RESUMO
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.
Assuntos
Terapia Genética/métodos , Pneumopatias/terapia , Pulmão/metabolismo , Transfecção/métodos , Resinas Acrílicas , Animais , Ânions , Expressão Gênica , Engenharia Genética , Lipossomos , Luciferases/genética , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microinjeções , Polietilenoimina , Células Tumorais Cultivadas , beta-Galactosidase/genéticaRESUMO
Non-viral DNA-containing particles represent a potentially attractive alternative to viruses for in vivo gene therapy applications. However, the inability to form small particles that remain stable and non-aggregating in vivo has limited their usefulness to date. The relatively simple self-assembling complexes that function so efficiently for in vitro gene delivery fail to provide similar gene delivery capabilities in vivo. Now for the first time, significant advances are being made into defining many of the in vivo barriers preventing stable particle formation. This improved understanding has been manifested into a rational design approach to DNA particle formation. This review highlights many of the recent studies in which rational design was employed in an effort to form polymer/DNA particles (polyplexes) that resist in vivo aggregation and inactivation.
Assuntos
Terapia Genética/métodos , Animais , DNA/administração & dosagem , DNA/química , DNA/genética , Sistemas de Liberação de Medicamentos , Estabilidade de Medicamentos , Humanos , Microesferas , Polímeros/administração & dosagem , Polímeros/químicaRESUMO
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.
Assuntos
Ânions/metabolismo , Cátions/metabolismo , DNA/química , DNA/metabolismo , Eletrólitos/metabolismo , DNA/genética , DNA/isolamento & purificação , Portadores de Fármacos , Floculação , Microscopia de Força Atômica , Peso Molecular , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Plasmídeos/metabolismo , Polilisina/análogos & derivados , Polilisina/metabolismo , Solubilidade , Ultracentrifugação , ÁguaRESUMO
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.
Assuntos
DNA/química , Reagentes de Ligações Cruzadas , Cicloexanos/química , DNA/genética , Indicadores e Reagentes , Microscopia Eletrônica , Concentração Osmolar , Tamanho da Partícula , Plasmídeos/química , Plasmídeos/genética , Poliaminas/química , Soluções , Transfecção , UltracentrifugaçãoRESUMO
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.
Assuntos
DNA Circular/química , Fluorescência , Polilisina/farmacologiaRESUMO
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.
Assuntos
DNA/química , Polietilenoglicóis/química , Células 3T3 , Acrilatos , Animais , Antígenos Transformantes de Poliomavirus/genética , Sítios de Ligação , Reagentes de Ligações Cruzadas , DNA/genética , DNA/ultraestrutura , Dimerização , Genes Reporter , Luciferases/biossíntese , Luciferases/genética , Camundongos , Microscopia Eletrônica , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/ultraestrutura , Proteínas Recombinantes/biossíntese , Vírus 40 dos Símios/genética , Moldes Genéticos , TransfecçãoRESUMO
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.
Assuntos
Antígenos Transformantes de Poliomavirus/química , DNA/química , Vetores Genéticos/química , Sinais Direcionadores de Proteínas/química , Vírus 40 dos Símios/imunologia , Núcleo Celular/metabolismo , Reagentes de Ligações Cruzadas/química , Ciclopropanos/química , DNA/genética , Desoxirribonuclease I , Eletroforese em Gel de Ágar , Corantes Fluorescentes , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Células HeLa/citologia , Humanos , Indóis/químicaRESUMO
DNA can enter intact mammalian nuclei with varying degrees of efficiency in both transfected and microinjected cells, yet very little is known about the mechanism by which it crosses the nuclear membrane. Nucleocytoplasmic transport of fluorescently labeled DNA was studied using a digitonin-permeabilized cell system. DNA accumulated in the nucleus with a punctate staining pattern in over 80% of the permeabilized HeLa cells. Nuclear localization of the labeled DNA was energy dependent and occurred through the nuclear pore, but did not require the addition of soluble cytoplasmic protein factors necessary for protein import.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Núcleo Celular/metabolismo , DNA/farmacocinética , Digitonina , Indicadores e Reagentes , Ligação Competitiva/fisiologia , Extratos Celulares/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Núcleo Celular/química , Citoplasma/química , DNA/química , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Corantes Fluorescentes/farmacocinética , Células HeLa , Humanos , Lectinas/farmacologia , Microinjeções , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/farmacocinética , Fosfoproteínas/química , Fosfoproteínas/farmacocinética , Ficocianina/farmacocinética , Transfecção , Xantenos/farmacocinéticaRESUMO
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.
Assuntos
Histonas/farmacologia , Indicadores e Reagentes/farmacologia , Poliaminas/farmacologia , Transfecção , Células 3T3 , Animais , Células COS , Sobrevivência Celular/efeitos dos fármacos , Genes Reporter , Células HeLa , Humanos , CamundongosRESUMO
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.
