RESUMEN
We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector. This suggested that bacterial DNA sequences played a role in episomal transgene silencing. To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms. In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups. In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups. All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact. In all groups, the relative amount of vector DNA in liver remained similar. Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements.
Asunto(s)
ADN Bacteriano/genética , Silenciador del Gen , Animales , Elementos de Facilitación Genéticos/genética , Factor IX/genética , Factor IX/metabolismo , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Humanos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Plásmidos , Regiones Promotoras Genéticas/genética , Transgenes , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMEN
We have constructed plasmid DNA vectors that contain Epstein-Barr virus (EBV) sequences and the human gene (SERPINA1) encoding alpha1-Antitrypsin (AAT). We demonstrate that a plasmid carrying the full SERPINA1 on a 19-kb genomic fragment and the EBV gene EBNA1 and its family of repeats binding sites undergoes efficient extrachromosomal replication in dividing mammalian tissue culture cells. Therefore, use of a whole genomic therapeutic gene to provide both replication and gene expression may be an effective gene therapy vector design, if the target cells are dividing. The efficacy of this same vector for expression of AAT in vivo in the nondividing cells of mouse liver was determined by hydrodynamic injection of naked plasmid DNA by means of the tail vein. A single injection of an EBV/genomic SERPINA1 vector provided >300 microg/ml of AAT, which approached normal plasma levels and persisted for the >9-month duration of the experiment. These data exceed most previously reported values, probably due to sequences in the genomic DNA that resist silencing of gene expression, possibly in combination with favorable effects on expression provided by the EBV sequences. These results demonstrate that plasmid DNA with the correct cis-acting sequences can provide in vivo long-term expression of protein at high levels that are therapeutically relevant for gene therapy.
Asunto(s)
Técnicas de Transferencia de Gen , Vectores Genéticos , Herpesvirus Humano 4/genética , alfa 1-Antitripsina/genética , Animales , Southern Blotting , Línea Celular , Replicación del ADN/fisiología , Herpesvirus Humano 4/metabolismo , Humanos , Hígado/fisiología , Ratones , Ratones Endogámicos C57BL , Plásmidos , Inhibidores de Tripsina/biosíntesis , Inhibidores de Tripsina/genética , alfa 1-Antitripsina/biosíntesisRESUMEN
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/genética , Genoma Viral , Hígado/metabolismo , Recombinación Genética , Animales , División Celular , ADN Circular , ADN Viral , Femenino , Expresión Génica , Hepatectomía , Hepatocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Plásmidos , Factores de Tiempo , Transducción Genética , Transgenes , Integración ViralRESUMEN
The short duration of transgene expression remains a major obstacle for the implementation of nonviral DNA vectors in clinical gene therapy trials. Here, we demonstrate stable, long-term transgene expression in vivo by transfecting a linear DNA expression cassette (LDNA) into mouse liver. Interestingly, despite similar quantities and cellular distribution of injected DNAs in their livers, mice receiving LDNA encoding human alpha1-antitrypsin (hAAT) expressed approximately 10- to 100-fold more serum hAAT than mice injected with closed circular (cc) DNA for a period of 9 months (length of study). Furthermore, when a linear human factor IX expression cassette was delivered to factor IX-deficient mice, sustained serum concentrations of more than 4 microg/ml (80% of normal) of the human clotting factor and correction of the bleeding diathesis were obtained. Southern blot analyses indicate that, unlike ccDNA, LDNA rapidly formed large, unintegrated concatemers in vivo, suggesting that transgene persistence from plasmid-based vectors was influenced by the structure of the vector in transfected cells. No differences in transgene expression or DNA molecular structures were observed when AAV ITRs were included to flank the hAAT expression cassette in both ccDNA- and LDNA-treated animals. Linear DNA transfection provides an approach for achieving long-term expression of a transgene in vivo.
