RESUMEN
A phase-I trial to assess the safety and tolerability of human interleukin-12 (IL-12) plasmid (phIL-12) formulated with a synthetic lipopolymer, polyethyleneglycol-polyethyleneimine-cholesterol (PPC), was conducted on women with chemotherapy-resistant recurrent ovarian cancer. A total of 13 patients were enrolled in four dose-escalating cohorts and treated with 0.6, 3, 12 or 24 mg m(-2) of the formulated plasmid once every week for 4 weeks. Administration of phIL-12/PPC was generally safe and well-tolerated. Common side effects included low-grade fever and abdominal pain. Stable disease and reduction in serum CA-125 levels were clinically observed in some patients. Measurable levels of IL-12 plasmid were detectable in PF samples collected throughout the course of phIL-12/PPC treatment. In comparison, serum samples either did not contain detectable amounts of plasmid DNA or contained <1% of the amount found in the corresponding PF samples. Treatment-related increases in IFN-gamma levels were observed in PF but not in serum. These data demonstrate that IL-12 gene delivery with a synthetic delivery system is feasible for ovarian cancer patients.
Asunto(s)
Adenocarcinoma Papilar/terapia , Carcinoma Endometrioide/terapia , Terapia Genética/métodos , Interleucina-12/genética , Recurrencia Local de Neoplasia/terapia , Neoplasias Ováricas/terapia , Adulto , Anciano , Antígeno Ca-125/sangre , Colesterol/uso terapéutico , Femenino , Terapia Genética/efectos adversos , Humanos , Interferón gamma/sangre , Persona de Mediana Edad , Plásmidos/genética , Polietilenglicoles/uso terapéutico , Polietileneimina/uso terapéuticoRESUMEN
BACKGROUND: Systemic administration of non-viral gene therapy provides better access to tumors than local administration. Development of a promoter that restricts expression of cytotoxic proteins to the tumor vasculature will increase the safety of the system by minimizing expression in the non-dividing endothelial cells of the vasculature of non-target tissues. METHODS: Cell cycle promoters were tested for selective expression in dividing cells vs. non-dividing cells in vitro and promoter strength was compared to the cytomegalovirus (CMV) promoter. Successful promoter candidates were tested in vivo using two proliferating endothelium mouse models. Ovarectomized mice were injected with estradiol prior to lipoplex administration and expression levels were measured in the lungs and uterus 4 days after administration. The second model was a subcutaneous tumor model and expression levels were measured in the lungs and tumors. For both animal models, expression levels from the proliferating endothelium promoter were compared to that obtained from a CMV promoter. RESULTS: The results showed that the Cdc6 promoter yielded higher expression in proliferating vs. non-proliferating cells. Secondly, promoter strength could be selectively increased in endothelial cells by the addition of a multimerized endothelin enhancer (ET) to the Cdc6 promoter. Thirdly, comparison of expression levels in the lungs vs. uterus in the ovarectomized mouse model and lungs vs. tumor in the mouse tumor model showed expression was much higher in the uterus and the tumor than in the lungs for the ET/Cdc6 promoter, and expression levels were comparable to that of the CMV promoter in the hypervascularized tissues. CONCLUSIONS: These results demonstrate that the combination of the endothelin enhancer with the Cdc6 promoter yields selective expression in proliferating endothelium and can be used to express cytotoxic proteins to treat vascularized tumors.
