RESUMEN
The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.
Asunto(s)
Terapia Genética/métodos , Enfermedades Pulmonares/terapia , Pulmón/metabolismo , Transfección/métodos , Resinas Acrílicas , Animales , Aniones , Expresión Génica , Ingeniería Genética , Liposomas , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos ICR , Microinyecciones , Polietileneimina , Células Tumorales Cultivadas , beta-Galactosidasa/genéticaRESUMEN
Growing endothelial cells at sites of angiogenesis may be more sensitive than quiescent endothelial cells to toxin-VEGF fusion proteins, because they express higher numbers of VEGF receptors. We have constructed, expressed and purified a protein containing the catalytic A-subunit of Shiga-like toxin I fused to VEGF(121) (SLT-VEGF/L). SLT-VEGF/L inhibits protein synthesis in a cell-free translation system and induces VEGFR-2 tyrosine autophosphorylation in cells overexpressing VEGFR-2 indicating that both SLT and VEGF moieties are properly folded in the fusion protein. SLT-VEGF/L selectively inhibits growth of porcine endothelial cells expressing 2-3x10(5) VEGFR-2/cell with an IC(50) of 0.1 nM, and rapidly induces apoptosis at concentrations >1 nM. Similar results are observed with human transformed embryonic kidney cells, 293, engineered to express 2.5x10(6) VEGFR-2/cell. In contrast, SLT-VEGF/L does not affect three different types of endothelial cells (PAE/KDR(low), HUVE, MS1) expressing between 5x10(3) and 5x10(4) VEGFR-2/cell, and quiescent endothelial cells overexpressing VEGFR-2. Growth inhibition and induction of apoptosis by SLT-VEGF/L require intrinsic N-glycosidase activity of the SLT moiety, but occur without significant inhibition of protein synthesis. The selective cytotoxicity of SLT-VEGF proteins against growing endothelial cells overexpressing VEGFR-2 suggests that they may be useful in targeting similar cells at sites of angiogenesis.
Asunto(s)
Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Linfocinas/farmacología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores de Factores de Crecimiento/biosíntesis , Toxina Shiga/farmacología , Animales , Western Blotting , Caspasa 6 , Caspasas/biosíntesis , Supervivencia Celular/efectos de los fármacos , ADN/administración & dosificación , ADN/genética , Factores de Crecimiento Endotelial/administración & dosificación , Endotelio Vascular/efectos de los fármacos , Humanos , Linfocinas/administración & dosificación , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacología , Toxina Shiga/administración & dosificación , Porcinos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: High levels of expression in hepatocytes can be achieved after intraportal delivery of plasmid DNA vectors with up to 10% of all liver cells transfected. CMV promoter-driven expression is very high on Day 1 after injection, but is diminished strongly by Day 2. Expression slowly declines after 1 week. We describe experiments aimed at elucidating the reasons for this rapid decline in transgene expression. METHODS: Histological methods were used to determine the presence and extent of liver damage and hepatocyte proliferation. Viral and liver-specific promoters were tested to study promoter shut-off, Southern blotting was performed to determine the loss of the pDNA vector over time, and several mouse models were used to study the host immunological response. RESULTS: pDNA is lost rapidly early after injection, but remains at a relatively stable copy number after Day 4. Southern blotting experiments showed that plasmid DNA could be detected for at least 12 weeks after injection (0.2 copies per genome). The early rapid decline of expression is promoter dependent. A liver-specific albumin promoter resulted in similar levels of expression on Days 1 and 7, suggesting that promoter inactivation may be responsible for the instability of CMV promoter-driven expression. The slow decline in expression levels after 1 week appears to be the result of an immune response directed against the expressed transgene. Expression was much prolonged in immunosuppressed, immunodeficient, or antigen-tolerized mice. CONCLUSION: The present data suggest that if promoter inactivation can be overcome, intravascular delivery of plasmid DNA could be a highly efficient, simple and non-toxic liver gene therapy approach. Intravascular delivery of pDNA allows for the rapid screening of novel expression vectors in vivo.