Asunto(s)
ADN Recombinante , Factor IX/genética , Expresión Génica , Vectores Genéticos , Hígado/metabolismo , Transfección/métodos , alfa 1-Antitripsina/genética , Animales , ADN Recombinante/administración & dosificación , Terapia Genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Hemofilia A , Humanos , Ratones , Factores de Tiempo , Transgenes/efectos de los fármacos , Transgenes/genéticaAsunto(s)
Anticuerpos Monoclonales/farmacología , Supervivencia de Injerto/fisiología , Hepatocitos/trasplante , Proteínas Proto-Oncogénicas c-met/fisiología , Animales , Regulación de la Expresión Génica , Supervivencia de Injerto/genética , Humanos , Ratones , Proteínas Proto-Oncogénicas c-met/inmunología , Trasplante Heterólogo , Trasplante Heterotópico , alfa 1-Antitripsina/metabolismoRESUMEN
We systematically compared human factor IX gene expression from a variety of plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5- to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the in vivo studies. We found that a plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) sequence including a portion of the first intron (intron A), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 microg/ml (normal = 5 microg/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 microg/ml (10 to 40% of normal) for 225 days (length of experiment). These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.
Asunto(s)
Factor IX/genética , Hígado/metabolismo , Plásmidos/genética , Transfección , Regiones no Traducidas 3' , Animales , Bovinos , Línea Celular , Factor IX/metabolismo , Expresión Génica , Terapia Genética/métodos , Vectores Genéticos , Hemofilia B/sangre , Hemofilia B/genética , Hemofilia B/terapia , Humanos , Técnicas In Vitro , Inyecciones Intravenosas , Intrones , Región de Control de Posición , Ratones , Ratones Endogámicos C57BL , Plásmidos/administración & dosificación , Vena PortaRESUMEN
The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.
Asunto(s)
Elementos Transponibles de ADN/genética , Regulación de la Expresión Génica , Hemofilia B/genética , Transgenes/genética , Animales , Secuencia de Bases , Proteínas de Unión al ADN/genética , Susceptibilidad a Enfermedades , Dosificación de Gen , Técnicas de Transferencia de Gen , Células HeLa , Hemofilia B/terapia , Humanos , Inyecciones Intravenosas , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Datos de Secuencia Molecular , Plásmidos/administración & dosificación , Plásmidos/uso terapéutico , Transposasas/genéticaRESUMEN
Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.
Asunto(s)
Trasplante de Células , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B/patología , Hepatitis D/patología , Virus de la Hepatitis Delta/aislamiento & purificación , Hígado/citología , Trasplante Heterólogo , Animales , Modelos Animales de Enfermedad , Hepatitis B/transmisión , Hepatitis D/transmisión , Humanos , Hígado/patología , Hígado/virología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Proto-Oncogénicas c-met/inmunología , Factores de TiempoRESUMEN
Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.
Asunto(s)
Dependovirus , Factor IX/genética , Terapia Genética , Vectores Genéticos , Hemofilia B/terapia , Animales , Anticuerpos/sangre , Tiempo de Sangría , Transformación Celular Viral , Modelos Animales de Enfermedad , Perros , Humanos , Hígado , Ratones , Ratones Endogámicos C57BL , Recombinación GenéticaRESUMEN
NF-kappaB is a key regulator of the innate antiviral immune response, due in part to its transcriptional activation of cytokines and adhesion molecules, which, in turn, function in chemotaxis and activation of inflammatory cells. We reported earlier that viral gene expression in hepatocytes transduced with first-generation (E1-deleted) adenoviruses induced NF-kappaB activation, elevation of serum cytokines, and hepatocellular apoptosis during the first days postinfusion. These events did not occur in mice infused with an adenovirus vector deleted for E1, E2, E3, and late gene expression. In the present study, we used an adenovirus expressing an IkappaBalpha supersuppressor (Ad.IkappaBM) and bcl-2 transgenic mice to unravel the role of virus-induced NF-kappaB activation and apoptosis in the clearance of recombinant adenovirus vectors from the liver. The combined action of IkappaBM and Bcl-2 allowed for vector persistence in livers of C57BL/6 x C3H mice. In the absence of Bcl-2, IkappaBM expression in mouse livers significantly reduced NF-kappaB activation, cytokine expression, leukocyte infiltration, and the humoral immune response against the transgene product; however, this was not sufficient to prevent the decline of vector DNA in transduced cells. Infusion of Ad.IkappaBM caused extended apoptosis predominantly in periportal liver regions, indicating that NF-kappaB activation may protect transduced hepatocytes from apoptosis induced by adenovirus gene products. To confer vector persistence, bcl-2 transgene expression was required to block virus-induced apoptosis if NF-kappaB protection was inactivated by IkappaBM. Expression of gene products involved in early stages of apoptotic pathways was up-regulated in response to virus infusion in bcl-2 transgenic mice, which may represent a compensatory effect. Our study supports the idea that the suppression of innate defense mechanisms improves vector persistence.