Asunto(s)
Células Endoteliales/fisiología , Elementos de Facilitación Genéticos , Terapia Genética/métodos , Vectores Genéticos , Regiones Promotoras Genéticas , Animales , Bovinos , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , Células Endoteliales/metabolismo , Endotelinas/genética , Femenino , Expresión Génica , Humanos , Ratones , Células 3T3 NIH , Proteínas Nucleares/genética , Transfección , TrasplantesRESUMEN
The purpose of this research is to develop ligand-targeted plasmid based gene delivery systems for gene transfer to tumor endothelium. Cell adhesion assays were used to test the peptide inhibition of human endothelial cell adsorption to vitronectin-treated tissue culture plates. A series of RGD containing peptides were tested in linear form and with one and two disulfide bonds. The linear and two disulfide bond peptides yielded similar IC50 (approximately 1 x 10(-7) M). Substitution of two methionines for cysteines yielded a single disulfide bond that increased the IC50 by 10-fold. The single and double disulfide peptides were derivatized to N-succinyl-dioleoylphopsphatidylethanolamine and incorporated into 100 nm liposomes radiolabeled with H-cholesterylhexadecylether. Liposome uptake by human umbilical vein endothelial cells was tested as a function of lipopeptide surface density. Increase in membrane surface density from 5 to 20mol% increased human umbilical derived endothelial cell (HUVEC) uptake of the liposomes for both the single and double disulfide peptides. Liposome uptake by HUVECs was 3-fold greater for the double disulfide compared to the single disulfide. The single and double disulfide lipopeptides were then tested for gene transfer to HUVECs using DOTMA:Cholesterol cationic liposomes. The polyplexes were formed by rapidly mixing plasmid DNA with DOTMA:CHOL liposomes at a 3:1 charge ratio in 2% ethanol, 10% lactose. The ethanol was removed by lyophilization and upon rehydration, the lipoplexes had a mean diameter of approximately 100nm. HUVEC transfection studies showed that increasing the mol% of the single disulfide RGD lipopeptide to 20mol% increased gene transfer by 10-fold. This increase in transfection could be reduced to that obtained in the absence of lipopeptide by co-incubating the HUVECs with a 100-fold excess of the single disulfide RGD peptide, thus demonstrating lipopeptide mediated gene transfer to endothelial cells.
Asunto(s)
Colesterol , Células Endoteliales/metabolismo , Técnicas de Transferencia de Gen , Oligopéptidos/química , Péptidos/química , Compuestos de Amonio Cuaternario , Cationes , Adhesión Celular , Disulfuros/química , Células Endoteliales/fisiología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Integrina alfaVbeta3/biosíntesis , Ligandos , Liposomas , Luciferasas/biosíntesis , Luciferasas/genética , Oligopéptidos/metabolismo , Plásmidos , Transfección , Venas Umbilicales/citología , Venas Umbilicales/metabolismoRESUMEN
The impact of a localized application of ultrasound on gene transfer to primary tumors following systemic administration of cationic lipid based transfection complexes was investigated. We have previously shown that systemic administration of DOTMA (N-[(1-(2-3-dioleyloxy) propyl)]-N-N-N-trimethylammonium chloride):cholesterol-based transfection complexes to tumor-bearing mice resulted in expression in the tumor and other tissues, primarily the lungs. Application of ultrasound to the tumor before or after the injection resulted in a significant increase in gene transfer to the tumor with no increase observed in other tissues. The magnitude of increased expression ranged from three- to 270-fold depending upon the DNA dose. The following parameters were optimized for maximal increase: duration of ultrasound application, the time interval between plasmid injection and sonoporation, and plasmid dose. A combination of plasmid quantitation and fluorescence microscopy showed that ultrasound increased tumor uptake of the plasmid and that uptake was limited to the tumor vasculature. Using an IL- 12 expression plasmid, the combination of a single plasmid dose (10 microg) and ultrasound treatment produced significantly higher levels of IL-12 in tumor. This increased expression was sufficient to inhibit tumor growth compared with the control conditions. These data demonstrate the potential application of sonoporation as an effective method for enhancing the expression of systemically administered genes in tumor endothelium for cancer gene therapy.
Asunto(s)
Carcinoma de Células Escamosas/terapia , Terapia Genética/métodos , Interleucina-12/genética , Neoplasias Experimentales/terapia , Transfección/métodos , Ultrasonografía , Animales , Carcinoma de Células Escamosas/inmunología , Endotelio/inmunología , Femenino , Expresión Génica , Interleucina-12/análisis , Liposomas , Ratones , Ratones Endogámicos C3H , Microscopía Fluorescente , Neoplasias Experimentales/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisisRESUMEN
A cationic lipid-based gene delivery system composed of N-[(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride and cholesterol, at a 4:1 molar ratio, was developed for systemic administration. Plasmid biodistribution and expression were characterized in syngeneic mouse tumor model squamous cell carcinoma VII cells. A reporter gene expression plasmid was used for biodistribution of plasmid and expression. The results showed that lungs and primary tumors were transfected. Fluorescence microscopy showed that fluorescent-labeled transfection complexes were passively targeted to the tumor vasculature and that the endothelial cells internalized the plasmid. Transgene expression was characterized based on duration of expression and dosing schedule. In vivo gene transfer with an interleukin-12 expression plasmid yielded protein levels in blood, lungs, and primary tumor after intravenous administration. Efficacy studies showed that 15 microg of interleukin-12 plasmid was sufficient to produce a gene-specific inhibition of primary tumor growth. These results characterize the vascularity of the tumor model, characterize the in vivo gene transfer properties of the plasmid-based gene delivery system, and show that the transgene expression level was sufficient to elicit a biological response by inhibiting tumor growth.
