RESUMEN
Adeno-associated virus (AAV) -mediated gene therapy is a promising strategy to treat liver-based monogenic diseases. However, two major obstacles limit its success: first, vector dilution in actively dividing cells, such as hepatocytes in neonates/children, due to the non-integrating nature of the vector; second, development of an immune response against the transgene and/or viral vector. Crigler-Najjar Syndrome Type I is a rare monogenic disease with neonatal onset, caused by mutations in the liver-specific UGT1 gene, with toxic accumulation of unconjugated bilirubin in plasma, tissues and brain. To establish an effective and long lasting cure, we applied AAV-mediated liver gene therapy to a relevant mouse model of the disease. Repeated gene transfer to adults by AAV-serotype switching, upon neonatal administration, resulted in lifelong correction of total bilirubin (TB) levels in both genders. In contrast, vector loss over time was observed after a single neonatal administration. Adult administration resulted in lifelong TB levels correction in male, but not female Ugt1-/- mice. Our findings demonstrate that neonatal AAV-mediated gene transfer to the liver supports a second transfer of the therapeutic vector, by preventing the induction of an immune response and supporting the possibility to improve AAV-therapeutic efficacy by repeated administration.
Asunto(s)
Síndrome de Crigler-Najjar/terapia , Dependovirus/genética , Terapia Genética/métodos , Glucuronosiltransferasa/genética , Animales , Bilirrubina/metabolismo , Encéfalo/metabolismo , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Glucuronosiltransferasa/metabolismo , Células HEK293 , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , SerogrupoRESUMEN
Ex vivo gene transfer to the graft before transplantation is an attractive option for circumventing systemic side effects of chronic antirejection therapy. Gene delivery of the immunomodulatory protein cytotoxic T-lymphocyte-associated protein 4-immunoglobulin (CTLA4-Ig) prevented chronic kidney rejection in a rat model of allotransplantation without the need for systemic immunosuppression. Here we generated adeno-associated virus type 2 (AAV2) and AAV9 vectors encoding for LEA29Y, an optimized version of CTLA4-Ig. Both LEA29Y vectors were equally efficient for reducing T-cell proliferation in vitro. Serotype 9 was chosen for in vivo experiments owing to a lower frequency of preformed antibodies against the AAV9 capsid in 16 non-human primate tested sera. AAV9-LEA29Y was able to transduce the kidney of non-human primates in an autotransplantation model. Expression of LEA29Y mRNA by renal cells translated into the production of the corresponding protein, which was confined to the graft but not detected in serum. Results in non-human primates represent a step forward in maintaining the portability of this strategy into clinics.
Asunto(s)
Abatacept/genética , Dependovirus/genética , Terapia Genética/métodos , Rechazo de Injerto/terapia , Trasplante de Riñón/efectos adversos , Abatacept/metabolismo , Animales , Línea Celular Tumoral , Vectores Genéticos/genética , Rechazo de Injerto/etiología , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Ratas , Ratas Endogámicas Lew , Linfocitos T/inmunología , Trasplante Autólogo/efectos adversosRESUMEN
Tumor angiogenesis depends on the vascular endothelial growth factor (VEGF), which exists in multiple splicing isoforms, including the most abundant VEGF165 and VEGF121. We have previously shown that the differential capacity of these two VEGF isoforms to bind Neuropilin-1 accounts for their diverse ability to recruit Nrp1-expressing monocytes (NEMs), resulting in a different arteriogenic potential. Here we measure the expression of VEGF165 and VEGF121 in human cancer and their influence on tumor growth and vascularization. We measured the expression levels of VEGF165 and VEGF121 in human colorectal cancer and found that VEGF121 was more expressed than VEGF165, particularly in patients with extensive lymph node infiltration. Overexpressing either VEGF165 or VEGF121 in a cancer mouse model, we observed that the former decreased, whereas the latter increased tumor growth. In both clinical and experimental tumors, VEGF165 expression resulted in the recruitment of NEMs, paralleled by maturation of the tumor vascular network. Finally, hypoxia