Asunto(s)
Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/genética , Hígado/metabolismo , Luciferasas/genética , Plásmidos/genética , Transfección/métodos , Animales , Southern Blotting , ADN/administración & dosificación , ADN/metabolismo , Inyecciones Intravenosas , Hígado/citología , Luciferasas/análisis , Ratones , Ratones Endogámicos , Factores de TiempoRESUMEN
We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of foreign gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 +/- 187 ng/g for the arm and 1692 +/- 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of beta-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable gene expression in human muscle without substantial toxicity.
Asunto(s)
ADN/genética , Gónadas/enzimología , Músculo Esquelético/enzimología , Plásmidos/genética , Animales , Arteria Femoral , Expresión Génica , Vectores Genéticos , Miembro Posterior , Inmunohistoquímica , Inyecciones Intraarteriales , Luciferasas/genética , Luciferasas/metabolismo , Macaca mulatta , Pruebas de Mutagenicidad , Regiones Promotoras Genéticas , Conejos , Ratas , Ratas Sprague-Dawley , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Following the initial demonstration that intramuscularly-injected naked plasmid DNA (pDNA) is expressed in myofibers, it was shown that pDNA can be used for vaccination purposes. More recent studies have indicated that naked pDNA can also achieve high levels of transgene expression in vivo. This efficiency of naked pDNA expression, especially via intravascular route, is truly astounding. In this prospective review, we examine the possible mechanisms of naked pDNA uptake. The possible mechanisms; (a) large membrane disruption, (b) small membrane pores, and (c) receptor-mediated endocytosis, are considered in turn. Some recent original laboratory data relevant to these hypotheses are also presented.
Asunto(s)
Técnicas de Transferencia de Gen , Plásmidos/metabolismo , Receptores de Superficie Celular/fisiología , Animales , Endocitosis , Inyecciones Intravenosas , Hígado/citología , Hígado/metabolismo , Plásmidos/administración & dosificación , Polielectrolitos , Polímeros/farmacologíaRESUMEN
DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.
Asunto(s)
Aniones/metabolismo , Cationes/metabolismo , ADN/química , ADN/metabolismo , Electrólitos/metabolismo , ADN/genética , ADN/aislamiento & purificación , Portadores de Fármacos , Floculación , Microscopía de Fuerza Atómica , Peso Molecular , Conformación de Ácido Nucleico , Plásmidos/química , Plásmidos/genética , Plásmidos/aislamiento & purificación , Plásmidos/metabolismo , Polilisina/análogos & derivados , Polilisina/metabolismo , Solubilidad , Ultracentrifugación , AguaRESUMEN
We have previously shown that the intramuscular injection of naked plasmid DNA enables foreign gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this technique required injection directly into the liver vessels (portal vein, hepatic vein, or bile duct) and occlusion of outflow. The present study now demonstrates that high levels of plasmid DNA expression in hepatocytes can be easily obtained by tail vein injections. The highest levels of expression are achieved by rapidly injecting the plasmid DNA in large volumes, approximately 2.5 ml. This technique has great potential for a wide variety of laboratory studies.
Asunto(s)
Expresión Génica , Hígado , Plásmidos , Animales , Femenino , Genes Reporteros , Humanos , Inyecciones Intravenosas , Hígado/citología , Luciferasas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , beta-Galactosidasa/genéticaRESUMEN
The assembly of DNA into compact particles that do not aggregate in physiologic salt solution occurs naturally in chromatin and viral particles but has been challenging to duplicate using artificial constructs. Cross-linking amino-containing polycations in the presence of DNA with bisimidoester cross-linker leads to the formation of caged DNA particles that are stable in salt solutions. This first demonstration of caged DNA provides insight into how natural condensation processes avoid aggregation and a promising avenue for developing nonviral gene therapy vectors.
Asunto(s)
ADN/química , Reactivos de Enlaces Cruzados , Ciclohexanos/química , ADN/genética , Indicadores y Reactivos , Microscopía Electrónica , Concentración Osmolar , Tamaño de la Partícula , Plásmidos/química , Plásmidos/genética , Poliaminas/química , Soluciones , Transfección , UltracentrifugaciónRESUMEN
A fluorescent method is proposed for assessing DNA condensation in aqueous solutions with variety of condensing agents. The technique is based on the effect of concentration-dependent self-quenching of covalently bound fluorophores upon DNA collapse. The method allows a more precise determination of charge equivalency in titration experiments with various polycations. The technique's ability to determine the number of DNA molecules that are condensed together in close proximity is under further investigation.