Asunto(s)
Carcinoma de Células Escamosas/terapia , Terapia Genética , Lípidos/administración & dosificación , Animales , Secuencia de Bases , Carcinoma de Células Escamosas/patología , Cationes , División Celular , Cartilla de ADN , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C3H , Plásmidos , TransfecciónRESUMEN
The success of gene therapy relies on the ability of gene delivery systems to selectively deliver therapeutic genes to a sufficient number of target cells yielding expression levels that impact the diseased state. The gene delivery systems can be divided into two classes: viral and nonviral (or plasmid DNA-based). The present gene delivery technology being used in clinics today can be considered first generation, in that they possess the ability to transfect or infect target cells through their inherent chemical, biochemical, and molecular biological properties. Relying on these sole properties, however, limits therapeutic applications. Expansion of therapeutic applications or increased effectiveness of current therapies can be achieved by increasing the number of cells and cell types susceptible to gene transfer. This can be achieved through physical targeting and molecular biological targeting. Physical targeting relies on the attachment to the delivery vehicle of ligands that bind to cell surface receptors unique to the target cells. Molecular biological targeting refers to selective expression of the therapeutic gene by the target cell through the use of selective promoters. Selective expression can be further achieved by the use of expression systems controlled by extrinsic induction molecules. This review will describe in detail the advances that have been made in each of these areas of gene targeting.
Asunto(s)
Técnicas de Transferencia de Gen , Animales , Terapia Genética , Vectores Genéticos , HumanosRESUMEN
PURPOSE: To design, synthesize, and test synthetic glycopeptide-based delivery systems for gene targeting to hepatocytes by systemic administration. METHODS: All peptides were synthesized by the solid phase method developed using Fmoc chemistry on a peptide synthesizer. The binding of galactosylated peptides to HepG2 cells and accessibility of the galactose residues on particle surface was demonstrated by a competition assay using 125I-labeled asialoorosomucoid and RCA lectin agglutination assay, respectively. DNA plasmid encoding chloramphenicol acetyl transferase (CAT) gene was complexed with a tri-galactosylated peptide (GM245.3) or tri-galactosylated lipopeptide (GM246.3) in the presence of an endosomolytic peptide (GM225.1) or endosomolytic lipopeptide (GM227.3) to obtain DNA particles of 100-150 nm in size. The plasmid/peptide complexes were added to HepG2 cell cultures or intravenously administered by tail vein injection into normal mice or rats. Plasmid uptake and expression was quantified by qPCR and ELISA, respectively. RESULTS: Multiple antennary glycopeptides that have the ability to condense and deliver DNA plasmid to hepatocytes were synthesized and complexed with DNA plasmid to obtain colloidally stable DNA/peptide complexes. Addition of DNA/GM245.3/GM225.1 peptide complexes (1:3:1 (-/+/-)) to HepG2 cell cultures yielded CAT expression in transfected cells. The transfection efficiency was significantly reduced in the absence of galactose ligand or removal of endosomolytic peptide. Intravenous administration of DNA/GM245.3 peptide complexes (1:0.5 (-/+)) into the tail vein of normal rats yielded DNA uptake in the liver. Substitution of GM245.3 by galactosylated lipopeptide GM246.3 resulted in more stable DNA particles, and a 10-fold enhancement in liver plasmid uptake. CAT expression was detectable in liver following intravenous administration of DNA/GM246.3 complexes. Addition of endosomolytic lipopeptide GM227.3 into the complexes (DNA/ GM246.3/GM227.3 (1:0.5:1 (-/+/-))) yielded a 5-fold increase in CAT expression. Liver expression was 8-fold and 40-fold higher than lung and spleen, respectively, and localized in the hepatocytes only. The transfection efficiency in liver was enhanced by increasing DNA dose and injection volume. The plasmid uptake and expression in liver using DNA/GM246.3/GM227.3 complexes was 100-200-fold higher than DNA formulated in glucose. Tissue examination and serum biochemistry did not show any adverse effect of the DNA/GM246.3/ GM227.3 (1:0.5:1 (-/+/-)) complexes after intravenous delivery. CONCLUSIONS: Gene targeting to hepatocytes was achieved by systemic administration of a well-tolerated synthetic glycopeptide-based delivery system. The transfection efficiency of this glycopeptide delivery system was dependent on peptide structure, endosomolytic activity, colloidal particle stability, and injection volume.
