-HDCA)) showed a slight influence on pDNA in 0.1M NaHCO(3) (pH 8.0) when its concentration increased from 0.5 to 4% (w/v). TF-PEG-nanoparticles loading pDNA were spherical in shape with size under 200nm and entrapment efficiency 35-50%. 0.1M NaHCO(3) with 3% PVA (w/v) could largely reduce the damage of pDNA during microencapsulation. TF-PEG-nanoparticles bore 1-3% of the total PEG chains conjugated to TF molecules, and exhibited the burst effect with over 30% drug release within 1 day. After the first phase, pDNA release profiles displayed a sustained release. The amount of cumulated pDNA release over 7 days was: 86.3, 81.5 and 74.4% for 1, 2 and 4% polymer nanoparticles, respectively. The degree of target K562 cell binding of TF-PEG-nanoparticles was greater than that of non-targeted PEG-nanoparticles at 4 degrees C. The presence of free TF decreased significantly the degree of cell binding of TF-PEG-nanoparticles, which revealed that the binding of TF-PEG-nanoparticles to K562 cells was indeed receptor specific. These results suggested that TF-PEG-nanoparticles were useful for delivery of pDNA to target cells.

Buffers↗

PubMed2003

Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 - new vectors for in vitro and in vivo delivery.

BACKGROUND: Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells. RESULTS: We describe a series of retrofitting plasmids and a protocol for in vivo loxP/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an oriP/EBNA-1 cassette and pRetroNeoOE carries G418 resistance and oriP/EBNA-1. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice in vivo. We also show that a BAC carrying GFP, oriP and EBNA-1 can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks. CONCLUSION: The pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or oriP/EBNA-1 using in vivo expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle in vivo. OriP/EBNA-1 allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks.

Animals↗

PubMed2003

[Rehabilitation of rheumatic diseases].

Rehabilitation aims at restoration of function and integration of the disabled or chronically ill patient in his social environment. Cooperation of health professionals in a multidisciplinary rehabilitation team is required. Functional deficits are defined on the levels of impairment, activity, and participation. A rehabilitation concept tailored to the individual patient and the use of outcome measures are mandatory. The principles of rehabilitation of rheumatoid arthritis and osteoarthritis are outlined.

Activities of Daily Living↗

PubMed2003

Reactivation of the mitosis-promoting factor in postmitotic cardiomyocytes.

Cardiomyocytes cease to divide shortly after birth and an irreversible cell cycle arrest is evident accompanied by the downregulation of cyclin-dependent kinase activities. To get a better understanding of the cardiac cell cycle and its regulation, the effect of functional recovery of the mitosis-promoting factor (MPF) consisting of cyclin B1 and the cyclin-dependent kinase Cdc2 was assessed in primary cultures of postmitotic ventricular adult rat cardiomyocytes (ARC). Gene transfer into ARC was achieved using the adenovirus-enhanced transferrinfection system that was characterized by the absence of cytotoxic events. Simultaneous ectopic expression of wild-type versions of cyclin B1 and Cdc2 was sufficient to induce MPF activity. Reestablished MPF resulted in a mitotic phenotype, marked by an abnormal condensation of the nuclei, histone H3 phosphorylation and variable degree of decay of the contractile apparatus. Although a complete cell division was not observed, the results provided conclusive evidence that cell cycle-related events in postmitotic cardiomyocytes could be triggered by genetic intervention downstream of the G1/S checkpoint. This will be of importance to design novel strategies to overcome the proliferation arrest in adult cardiomyocytes.

Animals↗

PubMed2003

Technology evaluation: TNFerade, GenVec.

TNFerade is a new gene therapy drug under development by GenVec that employs a replication-deficient adenovector carrying the gene for human tumor necrosis factor (TNF)-alpha, regulated by a radiation-sensitive promoter. TNFerade is currently undergoing phase II trials for the potential treatment of cancer.

Animals↗

PubMed2002

Treatment of psoriatic arthritis and psoriasis vulgaris with the tumor necrosis factor inhibitor infliximab.

OBJECTIVE: The aim was to evaluate the efficacy and safety of multiple infusions with achimeric, anti-tumor necrosis factor (TNF)alpha monoclonal antibody (infliximab) in patients with psoriatic arthritis (PsA) and psoriasis vulgaris. METHODS: Over 22 weeks, nine patients with both active psoriasis and PsA received five infusions of 3 mg/kg infliximab. The endpoints included changes in the swollen and tender joints counts, American College of Rheumatology (ACR) preliminary criteria for improvement response rates 20, 50, and 70, and improvement in the psoriasis area and severity index (PASI). RESULTS: The swollen count (SJC) and tender joint count (TJC) fell from means of 5.33+/-2.22 and 17.80+/-4.21 to 1.44+/-1.09 and 9.77+/-0.92, respectively, by week 2 ( P=0.02, P=0.02). This benefit was sustained through week 22 (2.00+/-1.12/7.77+/-3.68, P=0.05/ P=0.002). The ACR 20/50/70 response was achieved in 89%/56%/22% of cases. The mean PASI score improved from 19.04+/-5.41 to 4.91+/-2.51 ( P=0.002). CONCLUSION: Multiple infusions of infliximab were effective and well tolerated in patients with active psoriasis and PsA.

Aged↗