induced a shift toward the VEGF165 isoform in the central core of human cancers, as well as in various types of cultured cells. These results demonstrate that the two VEGF splicing isoforms are differentially expressed in colorectal cancers, exerting opposite effects on tumor growth and vessel maturation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/patología , Neovascularización Patológica/genética , Factor A de Crecimiento Endotelial Vascular/genética , Empalme Alternativo , Animales , Línea Celular Tumoral , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Inmunohistoquímica , Metástasis Linfática , Melanoma Experimental , Ratones , Neoplasias/metabolismo , Neoplasias/terapia , Neovascularización Patológica/metabolismo , Isoformas de Proteínas , Carga Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at â¼3 weeks after injection and persisting for almost a year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified monoclonal antibody 14F7.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Gangliósido G(M3)/inmunología , Secuencia de Aminoácidos , Animales , Dependovirus/genética , Dependovirus/metabolismo , Células HEK293 , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The Neuregulin/ErbB system plays an important role in the peripheral nervous system, under both normal and pathological conditions. We previously demonstrated that expression of soluble ecto-ErbB4, the released extracellular fragment of the ErbB4 receptor, stimulated glial cell migration in vitro. In this study we examined the possibility of manipulating this system in vivo in order to improve injured peripheral nerve regeneration. Transected rat median nerves of adult female Wistar rats were repaired with a 10-mm-long graft made by muscle-in-vein combined nerve guide previously transduced with either the adeno-associated viral (AAV) vector AAV2-LacZ or AAV2-ecto-ErbB4. Autologous nerve grafts were used as control. Both stereological and functional analyses were performed to assess nerve regeneration. Data show that delivery of soluble ecto-ErbB4 by gene transfer in the muscle-in-vein combined nerve guide has a positive effect on fiber maturation, suggesting that it could represent a potential tool for improving peripheral nerve regeneration.
Asunto(s)
Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/terapia , Nervios Periféricos/fisiología , Receptor ErbB-4/genética , Animales , Axones/fisiología , Dependovirus/genética , Femenino , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Fibras Nerviosas/fisiología , Regeneración Nerviosa/genética , Procedimientos Neuroquirúrgicos/métodos , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Receptor ErbB-4/biosíntesisRESUMEN
Functional recovery after peripheral nerve injury depends on both improvement of nerve regeneration and prevention of denervation-related skeletal muscle atrophy. To reach these goals, in this study we overexpressed vascular endothelial growth factor (VEGF) by means of local gene transfer with adeno-associated virus (AAV). Local gene transfer in the regenerating peripheral nerve was obtained by reconstructing a 1-cm-long rat median nerve defect using a vein segment filled with skeletal muscle fibers that have been previously injected with either AAV2-VEGF or AAV2-LacZ, and the morphofunctional outcome of nerve regeneration was assessed 3 months after surgery. Surprisingly, results showed that overexpression of VEGF in the muscle-vein-combined guide led to a worse nerve regeneration in comparison with AAV-LacZ controls. Local gene transfer in the denervated muscle was obtained by direct injection of either AAV2-VEGF or AAV2-LacZ in the flexor digitorum sublimis muscle after median nerve transection and results showed a significantly lower progression of muscle atrophy in AAV2-VEGF-treated muscles in comparison with muscles treated with AAV2-LacZ. Altogether, our results suggest that local delivery of VEGF by AAV2-VEGF-injected transplanted muscle fibers do not represent a rational approach to promote axonal regeneration along a venous nerve guide. By contrast, AAV2-VEGF direct local injection in denervated skeletal muscle significantly attenuates denervation-related atrophy, thus representing a promising strategy for improving the outcome of post-traumatic neuromuscular recovery after nerve injury and repair.