Asunto(s)
Marcación de Gen , Glicopéptidos/química , Hígado/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/genética , Expresión Génica , Glicopéptidos/síntesis química , Glicopéptidos/genética , Hígado/citología , Ratones , Datos de Secuencia Molecular , Plásmidos , Ratas , Ratas Sprague-DawleyRESUMEN
Intravenous (i.v.) administration of cationic lipid N-[( 1-(2-3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride (DOTMA)-based transfection complexes in mice with subcutaneous squamous cell tumors yielded plasmid delivery and expression in tumor lesions. The efficiency of gene transfer in tumors was significantly lower than in the lung. This was consistent with low plasmid levels associated with the tumor, suggesting that plasmid delivery to the tumor site was a limiting factor. Lowering the lipid/DNA charge ratio from 5:1 to 0.8:1 (+/-) did not change DNA levels in tumor but significantly reduced DNA levels in lung. However, expression levels were significantly reduced in both tissues at lower lipid/DNA charge ratios. Complexes prepared from small unilamellar liposomes gave significantly lower expression levels in the lungs but similar expression levels in tumors when compared to complexes prepared from larger unilamellar liposomes. The small liposome complexes were better tolerated than large liposome complexes. Varying the cationic lipid to colipid (cholesterol or DOPE) molar ratio from 4: 1 to 1: 1 significantly reduced expression levels in both tumor and lung. Cationic lipid substitution, using a cholesterol cationic lipid, diethyldiamino-carbamyl-cholesterol instead of DOTMA, produced reduced expression in all other tissues except tumor. Incorporation of PEG into preformed transfection complexes reduced DNA delivery to lung, increased circulation half-life, and enhanced DNA delivery to tumor. In a lung metastatic mouse tumor model, where the accessibility of the i.v. administered transfection complexes to tumor lesions should be less challenging, DOTMA: CHOL complexes (4: 1 lipid to colipid molar ratio, 3: 1 +/- lipid to plasmid charge ratio) were preferentially localized in tumor lesions. These data demonstrate that systemic gene transfer to distal tumor sites by lipid/ DNA complexes may be limited by low plasmid delivery. Modifying the chemical surface properties of transfection complexes enhanced both DNA delivery and expression in tumor and is one approach that may overcome limitations.
Asunto(s)
Carcinoma de Células Escamosas/terapia , ADN/administración & dosificación , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Neoplasias Pulmonares/terapia , Neoplasias Cutáneas/terapia , Animales , Carcinoma de Células Escamosas/secundario , Cationes , ADN/química , ADN/farmacocinética , Cartilla de ADN , Portadores de Fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunohistoquímica , Inyecciones Intravenosas , Liposomas , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos C3H , Reacción en Cadena de la Polimerasa , Neoplasias Cutáneas/patologíaRESUMEN
PURPOSE: A plasmid-based gene expression system was complexed with protective, interactive, and non-condensing (PINC) polymer system and administered with Medi-Jector, a needle-free injection device (NFID), to achieve high and sustained levels of antigen-specific antibodies in blood circulation. METHODS: Human growth hormone (hGH) or bacterial beta-galactosidase gene expression plasmids driven by a cytomegalovirus (CMV) promoter were formulated in saline or complexed with a PINC polymer, polyvinylpyrrolidone (PVP), and intramuscularly or subcutaneously administered into dogs and pigs using a 22-gauge needle or a NFID. The hGH-specific IgG titers in serum were measured by an ELISA. Beta-galactosidase expression was measured in injected muscles by an enzymatic assay or immunohistochemistry. The effect of NFID on DNA stability and topology was assessed by gel electrophoresis. RESULTS: Intramuscular (i.m.) or subcutaneous (s.c.) injection of a hGH expression plasmid pCMV-hGH (0.05-0.5 mg/kg) in dogs and pigs elicited antigen-specific IgG antibody titers to expressed hGH. With both routes of injection, pDNA delivery by a NFID was superior to pDNA injection by needle. The magnitude of hGH-specific IgG titers with NFID was 15-20-fold higher than needle injection when pDNA was complexed with PVP, and only 3-4-fold higher with pDNA in saline. The transfection efficiency in the injected muscle, as measured by beta-galactosidase expression, following i.m. injection of pCMV-betagalactosidase/PVP, was not significantly different between needle and NFID-injected groups. CONCLUSIONS: These data demonstrate that the combination of pDNA/ PVP complexes and a NFID act synergistically to achieve high and sustained levels of antigen-specific IgG response to expressed antigen. This gene delivery approach may offer advantage over needle injection of naked DNA for the development of genetic vaccines.
