Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trials of gene transfer for cystic fibrosis (CF) are currently underway. However, direct application to the airways may be impeded by the presence of airway secretions. We have therefore assessed the effect of CF sputum on the expression of the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE in a number of cell lines in vitro. Transfection was markedly inhibited in the presence of sputum; the effect was concentration dependent and was only partially ameliorated by removal of sputum with phosphate-buffered saline (PBS) washing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 micrograms/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant alginase (0.5 U/ml) significantly (P < 0.005) improved gene transfer. Adenovirus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfection significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrated similar inhibition to that observed with sputum and was also ameliorated by pre-treatment of DNA-covered cells with rhDNase. In a separate series of experiments performed in the absence of added sputum or genomic DNA, increasing concentrations of rhDNase resulted in a concentration-related decline in transfection efficiency. However, even at the highest concentration (500 micrograms/ml of rhDNase), transfection efficiency remained more than 50% of control. Thus, pre-treatment of CF airways with rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.
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PMID:The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro. 953 69

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).
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PMID:A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy. 963 34

A new DNA delivery vector (the terplex system) based on a balanced hydrophobicity and net surface charge between stearyl-poly(L-lysine), low density lipoprotein (LDL), and genetic material (i.e. plasmid DNA or antisense oligonucleotide) was developed. The pSV-beta-gal plasmid in terplex system showed a 2-5-fold increase in beta-galactosidase expression on murine smooth muscle cells (A7R5) compared to Lipofectin. Delivery of unmodified c-myb antisense oligonucleotide to A7R5 cells was also facilitated significantly by the terplex system, requiring as little as 5.4 nM of antisense oligonucleotide to achieve a 50% antiproliferative effect. Similar antiproliferative effect was observed when the c-myb antisense/terplex formulation was tested on CCD-32 Lu human lung fibroblasts. Characterization of the physical properties of the terplex system was performed using various techniques. Plasmid DNA was condensed by addition of stearyl-PLL and LDL, resulting in the terplex system of about 100 nm in diameter as shown by atomic force microscopy. A strong hydrophobic interaction between stearyl-poly(L-lysine) and LDL was registered by 1H-NMR spectrometry, showing a significant decrease in the epsilon-methylene signal of poly(L-lysine) backbone when stearyl-poly(L-lysine) was mixed with LDL; however, this phenomenon was not observed with unmodified poly(L-lysine). Agarose gel electrophoresis revealed that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-poly(L-lysine), indicating that the surface charge of the terplex system became more positive by addition of stearyl-poly(L-lysine). Zeta-potential measurement showed that the terplex system exerted a slightly positive charge (+2 mV) at a 1:1:1 weight ratio of plasmid DNA:LDL:stearyl-poly(L-lysine). The obtained results will be utilized in the design of more efficient and safer DNA delivery vectors for in vivo gene therapy.
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PMID:A new non-viral DNA delivery vector: the terplex system. 974 25

DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.
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PMID:Polycation-DNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids. 974 37

We have investigated the nuclear transport of the replacement histone H1(0) and have searched for its nuclear localization sequence (NLS). The lysine-rich H1(0) histone differs from the other H1 histones with respect to its mode of expression and to the processing of the respective mRNA. Using the digitonin-permeabilized cell import assay we demonstrate that H1(0) is transported into the nucleus in an energy- and temperature-dependent manner. In competition experiments we show that the transport of H1(0) from the cytoplasm into the nucleus is competed by the SV40 T-antigen-NLS-peptide coupled to HSA, an established substrate of the importin pathway. In transfection studies we have expressed in HeLa cells a series of plasmid constructs containing different fragments of the coding region of the H1(0) histone gene that were fused to the beta-galactosidase gene, and we have determined the subcellular localization of each fusion protein. The results show that H1(0) contains multiple transport-competent sequence elements that can function as NLS and that H1(0) meets the requirements for a transport into the nucleus by an importin-dependent pathway.
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PMID:The histone H1(0) contains multiple sequence elements for nuclear targeting. 977 Mar 63