Assuntos
DNA/farmacologia , Técnicas de Transferência de Genes , Terapia Genética , Histonas/química , Células 3T3 , Animais , DNA Super-Helicoidal/farmacologia , Portadores de Fármacos , Lipossomos/química , Camundongos , Microscopia Eletrônica , Plasmídeos/química , TransfecçãoRESUMO
The GP1G gene codes for three of the four abundant androgen-regulated secretory proteins produced by the guinea pig seminal vesicle. Sequencing of the entire 6.3-kilobase gene and comparison with other mammalian seminal vesicle secretory protein genes reveals a common three-exon, two-intron organization. However, significant sequence similarity between this group of genes is largely limited to their 5'-flanking regions and first exons, which code almost exclusively for signal peptides in each case. The first intron of GP1G does contain a region with high similarity to the coding exon of a human seminal vesicle secretory protein gene, semenogelin II. The 3' half of the GP1G gene appears to share a common ancestry with the human SKALP/elafin gene. Sequences related to the elafin promoter, coding, untranslated regions, and introns are clearly identifiable within the GP1G sequence. The elafin gene codes for a serine protease inhibitor and is expressed in a variety of different human tissues. To determine if the GP1G gene was also active outside of the seminal vesicle, RNA from a variety of guinea pig tissues was hybridized to a GP1G cDNA probe. At least three novel RNA bands hybridizing to the GP1G probe were detected in testis RNA samples, and GP1G-related mRNAs were also found in other tissues. These data suggest that these seminal vesicle secretory proteins may have functional roles outside the reproductive system.
Assuntos
Evolução Biológica , Éxons , Proteínas/genética , Proteínas/metabolismo , Proteínas Secretadas pela Vesícula Seminal , Glândulas Seminais/enzimologia , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA , Cobaias , Humanos , Masculino , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The mechanism by which striated muscle internalizes plasmid DNA is unknown. A survey of nonnuclear membrane-associated DNA binding proteins from both skeletal and cardiac muscle revealed several sarcoplasmic reticulum restricted DNA binding species. 22P-DNA overlay and DNA-cellulose chromatography were used to identify membrane-associated DNA binding proteins that may mediate the uptake and expression of plasmid DNA by striated muscle. A total membrane vesicle fraction prepared from rabbit skeletal muscle contained 95-, 60-, and 28-kDa proteins that bound double-strand DNA specifically and with high affinity. The DNA binding proteins appear to originate from the sarcoplasmic reticulum because of their absence in purified sarcolemma vesicles and codistribution with several sarcoplasmic reticulum markers after subcellular fractionation. Several distinguishing biochemical features as well as cross-reactivity with triadin-specific antibodies indicated that the 95- and 60-kDa DNA binding proteins are triadin or proteolytic fragments of triadin, respectively. The role of these sarcoplasmic reticulum proteins in the transport of plasmid DNA is discussed.
Assuntos
Proteínas de Ligação a DNA/análise , Técnicas de Transferência de Genes , Músculo Esquelético/química , Animais , Ligação Competitiva , Membrana Celular/química , Cromatografia de Afinidade , DNA/metabolismo , Hexosaminidases/metabolismo , Peso Molecular , Plasmídeos/metabolismo , CoelhosRESUMO
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.
Assuntos
Vetores Genéticos , Lipossomos , Macrolídeos , Transfecção/métodos , Células 3T3 , Animais , Antibacterianos/farmacologia , Brefeldina A , Cátions , Cloroquina/farmacologia , Ciclopentanos/farmacologia , Endossomos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , CamundongosRESUMO
These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear pore by a process common to other large karyophilic macromolecules. The majority of the injected plasmid DNA was sequestered by cytoplasmic elements. This understanding of plasmid DNA nuclear transport provides a basis for increasing the efficiency of gene transfer.
Assuntos
Músculos/metabolismo , Plasmídeos/metabolismo , Animais , Animais Recém-Nascidos , Sequência de Bases , Ciclo Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Besouros/enzimologia , Primers do DNA , Escherichia coli/enzimologia , Vetores Genéticos , Cinética , Luciferases/biossíntese , Microinjeções , Mitose , Dados de Sequência Molecular , Músculos/citologia , Plasmídeos/administração & dosagem , Ratos , Ratos Sprague-Dawley , beta-Galactosidase/biossínteseRESUMO
The guinea pig seminal vesicle epithelium synthesizes and secretes four major secretory proteins (SVP-1-4). Previous work has established that these four proteins are cleaved from two primary translation products in a complex series of protein processing reactions. The present studies suggest that these protein processing reactions are regulated by androgens. In vitro labeling of seminal vesicle proteins revealed significant differences in the patterns of secretory protein intermediates produced by tissue from intact and castrated animals. Seminal vesicle tissue explants from castrated animals secreted a subset of the processing intermediates secreted by tissue from intact animals. The changes in the patterns of secretory protein intermediates became more pronounced with increasing time after castration, and were fully reversible by treatment of castrated animals with testosterone, suggesting that androgens were affecting the processing or secretion of secretory protein precursors. Amino-terminal protein sequencing of secretory protein processing intermediates that accumulate in the seminal vesicle lumen after castration suggests that the guinea pig seminal vesicle contains an androgen-regulated proteolytic processing activity.