Asunto(s)
Plásmidos , Povidona , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Animales , Perros , Femenino , Vectores Genéticos/administración & dosificación , Hormona de Crecimiento Humana/administración & dosificación , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/inmunología , Humanos , Inmunoglobulina G/inmunología , Inyecciones a Chorro , Plásmidos/genética , Polímeros/administración & dosificación , Povidona/administración & dosificación , Povidona/química , Porcinos , Vacunas de ADN/inmunología , beta-Galactosidasa/biosíntesisRESUMEN
The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.
Asunto(s)
Expresión Génica/genética , Técnicas de Transferencia de Gen , Metabolismo de los Lípidos , Plásmidos/metabolismo , Animales , Carbohidratos/farmacología , Cloranfenicol O-Acetiltransferasa/genética , Factor IX/genética , Hormona de Crecimiento Humana/genética , Inyecciones Intravenosas , Liposomas/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos , Microscopía Fluorescente , Plásmidos/genética , Factores de TiempoRESUMEN
We have developed protective interactive noncondensing (PINC) polymers, such as poly(N-vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA), to protect plasmids from extracellular nuclease degradation while allowing the flexible complex to diffuse throughout the muscle tissue. Molecular modeling, zeta potential modulation, and ethidium bromide intercalation studies were performed to assess the mechanism of interaction between PVP and plasmid. The effect of salt concentration, pH, and polymer-plasmid ratios were investigated. We have correlated these variables with beta-galactosidase (beta-gal) expression after intramuscular administration to rats. PVP can form hydrogen bonds with the base pairs within the major groove of DNA at pH 4.0. The PVP-plasmid interaction results in a complex that is more hydrophobic (less negatively charged) than the uncomplexed plasmid due to the vinyl backbone of PVP. Up to a ten-fold enhancement in gene expression in rat muscle over the use of 'naked' DNA has been demonstrated using these systems. A linear structure-activity relationship (SAR) was found between the percent vinyl pyrrolidone monomer content in poly (vinyl pyrrolidone-covinyl acetate) polymers and beta-gal expression in muscle. Modulation of the interaction between PINC polymers and plasmid directly impacts the levels of gene expression in vivo. The linear SAR is being used to design novel PINC polymers with enhanced binding affinity to plasmids.
Asunto(s)
Músculo Esquelético/metabolismo , Plásmidos , Alcohol Polivinílico/farmacología , Povidona/farmacología , Animales , Etidio , Expresión Génica , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Masculino , Modelos Moleculares , Ratas , Ratas Endogámicas F344 , beta-Galactosidasa/metabolismoRESUMEN
A muscle-specific gene medicine is described that provides for long-term secretion of biologically active human growth hormone (hGH) from skeletal muscle into the systemic circulation. The hGH gene medicine is composed of a muscle-specific hGH plasmid expression system complexed with a protective, interactive, non-condensing (PINC) delivery system. The muscle-specific gene expression system, pSK-hGH-GH, was constructed by linking the promoter/enhancer regions of chicken skeletal alpha-actin to hGH gene. C2C12 myoblast transfection with pSK-hGH-GH resulted in the synthesis of hGH in a muscle-specific manner. Direct injection into rat tibialis cranialis muscle of pSK-hGH-GH complexed with a polymeric PINC delivery system, polyvinylpyrrolidone (PVP), produced hGH levels in muscle that were 10- to 15-fold higher compared with plasmid formulated in saline at 14 days post-injection. Intratracheal instillation in rat lung of pSK-hGH-GH did not produce significantly detectable levels of hGH. In hypophysectomized rats, a single intramuscular dose of the pSK-hGH-GH/PVP complex resulted in hGH expression and a subsequent increase in serum levels of rat IGF-I and growth. hGH expression and effects on rat serum IGF-I levels were detectable up to 28 days after injection of formulated plasmid and effects on growth were detectable unto 21 days. Anti-hGH antibodies were detectable in serum at 14 days post-injection, reached a plateau at 21 days, and remained elevated through the study period. Cyclosporin treatment of the pSK-hGH-GH/PVP-injected animals completely inhibited the antibody response and resulted in increased hGH expression.