Asunto(s)
Adenoviridae/genética , Genes bcl-2 , Vectores Genéticos , Proteínas I-kappa B , Hígado/virología , FN-kappa B/metabolismo , Adenoviridae/inmunología , Animales , Formación de Anticuerpos , Apoptosis , Secuencia de Bases , Citocinas/biosíntesis , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Eliminación de Gen , Expresión Génica , Genes Virales , Terapia Genética , Humanos , Inflamación/patología , Hígado/patología , Hígado/fisiopatología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidor NF-kappaB alfa , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/inmunologíaRESUMEN
Recombinant retroviral vectors represent an attractive means of transferring genes into the liver because they integrate in the host cell genome and result in permanent gene expression. However, efficient gene transfer is limited by the requirement of active cell division for integration. Surgical partial hepatectomy has been the traditional method of inducing hepatocellular proliferation, but this invasive approach would be difficult to justify in clinical gene therapy. As an alternative, we used a recombinant adenovirus expressing a nonsecreted form of urokinase plaminogen activator (Ad.PGKmuPA), which results in liver regeneration over a period of 8 days. When a high-titer retroviral vector was continuously infused into the portal vein of mice during this period of hepatocyte proliferation, 33.5% of hepatocytes were stably transduced. In addition, high-level expression of a secreted transgene reporter was sustained for at least 48 weeks (length of experiment). We investigated the influence of vector titer on the in vivo transduction efficiency in our system, and found that the total number of infectious retroviral particles delivered per target cell is a critical factor. The results presented here demonstrate the ability to obtain a high gene transfer efficiency and long-term gene expression in hepatocytes in vivo without the need for surgical hepatectomy. The two-vector system described here may be of clinical relevance, as the level of hepatic gene transfer achieved has potential to be curative for a large number of genetic liver diseases.
Asunto(s)
Adenoviridae/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Regeneración Hepática , Retroviridae/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Animales , Femenino , Vectores Genéticos/genética , Infusiones Intravenosas , Regeneración Hepática/genética , Ratones , Ratones Endogámicos C57BL , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Systemic application of first-generation adenovirus induces pathogenic effects in the liver. To begin unraveling the mechanisms underlying early liver toxicity after adenovirus infusion, particularly the role of macrophage activation and expression of viral genes in transduced target cells, first-generation adenovirus or adenovirus vectors that lacked most early and late gene expression were administered to C3H/HeJ mice after transient depletion of Kupffer cells by gadolinium chloride treatment. Activation of NF-kappaB, and the serum levels of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) were studied in correlation with liver damage, apoptosis, and hepatocellular DNA synthesis. While Kupffer cell depletion nearly eliminated adenovirus-induced TNF release, it resulted in a more robust IL-6 release. These responses were greatly reduced in animals receiving the deleted adenovirus. Although there were quantitative differences, NF-kappaB activation was observed within minutes of first-generation or deleted adenovirus vector administration regardless of the status of the Kupffer cells, suggesting that the induction is related to a direct effect of the virus particle on the hepatocyte. Early liver toxicity as determined by serum glutamic-pyruvic transaminase elevation and inflammatory cell infiltrates appeared to be dependent on adenovirus-mediated early gene expression and intact Kupffer cell function. Kupffer cell depletion had little effect on adenovirus-mediated hepatocyte apoptosis but did increase hepatocellular DNA synthesis. Finally, Kupffer cell depletion decreased the persistence of transgene (human alpha1-antitrypsin [hAAT]) expression that was associated with a more pronounced humoral immune response against hAAT. The elucidation of these events occurring after intravenous adenovirus injection will be important in developing new vectors and transfer techniques with reduced toxicity.