The "Fab immunogene" is a novel gene transfer vehicle in which the Fab fragment of anti-human epidermal growth factor (EGF) receptor antibody B4G7 is conjugated with poly-L-lysine to form an affinity complex with DNA. It was developed to target delivery of therapeutic genes into EGF receptor-hyperproducing tumor cells. Various characteristic features of the immunogene have been documented (Chen et al., 1998). Here we add further evidence to prove that in vitro transfer of beta-galactosidase/Fab immunogene is exclusively to EGF receptor-positive cells and that the herpes simplex virus thymidine kinase (TK)/Fab immunogene induces substantial suicide effects on A431 tumor cells when treated together with ganciclovir. The in vivo specificity of the immunogene transfer was examined using A431 tumor-bearing nude mice. When these nude mice were injected intraperitoneally with the chloramphenicol acetyltransferase (CAT)/Fab immunogene, CAT DNA was detected in the tumors as well as in liver and kidney but not brain, whereas CAT mRNA and enzyme activity were detected only in the tumors. Local and intraperitoneal injection of the TK/Fab immunogene and subsequent administration of ganciclovir effectively suppressed the growth of A431 tumors transplanted on the backs of nude mice. These observations suggest a possible application of the Fab immunogene system in cancer gene therapy.
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PMID:Targeted in vivo delivery of therapeutic gene into experimental squamous cell carcinomas using anti-epidermal growth factor receptor antibody: immunogene approach. 987 65

We previously developed the "immunogene" approach toward cancer gene therapy using epidermal growth factor receptor (EGFR)-mediated endocytosis. Here, we describe an improved immunogene system, in which the antigen-binding (Fab) fragments of the monoclonal antibody (Ab) B4G7 against the human EGFR were conjugated with poly-L-lysine to form a gene delivery vehicle (designated Fab "immunoporter"). Within 12 hours, the beta-galactosidase beta-gal) gene was transferred via the Fab immunoporter to virtually all of the nuclei of human squamous carcinoma A431 cells that overproduce the EGFR, and the beta-gal enzyme activity was detected within 24 hours and retained for more than 3 days. The beta-gal gene was not transferred into human and mouse cells that were deficient in EGFRs, but it was delivered if those mouse cells were transformed with human EGFR genes. Beta-gal gene transfer via the Fab immunoporter was inhibited by pretreatment with excess amounts of the Fab fragment. The transfer efficiency of the beta-gal gene to A431 cells via the Fab immunoporter was approximately 2%, which is as high as the lipofection method and 20- to 100-fold higher than the whole Ab immunoporter. The transfer of the herpes simplex virus thymidine kinase gene into A431 tumor cells as a form of the thymidine kinase/Fab immunogene was successful, and subsequent treatment with ganciclovir induced remarkable suicide effects which conferred 1000-fold higher drug sensitivity. Thus, the Fab immunogene was substantially improved with regard to the whole Ab immunogene and could be used as a potent gene transfer vehicle for the in vivo targeting of EGFR-hyperproducing tumor cells.
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PMID:Receptor-mediated gene delivery using the Fab fragments of anti-epidermal growth factor receptor antibodies: improved immunogene approach. 991 90

Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells. We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 microgram/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes: (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency. Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of foreign genes and inhibition of specific nucleases may facilitate transfection.
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PMID:Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor. 993 Mar 26

Gene transfer to provide long-term expression of a therapeutic product, without introducing unwelcome genetic information, is a goal being sought for therapy of both hereditary and acquired diseases. Polyoma virus pseudocapsids, generated from a VP1-expressing recombinant baculovirus, lack viral DNA and have been successfully used to introduce small exogenous genes stably into cells in vitro by a process designated 'pseudofection'; although pseudocapsids protect only about 3 kbp of exogenous DNA, low efficiency transfer of a larger fragment (6.2 kbp) has been observed. Here, expression of a 7.2 kbp plasmid (pCMV beta) encoding the beta-galactosidase gene was assessed to monitor not only efficiency, but the ability of pseudocapsids to transfer larger-sized DNA on their own, or in the presence of the polycation, poly-L-lysine, added to protect nonencapsidated DNA. When complexed to pseudocapsids only, the efficiency of expression of the transferred beta-galactosidase gene (in human or rodent cells), although low, appeared to stabilise with time. In the presence of polylysine, unencapsidated DNA was shown to be protected against DNase activity, but electron microscopy (EM) revealed the formation of large mixed aggregates. The addition of pseudocapsids to these aggregates, and measurement of mobilities of the complexes in CsCl equilibrum centrifugation, indicated that they contained negligible amounts of VP1. For subsequent pseudofection experiments, DNA was complexed first with pseudocapsids, then polylysine was added. The latter did not appear to displace pseudocapsids from DNA, and was found to increase the efficiency of short-term expression both in in vitro and in vivo experiments. Gene expression, analysed histochemically or by the polymerase chain reaction, revealed transcriptional activity of the input gene, with expression first diminishing, then stabilising over time. The presence of pseudocapsids, in complexes with DNA with or without polylysine, allowed for stable and persistent gene expression.
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PMID:Enhancement by polylysine of transient, but not stable, expression of genes carried into cells by polyoma VP1 pseudocapsids. 993 Mar 47

Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-L-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-L-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-L-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-L-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a beta-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-L-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-L-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-L-lysine.
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PMID:Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo. 998 33


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