Asunto(s)
Terapia Genética , Hormona del Crecimiento/genética , Músculo Esquelético/metabolismo , Actinas/genética , Animales , Anticuerpos/inmunología , Pollos , Ciclosporina , Sistemas de Liberación de Medicamentos , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos , Hormona del Crecimiento/administración & dosificación , Hormona del Crecimiento/biosíntesis , Hormona del Crecimiento/inmunología , Humanos , Hipofisectomía , Inyecciones Intramusculares , Especificidad de Órganos , Plásmidos/administración & dosificación , Polímeros , Ratas , Ratas Sprague-DawleyRESUMEN
Recent evidence has shown that insulin-like growth factor-I (IGF-I) plays an important role in the development, maintenance, and regeneration of peripheral nerves and skeletal muscle. IGF-I offers the potential to treat neuromuscular diseases in humans. We have developed a nonviral gene therapy method to express and produce localized and sustained therapeutic levels of IGF-I within target muscles by intramuscular injection of formulated plasmids. The purpose of the present study was to demonstrate that intramuscular injection of a plasmid encoding human IGF-I (hIGF-I) and engineered to restrict expression to skeletal muscle produces sustained local concentrations of biologically active hIGF-I. Normal rats received a single intramuscular injection of plasmids formulated as a complex with polyvinylpyrrolidone (PVP). Results show that hIGF-I mRNA and hIGF-I protein were detectable in the injected muscles for the duration of the study (28 days), whereas the hIGF-I protein was not detected in blood. Biological activity of hIGF-I was determined by immunodetection of a nerve-specific growth-associated protein, GAP-43, an indicator of motor neuron sprouting. Placement of human growth hormone (hGH) 3' untranslated region enhanced GAP-43 staining, probably due to improved secretion of hIGF-I. Enhanced immunoreactivity of GAP-43 was observed in muscles injected with the formulated hIGF-I plasmid when compared to controls. These results demonstrate that intramuscular injection of hIGF-I plasmid formulated as a complex with PVP produces a localized and sustained level of biologically active hIGF-I.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , Factor I del Crecimiento Similar a la Insulina/genética , Plásmidos , Animales , Línea Celular , Expresión Génica , Humanos , Inyecciones Intramusculares , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Masculino , Ratones , Plásmidos/administración & dosificación , Ratas , Ratas Endogámicas F344RESUMEN
PURPOSE: DNA plasmids (pDNA) can be taken up by and expressed in striated muscle after direct intramuscular injection. We have developed interactive polymeric gene delivery systems that increase pDNA bioavailability to muscle cells by both protecting pDNA from nucleases and controlling the dispersion and retention of pDNA in muscle tissue. METHODS: A DNA plasmid, containing a CMV promoter and a galactosidase reporter gene (CMV-beta-gal), was injected either in saline or formulated in polyvinyl pyrrolidone (PVP) and polyvinyl alcohol (PVA) solutions. Interactions between PVP and pDNA were assessed by dynamic dialysis, Isothermal Titration Calorimetry (ITC), and Fourier Transformed Infra Red (FT-IR) spectroscopy. Formulations (50 mu l) were injected into rat tibialis muscles after surgical exposure. Immunohistochemistry for beta-gal was used to visualize the sites of expression in muscle. RESULTS: Beta-gal expression using pDNA in saline reached a plateau while beta-gal expression using PVP formulations increased linearly in the dose range studied (12.5-150 mu g pDNA injected) and resulted in an increase in the number and distribution of cells expressing beta-gal. The interaction between PVP and pDNA was found to be an endothermic process governed largely by hydrogen-bonding and results in protection of pDNA from extracellular nucleases. CONCLUSIONS: Significant enhancement of gene expression using interactive polyvinyl-based delivery systems has been observed. The improved tissue dispersion and cellular uptake of pDNA using polyvinyl-based systems after direct injection into muscle is possibly due to osmotic effects.