Asunto(s)
Terapia Genética/métodos , Atrofia Muscular/terapia , Regeneración Nerviosa , Nervios Periféricos/fisiología , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Vectores Genéticos , Desnervación Muscular , Fibras Musculares Esqueléticas , Atrofia Muscular/patología , Traumatismos de los Nervios Periféricos/terapia , Ratas , Ratas WistarRESUMEN
Thanks to the safety of administration, efficiency of in vivo transduction and persistence of transgene expression, vectors based on the adeno-associated virus (AAV) are extensively utilized in both preclinical and clinical experimentation. Here we thoroughly explore the potential of AAV-mediated antigen delivery for tumour vaccination. A recombinant AAV vector (rAAV) encoding a lymphoma idiotype (Id) in a single-chain variable fragment format was found to induce an efficient anti-Id immune response upon injection in immunocompetent animals. The intensity of the immune response and the protective effect of rAAV administration in vivo were systematically compared with those elicited by simple injection of naked DNA or biolistic immunization. The results indicate that Id delivery via rAAV enhances the intensity of immune response compared with injection of naked DNA, while anti-idiotypic antibodies titres are not considerably increased compared with biolistic vaccination. On the contrary, a prime-boost vaccination strategy combining biolistic and AAV DNA delivery results in a major increase in anti-Id antibody response compared with the repetitive biolistic immunization. This increased anti-Id humoral response strictly correlated with a significant improvement on tumour protection in vivo.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Biolística/métodos , Vacunas contra el Cáncer/inmunología , Dependovirus/genética , Idiotipos de Inmunoglobulinas/genética , Linfoma de Células B/terapia , Animales , Antígenos de Neoplasias/inmunología , Vectores Genéticos , Humanos , Idiotipos de Inmunoglobulinas/administración & dosificación , Idiotipos de Inmunoglobulinas/inmunología , Inyecciones Intramusculares , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Linfoma de Células B/inmunología , Ratones , Transducción Genética , Vacunas de ADNRESUMEN
Delivery of therapeutic genes represents an appealing possibility to accelerate healing of wounds that are otherwise difficult to treat, such as those in patients with metabolic disorders or infections. Experimental evidence indicates that in such conditions potentiation of neo-angiogenesis at the wound site might represent an important therapeutic target. Here we explore the efficacy of gene therapy of wound healing with an adeno-associated virus (AAV) vector expressing the 165 amino acid isoform of vascular endothelial growth factor-A (VEGF-A). By gene marker studies, we found that AAV vectors are highly efficient for gene transfer to the rat skin, displaying an exquisite tropism for the panniculus carnosus. Gene expression from these vectors is sustained and persistent over time. Delivery of VEGF165 to full thickness excisional wounds in rats resulted in remarkable induction of new vessel formation, with consequent reduction of the healing time. Histological examination of treated wounds revealed accelerated remodeling of epidermis and dermis, with formation of a thick granular layer, containing numerous newly formed capillaries, as well as vessels of larger size. These data underline the importance of neo-angiogenesis in the healing process and indicate that VEGF gene transfer might represent a novel approach to treat wound healing disorders.
Asunto(s)
Dependovirus/genética , Factores de Crecimiento Endotelial/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Linfocinas/genética , Piel/lesiones , Cicatrización de Heridas , Animales , Vectores Genéticos/genética , Masculino , Neovascularización Fisiológica , Ratas , Ratas Wistar , Piel/irrigación sanguínea , Transducción Genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Unlike postmitotic tissues in vivo, transduction of cultured cells is poor with recombinant adeno-associated virus (rAAV). The ability of rAAV to transduce cells is greatly enhanced by a variety of agents that induce DNA damage and is elevated in cells defective in the ataxia telangiectasia gene product (ATM), showing increased genomic instability. Here we show that DNA double-stranded break (DSB) repair pathways are involved in the regulation of rAAV transduction efficiency. By quantitative chromatin immunoprecipitation, we found that Ku86 and Rad52 proteins associate with viral DNA inside transduced cells. Both proteins are known to competitively recognize hairpin structures and DNA termini and to promote repair of DSBs, the former by facilitating nonhomologous end joining and the latter by initiating homologous recombination. We found that rAAV transduction is increased in Ku86-defective cells while it is inhibited in Rad52 knockout cells. These results suggest that binding of Rad52 to the rAAV genome might be involved in processing of the vector genome through a homologous recombination pathway.