Asunto(s)
ADN Circular/química , Fluorescencia , Polilisina/farmacologíaRESUMEN
The self-assembly of supramolecular complexes of nucleic acids and polymers is of relevance to several biological processes including viral and chromatin formation as well as gene therapy vector design. We now show that template polymerization facilitates condensation of DNA into particles that are <150 nm in diameter. Inclusion of a poly(ethylene glycol)-containing monomer prevents aggregation of these particles. The DNA within the particles remains biologically active and can express foreign genes in cells. The formation or breakage of covalent bonds has until now not been employed to compact DNA into artificial particles.
Asunto(s)
ADN/química , Polietilenglicoles/química , Células 3T3 , Acrilatos , Animales , Antígenos Transformadores de Poliomavirus/genética , Sitios de Unión , Reactivos de Enlaces Cruzados , ADN/genética , ADN/ultraestructura , Dimerización , Genes Reporteros , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Microscopía Electrónica , Plásmidos/química , Plásmidos/genética , Plásmidos/ultraestructura , Proteínas Recombinantes/biosíntesis , Virus 40 de los Simios/genética , Moldes Genéticos , TransfecciónRESUMEN
Previous studies have demonstrated that muscle can take up and express naked DNA or RNA. This study demonstrates that the pDNA can be delivered to and expressed within skeletal muscle when injected rapidly, in a large volume and when all blood vessels leading into and out of the hindlimb are occluded. The additional use of collagenase, papaverine and ischemia raised expression moderately but was not critical. These results demonstrate that a nonviral method can lead to high levels of expression in the muscles of adult animals large than mice.
Asunto(s)
ADN/administración & dosificación , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Músculo Esquelético/metabolismo , Animales , Colagenasas/administración & dosificación , Expresión Génica , Inyecciones Intramusculares , Inyecciones Intravenosas , Isquemia , Músculo Esquelético/irrigación sanguínea , Papaverina/administración & dosificación , Plásmidos , Ratas , Ratas Sprague-DawleyRESUMEN
The nuclear entry of exogenous DNA in mammalian cells is critical for efficient gene transfer. A novel technique was developed for the covalent attachment of cationic peptides to double-stranded DNA using a cyclo-propapyrroloindole cross-linker. The attachment of the SV40 large T antigen nuclear localization signal peptide induced the nuclear accumulation of the conjugated DNA in digitonin-permeabilized cells via the classical pathway for the nuclear transport of karyophilic proteins. Increased nuclear uptake of the modified DNA, however, did not occur after it was microinjected into the cytoplasm of cultured cells. This demonstration that the covalent modification of DNA with a signal peptide alters its behavior and interaction with other cellular factors portends the potential of DNA vector chemistry to enhance the efficiency of cellular gene transfer.
Asunto(s)
Antígenos Transformadores de Poliomavirus/química , ADN/química , Vectores Genéticos/química , Señales de Clasificación de Proteína/química , Virus 40 de los Simios/inmunología , Núcleo Celular/metabolismo , Reactivos de Enlaces Cruzados/química , Ciclopropanos/química , ADN/genética , Desoxirribonucleasa I , Electroforesis en Gel de Agar , Colorantes Fluorescentes , Técnicas de Transferencia de Gen , Vectores Genéticos/metabolismo , Células HeLa/citología , Humanos , Indoles/químicaRESUMEN
A variety of reporter genes within plasmid constructs were injected into the afferent and efferent vessels of the liver in mice, rats, and dogs. Efficient plasmid expression was obtained following delivery via the portal vein, the hepatic vein, and the bile duct. The use of hyperosmotic injection solutions and occlusion of the blood outflow from the liver substantially increased the expression levels. Combining these surgical approaches with improved plasmid vectors enabled uncommonly high levels of foreign gene expression in which over 15 microg of luciferase protein/liver was produced in mice and over 50 microg in rats. Equally high levels of beta-galactosidase (beta-Gal) expression were obtained, in that over 5% of the hepatocytes had intense blue staining. Expression of luciferase or beta-Gal was evenly distributed in hepatocytes throughout the entire liver when either of the three routes were injected. Peri-acinar hepatocytes were preferentially transfected when the portal vein was injected in rats. These levels of foreign gene expression are among the highest levels obtained with nonviral vectors. Repetitive plasmid administration through the bile duct led to successive events of foreign gene expression. The integration of these findings into laboratory and clinical protocols is discussed.