Asunto(s)
Adenoviridae/patogenicidad , Macrófagos del Hígado/inmunología , Hepatopatías/virología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Citocinas/metabolismo , ADN/biosíntesis , Gadolinio/farmacología , Regulación Viral de la Expresión Génica , Vectores Genéticos/efectos adversos , Hepatopatías/inmunología , Ratones , Ratones Endogámicos C3H , Ratones SCID , Transgenes/genéticaRESUMEN
The administration of soluble muCTLA4Ig around the time of adenovirus vector mediated gene transfer into murine hepatocytes has been shown to markedly prolong transgene expression, diminish the formation of adenovirus neutralizing antibody, decrease T cell proliferative response and infiltration into the liver without causing irreversible systemic immunosuppression. In this study, an E1/E3-deleted adenovirus vector constitutively expressing murine CTLA4Ig (Ad.RSV-muCTLA4Ig) was constructed in order to determine if production of muCTLA4Ig from within transduced cells (i.e. hepatocytes) would provide a more specific/localized interference with the CD28/B7-1 and B7-2 signaling pathways, and thus result in prolonged transgene expression in vivo at nonimmunosuppressive serum concentrations. In contrast to C3H mice receiving a control adenovirus, transduction with 6 x 10(9) p.f.u. of Ad.RSV-muCTLA4Ig and a reporter adenovirus (2 x 10(9) p.f.u. of Ad.PGK-hAAT) resulted in prolonged reporter gene expression, reduced anti-adenovirus and anti-hAAT antibody production, and attenuated T cell proliferation and IFN-gamma production in response to adenoviral vector. Mice given a constant total amount of adenovirus with diminishing amounts of Ad.RSV-muCTLA4Ig and a constant amount of reporter virus (2 x 10(9) p.f.u. of Ad.PGK-hAAT) demonstrated prolonged reporter gene expression and decreased anti-adenovirus and anti-hAAT antibody production only when high serum levels of muCTLA4Ig were produced. Taken together, these findings suggest that a certain threshold of muCTLA4Ig must be achieved to alter the immune responses and prolong transgene expression from adenoviral vectors.
Asunto(s)
Adenoviridae , Antígenos de Diferenciación/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Inmunoconjugados , Hígado/inmunología , Abatacept , Animales , Formación de Anticuerpos/genética , Antígenos CD , Antígeno B7-1/inmunología , Antígenos CD28/inmunología , Antígeno CTLA-4 , División Celular , Perros , Factor IX/análisis , Femenino , Expresión Génica , Interferón gamma/fisiología , Ratones , Ratones Endogámicos C3H , Linfocitos T/fisiología , Transgenes , alfa 1-Antitripsina/inmunologíaRESUMEN
Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.
Asunto(s)
Dependovirus/genética , Factor IX/genética , Técnicas de Transferencia de Gen , Vectores Genéticos , Hemofilia B/terapia , Hígado/metabolismo , Alanina Transaminasa/sangre , Alanina Transaminasa/metabolismo , Animales , División Celular , ADN sin Sentido/genética , ADN sin Sentido/metabolismo , Factor IX/metabolismo , Expresión Génica , Terapia Genética , Hemofilia B/genética , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Although recombinant adenovirus vectors offer a very efficient means by which to transfer genetic information into cells in vivo, antigen-dependent immunity limits the duration of gene expression and prevents retreatment. Recombinant murine CTLA4Ig and anti-CD40 ligand antibody block costimulatory interactions between T cells and antigen presenting cells. We previously reported that murine CTLA4Ig prolongs adenoviral-mediated gene transfer, but does not allow for secondary expression after readministration of the vector. In studies described here, when anti-CD40 ligand and recombinant murine CTLA4Ig were coadministered around the time of primary vector administration (i) prolonged adenovirus-mediated gene expression (length of experiment up to 1 year) from the livers of >90% of treated mice was observed, and (ii) secondary adenovirus-mediated gene transfer was achieved in >50% of the mice even after the immunosuppressive effects of these agents were no longer present. Nearly two-thirds of these mice had persistent secondary gene expression lasting for at least 200-300 days. Neither agent alone allowed transduction after secondary vector administration. Treated mice had decreased immune responses to the vector as shown by markedly decreased production of neutralizing antibodies, diminished spleen proliferation responses and IFN-gamma production in vitro, and reduced T cell infiltrates in the liver. These results suggest that it may be possible to obtain persistence as well as secondary adenoviral-mediated gene transfer with transient immunosuppressive therapies.