Asunto(s)
Daño del ADN , ADN Viral/metabolismo , Proteínas de Unión al ADN/fisiología , Dependovirus/genética , Recombinación Genética , Animales , Células CHO , Cricetinae , Reparación del ADN , Unión ProteicaRESUMEN
We have comparatively evaluated the efficiency of a series of retroviral vectors transducing the gp91-phox gene, whose defects are responsible for impaired production of superoxide anion (O2-) by phagocytic cells and lead to the X-linked form of chronic granulomatous disease (X-CGD). These vectors included four constructs based on the MoMuLV backbone and expressing gp91-phox from the viral long terminal repeat (LTR) or from internal promoters, and one construct based on the myelotropic FMEV vector. Expression of the therapeutic gene from the MoMuLV LTR was unsatisfactory after transduction of the PLB985 X-CGD knockout cell line and of primary CD34+ hematopoietic progenitors from X-CGD patients. The presence of either constitutive or inducible internal promoters did not result in important improvements in the efficiency of O2- production and lowered the titers of the viral preparations. In contrast, sustained levels of superoxide generation were obtained upon transduction with the FMEV vector. To analyze the efficiency of transgene expression at the single cell level, over 150 cellular clones were generated from bulk cultures of PLB985 X-CGD cells transduced with this vector, each one representative of an individual transduction event. These clones revealed a markedly heterogeneous pattern of gp91-phox expression, ranging from complete silencing to full restoration of superoxide production. Within each clone, expression of the therapeutic gene correlated with the number of expressing cells rather than with the average levels of expression from each cell, indicating that at the single cell level, the proviral promoter is regulated by a binary, on/off mechanism. Moreover, both transduced bulk and clonal cell populations displayed a tendency to a progressive extinction of expression over time, with a mechanism involving LTR methylation. The design of novel retroviral vectors escaping silencing is highly desirable for efficient gene therapy.
Asunto(s)
Vectores Genéticos/genética , Enfermedad Granulomatosa Crónica/genética , Retroviridae/genética , Expresión Génica , Técnicas de Transferencia de Gen , Enfermedad Granulomatosa Crónica/enzimología , Enfermedad Granulomatosa Crónica/virología , Células HL-60 , Células HeLa , Humanos , NADPH Oxidasas/metabolismo , Provirus/genética , Transgenes/genéticaRESUMEN
In chronic granulomatous disease, interferon-gamma (IFN-gamma) significantly reduces the incidence and severity of recurrent infections, but its effectiveness administered ex novo during acute infection has been reported in only one case. In this report, we describe two adult brothers with chronic granulomatous disease treated successfully with IFN-gamma for acute liver abscesses. Two brothers with severe recurrent infections of unknown origin were hospitalized for septic fever, malnutrition, and ultrasonographic evidence of liver abscess. Autosomal recessive chronic granulomatous disease was diagnosed based on lack of superoxide anion production by phagocytes and absence of p47-phox protein. An antibiotic regimen specifically directed against Staphylococcus aureus was ineffective, whereas treatment with 50 microg/m2 IFN-gamma s.c. thrice weekly induced complete healing with scarring within 3 months. No septic recurrence was observed during a 4-year follow-up period. In chronic granulomatous disease, IFN-gamma is effective not only in preventing but also in healing life-threatening acute infections.
Asunto(s)
Enfermedad Granulomatosa Crónica/tratamiento farmacológico , Interferón gamma/uso terapéutico , Absceso Hepático/tratamiento farmacológico , Adolescente , Adulto , Genes Recesivos , Enfermedad Granulomatosa Crónica/genética , Humanos , Absceso Hepático/complicaciones , Mediciones Luminiscentes , MasculinoRESUMEN
This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67phox, p40phox, p22phox, and gp91phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47phox, p67phox, and p40phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22phox in the absence of gp91phox, p47phox, p67phox, and p40phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22phox was normally expressed and gp91phox was present but lacked five amino acids, translocation of p47phox to the membranes was unaffected, but p67phox and p40phox were poorly translocated, and the production of O2- was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O2- in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22phox on the membranes in the absence of gp91phox; 3) p47phox can translocate to the membranes independently of p67phox and p40phox; and 4) gp91phox may have a role in mediating and/or stabilizing the binding of p67phox and p40phox to the membranes of activated cells.