Asunto(s)
Conductos Biliares/metabolismo , Expresión Génica , Vectores Genéticos , Venas Hepáticas/metabolismo , Hígado/metabolismo , Plásmidos/administración & dosificación , Vena Porta/metabolismo , Animales , Perros , Genes Reporteros , Humanos , Inyecciones , Luciferasas/genética , Ratones , Ratones Endogámicos ICR , Plásmidos/genética , Plásmidos/metabolismo , Ratas , Ratas Sprague-Dawley , beta-Galactosidasa/genéticaRESUMEN
Ternary complexes of plasmid DNA, histone H1 protein and amphipathic polyamines (PAPA) were able to mediate the efficient transfection of 3T3. HeLa and COS cells in culture. Using both the beta-galactosidase and luciferase reporter gene systems, the transfection efficiency of PAPA complexes was comparable to that of DOSPA/PE cationic liposomes, considered to be a highly-efficient transfection reagent. Using three different assays of cellular toxicity (propidium iodide, BCECF-AM and Trypan Blue), the PAPA complexes caused minimal cellular toxicity. These results indicate that PAPA complexes are useful transfection reagents for the study of gene expression and function in cultured cells.
Asunto(s)
Histonas/farmacología , Indicadores y Reactivos/farmacología , Poliaminas/farmacología , Transfección , Células 3T3 , Animales , Células COS , Supervivencia Celular/efectos de los fármacos , Genes Reporteros , Células HeLa , Humanos , RatonesRESUMEN
Transfection competent complexes were assembled using a three component system. The constituents of the basic system were plasmid DNA, cationic DNA binding protein (NLS-H1) and anionic liposomes (dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylserine (PS)). In contrast to cationic liposome/DNA binary complexes, all of the DNA in these ternary complexes was sensitive to DNase I degradation and ethidium bromide intercalation. Transmission electron microscopy revealed that these ternary complexes formed unique structures in which the DNA was located either on the outside of individual liposomes or bridging two or more liposomes. This provides evidence that plasmid DNA encapsulation is not essential for transfection competency.
Asunto(s)
ADN/farmacología , Técnicas de Transferencia de Gen , Terapia Genética , Histonas/química , Células 3T3 , Animales , ADN Superhelicoidal/farmacología , Portadores de Fármacos , Liposomas/química , Ratones , Microscopía Electrónica , Plásmidos/química , TransfecciónRESUMEN
Naked plasmid DNA in hypertonic solutions was injected intraportally in mice whose hepatic veins were transiently occluded. High levels of luciferase expression and beta-galactosidase expression in 1% of the hepatocytes throughout the entire liver were achieved using 100 micrograms of the respective plasmid vector. Two days after the intraportal injection of 100 micrograms of pCMVGH, the mean hGH serum concentration was 65 ng/ml +/- 26 (n = 7) which is approximately 50-fold above normal baseline levels. These unprecedented levels of foreign gene expression from naked plasmid DNA document the ability of parenchymal cells in vivo to take up naked DNA following intravascular delivery.
Asunto(s)
Factor de Crecimiento Epidérmico/genética , Técnicas de Transferencia de Gen , Hígado/metabolismo , Luciferasas/genética , beta-Galactosidasa/genética , Animales , ADN , Expresión Génica , Humanos , Inyecciones , Hígado/citología , Ratones , TransfecciónRESUMEN
We describe the use of cationic, pH-sensitive liposomes to mediate the efficient transfer of DNA into a variety of cells in culture. Cationic lipids, containing an amine with a pK within the physiologic range of 4.5 to 8, were synthesized and incorporated with dioleoylphosphatidylethanolamine into liposomes. Acid conditions promoted DNA-binding, DNA-incorporation, and DNA-induced fusion by these cationic, pH-sensitive liposomes. Transfection efficiency in cultured cells was dependent on endosomal acidification in a manner akin to acidic-induced endosomal release of viruses. These liposomes constitute a promising new class of reagents for gene therapy.