Asunto(s)
ADN Recombinante/administración & dosificación , Marcación de Gen , Músculo Esquelético/metabolismo , Alcohol Polivinílico/química , Povidona/química , Animales , Portadores de Fármacos , Enlace de Hidrógeno , Inmunohistoquímica , Masculino , Músculo Esquelético/enzimología , Plásmidos , Ratas , Ratas Endogámicas F344 , Espectroscopía Infrarroja por Transformada de Fourier , beta-Galactosidasa/metabolismoRESUMEN
OBJECTIVES: The objectives of this study were to determine whether epidermal growth factor increases intracellular calcium and phosphoinositide turnover in human myometrial cells by a tyrosine kinase-mediated mechanism, to evaluate an obligatory role for arachidonic acid metabolites in these actions, and to compare the actions of epidermal growth factor and oxytocin. STUDY DESIGN: Intracellular calcium and phosphoinositide turnover were measured in a myometrial cell line after stimulation with epidermal growth factor (0.1 to 100 nmol/L) or oxytocin (20 nmol/L). The effects of nifedipine, thapsigargin, genestein and tyrphostin, the guanosine triphosphate binding protein antagonist GPA-7, indomethacin, and nordihydroguaiaretic acid were determined. Data were analyzed by analysis of variance and Duncan's multiple-range test. RESULTS: Epidermal growth factor stimulated phosphoinositide turnover and increased intracellular calcium in a dose-dependent manner (median effective concentration 2.6 nmol/L). In contrast to oxytocin, the effects of epidermal growth factor were inhibited by tyrosine kinase inhibitors but not by GPA-7. Indomethacin and nordihydroguaiaretic acid did not inhibit the epidermal growth factor-stimulated increase in intracellular calcium. CONCLUSIONS: The acute epidermal growth factor-stimulated increase in intracellular calcium in this myometrial cell line is primarily derived from release of calcium from intracellular stores, and it involves the activation of a tyrosine kinase, presumably the epidermal growth factor receptor. Arachidonic acid metabolites are not obligatory intermediates. Oxytocin increases phosphoinositide turnover and intracellular calcium by a distinctly different pathway.
Asunto(s)
Ácido Araquidónico/metabolismo , Calcio/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Miometrio/metabolismo , Fosfatidilinositoles/metabolismo , Secuencia de Aminoácidos , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Línea Celular Transformada , Inhibidores Enzimáticos/farmacología , Femenino , Proteínas de Unión al GTP/antagonistas & inhibidores , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Miometrio/citología , Miometrio/efectos de los fármacos , Oligopéptidos/farmacología , Oxitocina/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Terpenos/farmacología , TapsigarginaRESUMEN
Oxytocin has been identified in both non-human primate and human corpora lutea of the menstrual cycle by RIA, immunocytochemistry and HPLC. Evidence for the transcription of the oxytocin gene in this tissue using PCR is available. Oxytocin receptors have been characterized by biochemical procedures. However, there is some debate as to whether the oxytocin identified in these tissues is biologically active and has a role in luteal function. In this study we have demonstrated that oxytocin isolated by gel chromatography of tissue extracts from the baboon and the human corpus luteum is biologically active as determined in a rat uterine bioassay. Since both oxytocin and its receptors are present in these tissues, it is suggested that oxytocin in the human and non-human primate corpora lutea has a functional role.
Asunto(s)
Cuerpo Lúteo/metabolismo , Oxitocina/metabolismo , Papio/metabolismo , Animales , Bioensayo , Cromatografía en Gel , Cuerpo Lúteo/química , Femenino , Hormonas/farmacología , Humanos , Oxitocina/análisis , Oxitocina/farmacología , Ratas , Ratas Sprague-Dawley , Contracción Uterina/efectos de los fármacosRESUMEN
Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activity. Ca(2+)-activated K+ channels (KCa) play a role in the modulation of uterine contractions and myometrial Ca2+ homeostasis and have been implicated in the control of smooth muscle excitability. We now show that relaxin stimulates KCa channels in cell-attached patches in a cell line derived from term pregnant human myometrium. This effect was prevented by the protein kinase A (PKA) antagonist, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). After patch excision, the channel was activated by PKA and inhibited by alkaline phosphatase. These data suggest that relaxin may promote myometrial quiescence in part by stimulation of KCa channels via a PKA-mediated mechanism.
