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)

The present work utilizes electrospinning to fabricate synthetic polymer/DNA composite scaffolds for therapeutic application in gene delivery for tissue engineering. The scaffolds are non-woven, nano-fibered, membranous structures composed predominantly of poly(lactide-co-glycolide) (PLGA) random copolymer and a poly(D,L-lactide)-poly(ethylene glycol) (PLA-PEG) block copolymer. Release of plasmid DNA from the scaffolds was sustained over a 20-day study period, with maximum release occurring at approximately 2 h. Cumulative release profiles indicated amounts released were approximately 68-80% of the initially loaded DNA. Variations in the PLGA to PLA-PEG block copolymer ratio vastly affected the overall structural morphology, as well as both the rate and efficiency of DNA release. Results indicated that DNA released directly from these electrospun scaffolds was indeed intact, capable of cellular transfection, and successfully encoded the protein beta-galactosidase. When tested under tensile loads, the electrospun polymer/DNA composite scaffolds exhibited tensile moduli of approximately 35 MPa, with approximately 45% strain initially. These values approximate those of skin and cartilage. Taken together, this work represents the first successful demonstration of plasmid DNA incorporation into a polymer scaffold using electrospinning.
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PMID:Development of a nanostructured DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers. 1271 56

The epithelium of the epididymal tubule has different biological functions in different regions of the tubule. Each region is further organized into lobules or intra-regional segments surrounded by connective tissue septa (CTS). Epididymal segmentation has received little direct attention, yet there is considerable evidence that expression of mRNA and protein often begins or ends precisely at the CTS border of a segment. How such 'on-off' regulation occurs coincident with the passing of the tubule from one segment to the next is unknown. This study examined the segmentation of epididymides in rats and mice. The average adult Sprague-Dawley rat and C57BL/6 mouse caput, corpus and cauda epididymides has seven, two and four, and three, one and two segments, respectively. The apoptosis response of the caput epididymal epithelium to deprivation of lumicrine factors 24 h after efferent duct ligation in rats and the epididymal expression of a marker protein, beta-galactosidase, in mice were segmented precisely. This validated both at a general response and at a specific protein level that many epididymal functions are regulated within segments. Blue dextran (molecular weight 20000) and erythrocine red (molecular weight 880) dyes infused into the interstitial space of specific segments by micropuncture were retained by the CTS of the segments. In similar micropuncture experiments, [(3)H]H(2)O (molecular weight 18) was able to diffuse into an adjacent segment relatively freely whereas [(14)C]polyethylene glycol (molecular weight 4000) could not. These studies indicate that the interstitium of intra-regional segments is organized into different physiological compartments and that these compartments play a role in regulating the epididymal epithelium.
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PMID:Association of segmentation of the epididymal interstitium with segmented tubule function in rats and mice. 1277 10

Novel carbohydrate-based agents for the stabilization of ternary liposome:mu:DNA (LMD) nonviral vector systems are described. LMD vector systems comprise plasmid DNA (pDNA; D,7.5 kb) expressing a reporter gene (in this instance beta-galactosidase expressing gene) that is precondensed with the adenoviral core peptide mu (mu, M; MRRAHHRRRRASHRRMRGG) and then further packaged by means of DC-Chol:DOPE (3:2; m/m) cationic liposomes. Final optimized lipid:mu:pDNA ratio is typically 12:0.6:1 (w/w/w). We report the synthesis of a series of nine neoglycolipids prepared by coupling completely unprotected sugar monomers or oligomers (mannose, glucose, galactose, glucuronic acid, maltose, lactose, maltotriose, maltotetraose, and maltoheptaose) through their reducing-residue termini to an aminoxy-functionalized cholesterol-based lipid. Characterization of these novel neoglycolipids by (1)H NMR reveals that the coupling reaction has a major configurational preference for the beta-anomer. Unusually, even mannose coupling results in a neoglycolipid product with a predominantly beta-anomeric conformation (>85%). Formulation of neoglycolipids into LMD vector systems by incubation of LMD particles with neoglycolipid micelles results in the formation of a range of potential stabilized-LMD (sLMD) vector systems. Those potential sLMD systems prepared with longer chain neoglycolipids are found to have enhanced stabilities, with respect to aggregation in high ionic strength buffers, and enhanced transfection efficacies in comparison to the transfection properties of the naked first generation LMD vector system (i.e., gene delivery and expression). By contrast, when LMD vector systems are incubated with poly(ethylene glycol) DSPE-PEG micelles, resulting PEG-LMD vector systems are very stable with respect to colloidal instablility and aggregation in high ionic strength buffers and in serum, but are completely refractory to transfection. These data suggest that oligosaccharides could represent an alternative to PEG as a stealth polymer able to stabilize synthetic nonviral vector systems in some fluids but without impairing transfection efficiency. Furthermore, sLMD systems prepared with longer chain neoglycolipids appear to have sufficient useful characteristics to form the basis of viable second-generation LMD vector systems after further development.
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PMID:Synthesis and formulation of neoglycolipids for the functionalization of liposomes and lipoplexes. 1312 91