Asunto(s)
Vectores Genéticos , Liposomas , Macrólidos , Transfección/métodos , Células 3T3 , Animales , Antibacterianos/farmacología , Brefeldino A , Cationes , Cloroquina/farmacología , Ciclopentanos/farmacología , Endosomas/efectos de los fármacos , Concentración de Iones de Hidrógeno , RatonesRESUMEN
Nitroxyl free radical electron spin relaxation times for spin-labeled low-spin methemoglobins were measured between 6 and 120 K by two-pulse electron spin echo spectroscopy and by saturation recovery electron paramagnetic resonance (EPR). Spin-lattice relaxation times for cyano-methemoglobin and imidazole-methemoglobin were measured between 8 and 25 K by saturation recovery and between 4.2 and 20 K by electron spin echo. At low temperature the iron electron spin relaxation rates are slow relative to the iron-nitroxyl electron-electron spin-spin splitting. As temperature is increased, the relaxation rates for the Fe(III) become comparable to and then greater than the spin-spin splitting, which collapses the splitting in the continuous wave EPR spectra and causes an increase and then a decrease in the nitroxyl electron spin echo decay rate. Throughout the temperature range examined, interaction with the Fe(III) increases the spin lattice relaxation rate (1/T1) for the nitroxyl. The measured relaxation times for the Fe(III) were used to analyze the temperature-dependent changes in the spin echo decays and in the saturation recovery (T1) data for the interacting nitroxyl and to determine the interspin distance, r. The values of r for three spin-labeled methemoglobins were between 15 and 15.5 A, with good agreement between values obtained by electron spin echo and saturation recovery. Analysis of the nitroxyl spin echo and saturation recovery data also provides values of the iron relaxation rates at temperatures where the iron relaxation rates are too fast to measure directly by saturation recovery or electron spin echo spectroscopy. These results demonstrate the power of using time-domain EPR measurements to probe the distance between a slowly relaxing spin and a relatively rapidly relaxing metal in a protein.
Asunto(s)
Metahemoglobina/química , Conformación Proteica , Óxidos N-Cíclicos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Humanos , Matemática , Modelos Teóricos , Marcadores de Spin , TermodinámicaRESUMEN
In the present work it is shown that large unilamellar lecithin/cholesterol liposomes are able to sequester small negatively charged liposomes in the presence of divalent cations. Evidence is presented suggesting that the sequestration occurs via the formation of membrane invaginations transformed further into intraliposomal vesicles.
Asunto(s)
Calcio/fisiología , Liposomas/metabolismo , Ácido Edético/farmacologíaRESUMEN
During experimental CCl4 cirrhosis, an increase of membrane-associated factor stimulating 3T3 cell proliferation in vitro was observed. This stimulator is a 150-kD protein similar to one previously described. In situ perfusion released growth stimulatory activity, suggesting a peripheral plasma membrane protein localizing on basolateral surfaces. The activity increased with increasing number of CCl4 treatments, reaching a maximum at the 14th intoxication. It was faster than the proliferation of connective tissues determined histologically. Cessation of treatment caused a decrease in activity to that of the level of untreated liver, although the number of fibroblastlike cells remained large. This data, taken with the results of experiments with enriched hepatocyte fraction, may serve as an evidence in favor of hepatocyte origin of the factor. A factor inhibiting fibroblast proliferation was measured in detergent extracts from membranes, suggesting an integral membrane protein. The activity of the inhibitory factor increased in acute liver lesions, but at the stage of maximal fibrogenesis this factor is reduced to levels comparable to those of the intact liver. Therefore it is unlikely that this factor is involved in CCl4-induced fibrogenesis at the final stages. These factors may be common controls for various hepatic lesions causing fibrosis, both in clinical and experimental modeling.