Asunto(s)
Adenoviridae/inmunología , Técnicas de Transferencia de Gen , Inmunoconjugados , Terapia de Inmunosupresión/métodos , Hígado/virología , Abatacept , Adenoviridae/genética , Animales , Anticuerpos/farmacología , Anticuerpos Antivirales/sangre , Células Presentadoras de Antígenos/inmunología , Antígenos CD , Antígenos de Diferenciación/farmacología , Antígenos CD40/inmunología , Ligando de CD40 , Antígeno CTLA-4 , Femenino , Expresión Génica/efectos de los fármacos , Terapia Genética/métodos , Inmunosupresores/farmacología , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Linfocitos T/inmunologíaRESUMEN
Recombinant adenoviruses have received much attention as a potential vector for gene therapy because of their ability to transduce many cell types with high efficiencies in vivo. After intravenous infusion, the majority of the vector is found in hepatocytes, but vector DNA is found to varying degrees in other tissues. In an attempt to restrict adenovirus-mediated gene transfer to the liver, we developed a microsurgical method that allowed for vector administration directly into the biliary tract of a mouse. We demonstrate that gene transfer was 4- to 10-fold more restricted to the liver after biliary tract infusion than after intravascular infusion. Intravascular infusion of recombinant adenovirus elicits a powerful immune response that limits gene expression and the ability to readminister the vector. Biliary infusion resulted in a slightly lesser immune response as determined by the lower neutralizing antibody titers directed against the vector compared with animals treated by intravascular infusion. There was no difference in the persistence of gene expression, suggesting a similar cell-mediated immune response against the vector containing cells in animals administered vector by either method. As future-generation adenovirus vectors that are safer and less immunogenic become available, the more liver specific gene transfer via the biliary tract may offer advantages over intravenous infusion for hepatic gene therapy.
Asunto(s)
Adenoviridae/genética , Sistema Biliar , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Hígado , Animales , Anticuerpos Antivirales/sangre , Cateterismo , ADN Viral/análisis , Expresión Génica , Genes Reporteros/genética , Vectores Genéticos/genética , Hígado/metabolismo , Ratones , Ratones Endogámicos C3H , Especificidad de Órganos , alfa 1-Antitripsina/metabolismo , beta-Galactosidasa/genéticaRESUMEN
Recombinant adenovirus vectors are efficient at transferring genes into somatic tissues but are limited for use in clinical gene therapy by immunologic factors that result in the rapid loss of gene expression and inhibit secondary gene transfer. This study demonstrates that systemic coadministration of recombinant adenovirus with soluble CTLA4Ig, which is known to block co-stimulatory signals between T cells and antigen presenting cells, leads to persistent adenoviral gene expression in mice without long-term immunosuppression. This form of immunotherapy greatly enhances the likelihood that recombinant adenovirus vectors will be useful for human gene therapy.
Asunto(s)
Adenoviridae , Formación de Anticuerpos , Células Presentadoras de Antígenos/inmunología , Antígenos de Diferenciación/biosíntesis , Expresión Génica , Técnicas de Transferencia de Gen , Inmunoconjugados , Activación de Linfocitos , Linfocitos T/inmunología , Abatacept , Animales , Antígenos CD , Antígenos de Diferenciación/análisis , Antígenos de Diferenciación/genética , Antígeno CTLA-4 , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Terapia Genética/métodos , Vectores Genéticos , Humanos , Inmunoglobulina G/biosíntesis , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C3H , Bazo/inmunología , Factores de Tiempo , alfa 1-Antitripsina/biosíntesisRESUMEN
Retrovirus-mediated gene transfer into hepatocytes in vivo results in long-term gene expression. Limitations include the need to remove two-thirds of the liver and the relatively low frequency of gene transfer. To increase gene transfer without surgical hepatectomy, mouse hepatocytes were transduced in vivo with a recombinant adenovirus that transiently expressed urokinase, resulting in high rates of asynchronous liver regeneration. During the regenerative phase, in vivo retroviral-mediated gene transfer in hepatocytes resulted in 5- to 10-fold greater transduction efficiencies than that obtained by conventional partial hepatectomy. In 3-4 weeks, the architecture and microscopic structure of the recipient livers were normal. The two-viral system of achieving permanent transgene expression from hepatocytes in vivo offers an alternative approach to current ex vivo and in vivo gene-transfer models.