Assuntos
Proteínas Secretadas pela Próstata , Biossíntese de Proteínas , Precursores de Proteínas/metabolismo , Glândulas Seminais/efeitos dos fármacos , Testosterona/farmacologia , Animais , Separação Celular , Epitélio/metabolismo , Cobaias , Masculino , Orquiectomia , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas/metabolismo , Proteínas de Plasma Seminal , Glândulas Seminais/citologia , Glândulas Seminais/metabolismoRESUMO
The guinea pig seminal vesicle epithelium is an androgen-dependent tissue that synthesizes and secretes four major secretory proteins (SVP-1, SVP-2, SVP-3, and SVP-4). Sequencing of near full-length cDNA clones corresponding to the two most abundant mRNAs produced by the seminal vesicle reveals that all four secretory proteins are cleaved from two secretory protein precursors. Amino acid sequences from purified SVP-2 match the central region of the predicted amino acid sequences from the smaller cDNA clone, GP2 (581 nucleotides). Similar analysis demonstrates that the predicted amino acid sequence from the longer cDNA clone, GP1 (1368 nucleotides), codes for the related proteins SVP-3 and SVP-4 as well as SVP-1. The 43.2 kilodalton polyprotein precursor coded by GP1 contains two different sets of 24 amino acid tandemly repeated sequences. The two secretory protein precursors have extensive regions of peptide sequence homology, particularly in regions where protein processing must occur to produce the mature secretory proteins. Analysis of the predicted secondary structure of the two precursor polypeptides revealed a strong correlation between structural features and sites of protein processing.
Assuntos
Proteínas Secretadas pela Próstata , Biossíntese de Proteínas , Precursores de Proteínas/metabolismo , Glândulas Seminais/efeitos dos fármacos , Testosterona/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Genes , Cobaias , Masculino , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas/genética , RNA Mensageiro/genética , Sequências Repetitivas de Ácido Nucleico , Proteínas de Plasma Seminal , Glândulas Seminais/citologia , Glândulas Seminais/metabolismo , Homologia de Sequência do Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
Acetogenic bacteria fix CO or CO2 by a pathway of autotrophic growth called the acetyl-CoA (or Wood) pathway. Key enzymes in the pathway are a methyltransferase, a corrinoid/Fe-S protein, a disulfide reductase, and a carbon monoxide dehydrogenase. This manuscript describes the isolation of the genes that code for the methyltransferase, the two subunits of the corrinoid/Fe-S protein, and the two subunits of carbon monoxide dehydrogenase. These five genes were found to be clustered within an approximately 10-kilobase segment on the Clostridium thermoaceticum genome. The proteins were expressed at up to 5-10% of Escherichia coli cell protein, and isopropyl beta-D-thiogalactopyranoside had no effect on the levels of expression, implying that the C. thermoaceticum inserts contained transcriptional and translational signals that were recognized by E. coli. The methyltransferase is expressed in E. coli in a fully active dimeric form with a specific activity and heat stability similar to the enzyme expressed in C. thermoaceticum. However, both the corrinoid/Fe-S protein and carbon dioxide dehydrogenase, although expressed in high amounts and with identical subunit molecular weights in E. coli, are inactive and less heat stable than are the native enzymes from C. thermoaceticum.
Assuntos
Acetilcoenzima A/biossíntese , Aldeído Oxirredutases/genética , Clostridium/genética , Genes Bacterianos , Genes , Proteínas Ferro-Enxofre/genética , Metaloproteínas/genética , Metiltransferases/genética , Complexos Multienzimáticos , Família Multigênica , Vitamina B 12/metabolismo , Sequência de Aminoácidos , Clostridium/enzimologia , Corrinoides , DNA Bacteriano/biossíntese , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Plasmídeos , Mapeamento por RestriçãoRESUMO
Bone marrow aspirates from 90 patients suspected of having a hematologic disorder were processed by using different cytogenetic methods to determine if any procedure was more likely to reveal a chromosomally abnormal clone or produce better-quality metaphases. All specimens were processed by a direct technique and 24-hr culture without mitogens; 50 specimens were also processed by an amethopterin mitotic synchronization method. In each case, the microscope slides were coded by the processing technologist and analyzed by two other experienced cytogenetic technologists. The results were not known to any of the investigators until all 90 specimens were analyzed. With the exception of one specimen, in which a chromosomally abnormal clone was identified only in the direct preparation, no apparent differences were found in the karyotypes among the three methods. Also, the differences in the quality or number of metaphases found among the three methods were not statistically significant; however, 24-hr unstimulated cultures produced more metaphases than the mitotic synchronization procedure. The greatest source of discordance was caused by one test yielding either no metaphases or an uncertain result when the other tests produced a successful study. We suggest that in routine practice at least two different methods should be used, and it may be best if at least one of these methods is a direct technique.