Asunto(s)
Linfocitos B/enzimología , Enfermedad Granulomatosa Crónica/enzimología , Herpesvirus Humano 4/fisiología , Proteínas de Transporte de Membrana , NADPH Oxidasas/metabolismo , Linfocitos B/efectos de los fármacos , Transporte Biológico , Línea Celular Transformada , Membrana Celular/metabolismo , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Grupo Citocromo b/fisiología , Activación Enzimática , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/patología , Humanos , Sustancias Macromoleculares , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Mutación , NADPH Deshidrogenasa/deficiencia , NADPH Deshidrogenasa/genética , NADPH Deshidrogenasa/fisiología , NADPH Oxidasa 2 , Neutrófilos/inmunología , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Conformación Proteica , Estallido Respiratorio , Relación Estructura-Actividad , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Cromosoma X/genéticaRESUMEN
Chronic granulomatous disease (CGD) is an inherited immunodeficiency characterized by severe recurrent bacterial and fungal infections of several organs. The disease is due to the inability of phagocytic leukocytes to generate reactive oxygen species upon phagocytosis. The defect arises as a consequence of mutations of the genes encoding for the subunits of a membrane NADPH oxidase, which catalyzes the production of superoxide anion (O2-). CGD represents an ideal candidate disorder for gene therapy, since the disease has a recessive inheritance, its phenotype is exclusively expressed in phagocytic cells, and a partial correction is likely to be effective. Given the short half-life of mature phagocytes, the optimal target cell population for gene transfer is the pluripotent hematopoietic stem cell. Transduction of CD34+ hematopoietic progenitors with retroviral vectors carrying the cDNA of the defective gene results in the correction of the enzymatic defect in myeloid cells differentiated in vitro. Still, the effective development of a clinical gene therapy protocol for this disease will await a substantial improvement in our current technology for the identification and manipulation of hematopoietic stem cells, and in our understanding of their biological and molecular properties.
Asunto(s)
Terapia Genética , Enfermedad Granulomatosa Crónica/terapia , Adenoviridae , Antígenos CD34 , Técnicas de Transferencia de Gen , Vectores Genéticos , Enfermedad Granulomatosa Crónica/genética , Células Madre Hematopoyéticas , Humanos , NADPH Oxidasas/genética , Retroviridae , Transducción GenéticaRESUMEN
A quantitative polymerase chain reaction (PCR) procedure has been developed for rapid retrovirus titration. This procedure, which is based on the simultaneous amplification of the sample with known amounts of a competitor DNA fragment (competitive PCR), was used for the quantification of viral RNA genomes in retrovirus-producing cell clone supernatants and of proviral DNA molecules formed at 24 h after infection of different reference cell lines. The results obtained from the analysis of several samples indicated that proviral DNA quantification is in complete agreement with the number of selectable colonies in a standard colony assay. Conversely, the number of viral RNA genomes in the producer cell clone supernatants is a poor predictor of the actual efficiency of infection. Repeated competitive PCR experiments for provirus copy number determination at different times after transduction indicated that the number of proviral DNA molecules remains stable over time, suggesting stable integration into the host genome. The developed procedure is rapid and simple, is applicable to retroviral constructs not containing a selectable gene and can be used to directly measure the efficiency of infection of any target cell type, thus overcoming the problem of the dependency of retroviral titer determination on the rate of expression of a selectable gene and on the efficiency of colony formation of a reference cell line.
Asunto(s)
Vectores Genéticos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Retroviridae/aislamiento & purificación , Células 3T3 , Animales , Unión Competitiva , ADN Viral/metabolismo , Vectores Genéticos/genética , Células HeLa , Humanos , Ratones , Provirus/genética , Retroviridae/genética , Volumetría/métodosRESUMEN
The feasibility of correction of the disease phenotype by gene gene transfer was investigated in cells of four patients with X-linked chronic granulomatous disease. These patients carry point mutations of the gp91-phox gene, encoding for the large subunit of the catalytic core of the phagocytic cell NADPH oxidase. A retroviral vector expressing the gp91-phox protein was constructed and used to transduce lymphoblastoid cell lines established from the patients. Several transduced lymphoblastoid cell clones were investigated for mRNA and protein expression, and for functional reconstitution of oxidase activity. Although extensive quantitative variability was detected among different clones, functional reconstitution of O2- production was obtained in most cases, with oxidase function within the same range as in B cell lines derived from normal individuals. The same vector was also used for transduction of hematopoietic precursors from bone marrow or peripheral blood either with or without enrichment for CD34+ cells. A comprehensive analysis was performed on differentiated myeloid colonies, to evaluate the efficiency of transduction, the levels of gp91-phox expression, and the extent of functional reconstitution of oxidase activity. A high efficiency of transduction was obtained in most experiments, with 60-100% of colonies containing proviral DNA. Among the transduced colonies, an extensive variability in the levels of expression of the transduced gene and of functional restoration of NADPH oxidase activity was observed. These results represent a step toward the development of a gene therapy protocol for these patients.