Asunto(s)
Calcio/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Miometrio/metabolismo , Canales de Potasio/metabolismo , Relaxina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Conductividad Eléctrica , Femenino , Humanos , Miometrio/citología , Canales de Potasio/efectos de los fármacos , Embarazo , Relaxina/antagonistas & inhibidores , Tionucleótidos/farmacologíaRESUMEN
Oxytocin stimulates phosphoinositide turnover in myometrium. To elucidate whether the coupling mechanism involves the interaction of oxytocin receptor with GTP-binding proteins, we examined oxytocin stimulation of guanosine triphosphatase (GTPase) activity and phospholipase-C activity in rat and human myometrial membranes. Oxytocin consistently stimulated both GTPase and phospholipase-C activities, and both stimulations were attenuated by an antibody directed against the carboxyl-terminals of the GTP-binding proteins, G alpha q and G alpha 11. Neutralization of the antibody by preincubation with antigenic peptide reversed this inhibition. [Thr4,Gly7]oxytocin, a specific oxytocin receptor agonist, stimulated both GTPase and phospholipase-C activities, and the stimulations were also inhibited by anti-G alpha q/11 IgG. Immunoreactive GTP-binding proteins, G alpha q and G alpha 11, and phospholipase-C beta 3 isoforms were present in myometrial membranes. These results indicate that stimulation of phospholipase-C activity by oxytocin in myometrium is mediated via G alpha q, G alpha 11, or a closely related GTP-binding protein, probably coupling to phospholipase-C beta.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/fisiología , Miometrio/enzimología , Oxitocina/farmacología , Fosfolipasas de Tipo C/metabolismo , Animales , Anticuerpos/farmacología , Membrana Celular/enzimología , Femenino , Proteínas de Unión al GTP/inmunología , Humanos , Inmunoglobulina G/farmacología , Oxitocina/agonistas , Oxitocina/análogos & derivados , Ratas , Receptores de Oxitocina/fisiologíaRESUMEN
Oxytocin increases myometrial intracellular free calcium by promotion of calcium entry and release of calcium from intracellular stores. Calcium release from intracellular stores is secondary to an increase in phosphoinositide (PI) turnover and generation of IP3. We have explored the biochemical basis for the coupling of oxytocin (OT) to phospholipase C (PLC). Rat myometrial membranes contain PLC beta, gamma, and delta isoforms as well as the GTP-binding proteins G alpha(q) and G alpha(11). Oxytocin stimulates both GTPase and PLC activity in rat and human myometrial membranes. These data and available structural information suggest that the oxytocin receptor couples to PLC through a GTP-binding protein. In support of this hypothesis, an antibody generated against the specific C-terminal region of G alpha(q) and G alpha(11) inhibits both the oxytocin-stimulated GTPase and PLC activities. This inhibition is reversed by neutralization of the antibody with the antigenic peptide. The data indicate that the oxytocin receptor couples to PLC, presumably of the beta subclass, via interaction with proteins of the G alpha(q/11) subclass. In the nonpregnant, estrogen-primed rat, the stimulation of PI turnover by oxytocin is inhibited by the hormone relaxin and by pertussis toxin. The effects of both of these agents are mediated by the action of cAMP-dependent protein kinase. In plasma membranes, GTP-stimulated PLC activity can also be inhibited by treatment with protein kinase A. These data suggest that cAMP-dependent phosphorylation at a step involving GTP-binding protein/PLC coupling can exert a negative effect on the stimulation of IP3 formation by oxytocin and thereby affect contraction/relaxation in the myometrium.
Asunto(s)
Miometrio/metabolismo , Receptores de Oxitocina/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Calcio/metabolismo , Femenino , Proteínas de Unión al GTP/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Líquido Intracelular/metabolismo , Miometrio/efectos de los fármacos , Oxitocina/farmacología , Embarazo , Ratas , Receptores de Oxitocina/química , Fosfolipasas de Tipo C/químicaRESUMEN
Concentrations of Cu, Zn, Pb and Ni in the scalp hair of male and female donors, with an age range of 6-60 years, were determined by ICP atomic emission spectroscopy. The donors were drawn from the densely populated city of Lahore and the relatively less-populated capital city of Islamabad for comparative evaluation of the metal levels in relation to age, sex and location. Linear regression analyses and correlation between paired metals indicated a positive correlation between Cu and Zn for both sexes ( [Formula: see text] ) and between Pb and Ni ( [Formula: see text] ) for males and females of Lahore. Metal concentrations varied between the two cities and the two sexes. The highest mean concentration was found for Zn at 180.5 microg g(-1) for males and 202.4 microg g(-1) for females from Lahore, while for counterparts from Islamabad the values were 105.2 microg g(-1) and 206.6 microg g(-1). Copper showed an identical mean concentration (20.8 microg g(-1)) in the hair of both males and females from Lahore; however, relatively lower mean concentrations (7.7 and 10.8 microg g(-1)) were observed for donors from Islamabad. Mean Pb concentrations in hair of male donors from Lahore and Islamabad were 9.4 and 7.0 microg g(-1), respectively; in female groups the concentrations were 14.3 and 5.7 microg g(-1), respectively. Ni showed the lowest concentration range (4.3-4.5 microg g(-1)) of all the four metals for subjects from Lahore, and this was higher than the corresponding range (2.0-3.2 microg g(-1)) for subjects from Islamabad. The findings are discussed in relation to the available data from the literature.