DNA can be compacted using polyethylene glycol-substituted poly-L-lysine into discrete unimolecular (with respect to DNA) nanoparticles with minor diameter < 20 nm that are stable in normal saline for at least 23 months at 4 degrees C. We compared the activity of firefly luciferase in lungs of C57BL/6 mice that received 100 microg compacted plasmid in 25 microl saline (shown to be the optimal dose) via intratracheal or intranasal instillation with levels in animals given 100 microg naked plasmid or in untreated mice. Mice dosed with compacted DNA nanoparticles had peak activity of luciferase in lung at 2 days postinstillation, which declined in log-linear fashion with a half-life of 1.4 days. Luciferase activity in animals dosed with naked DNA was 200-fold less. Addition of polyethylene glycol to the complex was necessary for efficient gene transfer and animals that received DNA compacted with unmodified poly-L-lysine did not exhibit luciferase activity above background. Immunohistochemical staining for bacterial beta-galactosidase 2 days after administration of a compacted lacZ expression plasmid (n = 8) revealed expression predominantly in the dependent portions of the right lungs of mice, in alveolar and airway epithelial cells, though macrophages and sometimes endothelial cells also were transfected. No staining for beta-galactosidase was observed in uninjected animals (n = 4) or those dosed with naked lacZ plasmid (n = 7). Tissue survey for transgene expression shows expression only in lung and trachea following intranasal administration. Stable compacted DNA nanoparticles transfer exogenous genes to airway epithelium and show promise for lung gene therapy.
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PMID:Transfection of airway epithelium by stable PEGylated poly-L-lysine DNA nanoparticles in vivo. 1466 96

Oral induction of a disseminated mucosal immune response with polyplex-based DNA vaccines requires the delivery of intact polyplexes (polyelectrolyte complexes formed by self-assembly of plasmid DNA with a cationic polymer) to subepithelial lymphoid tissue (e.g. Peyer's patches) within the gastrointestinal tract. This work describes the formulation of a microparticle polyplex carrier allowing the potential of this approach to be realised. PEGylated PEI/DNA polyplexes (DNA concentration 20 microg/ml) formed at N/P 5:0 (defined as the ratio of polycation amino groups to DNA phosphates) were stable to salt-induced aggregation and could be concentrated to a final DNA concentration of 1 mg/ml without polyplex size increase. Polyplexes containing 1:1 polyethylene glycol (PEG)/polyethylenimine (PEI) ratio (mass/mass) gave similar levels of luciferase gene expression in B16F10 cells compared to non-PEG complexes. Poly-(D,L-lactide-co-glycolide) (PLGA) microparticles containing PEGylated polyplexes (approximately 17% DNA encapsulation efficiency) were formulated using a modified double emulsion solvent evaporation method. The microencapsulation and release of intact polyplexes from the microparticle carrier was demonstrated using polyanion (heparin sulfate and poly(aspartic acid) (PAA)) displacement techniques and electron microscopy. Microparticles containing PEGylated polyplexes (24 microg beta-galactosidase DNA) were given orally to Wistar rats. Significant transgene expression (compared to background) was found in peripheral tissue (spleen) 72 h after administration. This work demonstrates the potential application of microparticle carriers for mucosal polyplex-based vaccination.
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PMID:Formulation of a microparticle carrier for oral polyplex-based DNA vaccines. 1537 19

Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic.
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PMID:PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile. 1564 65

We have developed a novel polyethylenimine (PEI)-DNA vector formulation that is capable of efficient tumor-specific delivery after intravenous administration to nude mice. To further increase the specificity of delivery, we have attached the peptide CNGRC to the vector, which is specific for aminopeptidase N (CD13). The strategy for coupling this peptide to PEI was based on a novel method involving the strong affinity between phenyl(di)boronic acid (PDBA) and salicylhydroxamic acid (SHA) as well as a polyethylene glycol (PEG) linker to reduce steric hindrance between the vector and the peptide. In vitro assessment of targeting by the CNGRC/PEG/PEI/DNA vector carrying a beta-galactosidase (beta-Gal)-expressing plasmid showed as much as a 5-fold increase in transduction, relative to the untargeted PEG/PEI/DNA-betagal vector, of CD13-positive lung cancer, fibrosarcoma, bladder cancer, and human umbilical vein endothelial cells. Competition with free peptide resulted in up to a 90% reduction in delivery, indicating that gene delivery was specific for CD13-positive cells. Intravenous administration of the CNGRC/PEG/PEI/DNA-betagal vector to nude mice bearing subcutaneous tumors resulted in as much as a 12-fold increase in beta-Gal expression in tumors as compared with expression in either lungs or tumors from animals treated with the original PEI/DNA-betagal vector. In vivo transduction analysis using the CNGRC/PEG/PEI/DNA vector to target the intravenous delivery of a yellow fluorescence protein (YFP)-expressing plasmid to subcutaneous H1299 tumors confirmed delivery of YFP to both tumor cells and tumor endothelial cells. The use of this peptide to further increase tumor-specific delivery mediated by our novel PEI/DNA vector now provides a basis for developing tumor-targeted gene therapies for use in the clinical treatment of cancer.
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PMID:Tumor-specific gene delivery mediated by a novel peptide-polyethylenimine-DNA polyplex targeting aminopeptidase N/CD13. 1570 89

Extracellular and intracellular barriers typically prevent non-viral gene vectors from having an effective transfection efficiency. Formulation of a gene delivery vehicle that can overcome the barriers is a key step for successful tissue regeneration. We have developed a novel core-shelled DNA nanoparticle by invoking solvent-induced condensation of plasmid DNA (beta-galactosidase or GFP) in a solvent mixture [94% N,N-dimethylformamide (DMF) + 6% 1x TE buffer] and subsequent encapsulation of the condensed DNA globule in a triblock copolymer, polylactide-poly(ethylene glycol)-polylactide (L8E78L8), in the same solvent environment. The polylactide shell protects the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a nanofibrous non-woven scaffold using the same solution mixture. The bioactive plasmid DNA can then be released in an intact form from the scaffold with a controlled release rate and transfect cells in vitro.
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PMID:In vitro non-viral gene delivery with nanofibrous scaffolds. 1626 20

The cationic lipid mediated uptake of plasmid DNA by cells in monolayer culture was significantly enhanced with an aqueous solution of the block copolymer poly(p-dioxanone-co-L-lactide)-b-poly(ethylene glycol) (PPDO/PLLA-b-PEG). Plasmid uptake studies with DNA encoding the beta-galactosidase gene and cytotoxicity evaluations were performed on MCF-7, NIH 3T3 and CT-26 cell lines. Transfection yields and time courses for maximum release of FITC labeled DNA in MCF-7 cells were observed and quantified by beta-galactosidase assay and spectrofluorometry, respectively. The reported results suggest that the studied block copolymer might be useful for the enhancement of polycation mediated transfection and could find application in gene therapy.
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PMID:Novel block copolymer (PPDO/PLLA-b-PEG): enhancement of DNA uptake and cell transfection. 1670 79

Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.
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PMID:Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes. 1698 30


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