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos/fisiología , Enfermedad Granulomatosa Crónica/genética , NADH NADPH Oxidorreductasas/metabolismo , Retroviridae/genética , Células 3T3/enzimología , Células 3T3/fisiología , Animales , Linfocitos B/citología , Linfocitos B/fisiología , Secuencia de Bases , Callithrix , Línea Celular/enzimología , Línea Celular/fisiología , Enfermedad Granulomatosa Crónica/enzimología , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , NADPH Oxidasa 2 , NADPH Oxidasas , Nitroazul de Tetrazolio , Reacción en Cadena de la Polimerasa , Transducción GenéticaRESUMEN
We recently proposed a quantitative PCR procedure for the absolute measurement of c-erbB-2 oncogene amplification, based on the simultaneous polymerase chain reaction (PCR) amplification of the target gene and of a competitor DNA molecule acting as internal standard. To increase the number of assayable oncogenes and the accuracy of the quantitative comparison of gene amplification degree within the same tumor, we have now constructed a single synthetic competitor for int-2, c-myc, N-myc epidermal growth factor receptor, and c-erbB-2 genes, and for the reference gene beta-globin. This competitor was constructed by a two-step recombinant PCR procedure and inserted as a 297-bp sequence in a plasmid. The order of primer insertion was designed to obtain competitors of comparable sizes to those of the respective genomic targets, but still easily recognizable from the latter ones by gel electrophoresis. The clone competitor was tested to evaluate the linearity range for each assay. The present application of quantitative PCR based on a multiple competitor represents the first approach for the achievement of a single reagent for the evaluation of a panel of genes potentially amplified in human tumors.
Asunto(s)
Receptores ErbB/genética , Factores de Crecimiento de Fibroblastos/genética , Genes erbB , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Secuencia de Bases , Unión Competitiva , ADN/química , ADN/metabolismo , Cartilla de ADN/química , Factor 3 de Crecimiento de Fibroblastos , Genes myc , Globinas/genética , Humanos , Datos de Secuencia Molecular , Proteínas Tirosina Quinasas , Moldes Genéticos , Células Tumorales CultivadasRESUMEN
A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.
Asunto(s)
Replicación del ADN , Lamina Tipo B , Reacción en Cadena de la Polimerasa/métodos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN , Humanos , Laminas , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Virus 40 de los Simios/genéticaRESUMEN
We present an application of image analysis for the direct quantification of PCR products after gel electrophoresis and ethidium bromide staining of DNA. This procedure has been applied to the development of an assay based on competitive PCR for the measurement of the degree of amplification of c-erbB-2 oncogene in DNA from human tumours. In this method two DNA species (genomic and competitor) compete for PCR amplification. Since results are calculated from the final competitor/genomic ratio any variable affecting the rate of PCR amplification has no effect on the accuracy of the ratio measurement. Results are reported which show that even large variations in the experimental conditions (number of PCR cycles, sample volumes and extracted DNA quality) did not interfere with the precision of the measurement of the competitor/genomic ratio.
Asunto(s)
ADN de Neoplasias/metabolismo , Neoplasias/genética , Reacción en Cadena de la Polimerasa/métodos , Proto-Oncogenes , Receptor ErbB-2/biosíntesis , Unión Competitiva , Neoplasias de la Mama/genética , ADN de Neoplasias/análisis , Femenino , Humanos , Mediciones Luminiscentes , Neoplasias/metabolismo , Fotometría/métodos , Receptor ErbB-2/genéticaRESUMEN
We present an original application of competitive polymerase chain reaction (PCR) for measuring oncogene amplification in DNA from human tumors by simultaneous PCR amplification of genomic DNA with fixed amounts of an internal standard (competitor DNA). Competitors share the same sequence as the target genes but contain an additional 15- to 20-base-pair insert, which allows resolution of the amplified products after polyacrylamide gel electrophoresis and ethidium bromide staining. The gene copy number is derived from the ratio between the intensities of the bands corresponding to the amplified products. Using this procedure, we measured c-erbB-2 amplification in breast and bladder carcinomas in both fresh tumor tissues and paraffin-embedded tissue samples and assessed the precision, sensitivity, and accuracy of the assay. Competitive PCR is a simple, reliable, and accurate method for the evaluation of c-erbB-2 amplification and is potentially suitable for use in the clinical laboratory.