RESUMEN
BACKGROUND: A decrease in sarcoplasmic reticulum Ca(2+) pump (SERCA2) activity is believed to play a role in the impairment of diastolic function of the failing heart. Because the expression ratio of phospholamban (PL) to SERCA2 may be a target to improve contractile dysfunction, a PL antisense RNA strategy was developed under the control of either a constitutive cytomegalovirus (CMV) or an inducible atrial natriuretic factor (ANF) promoter. The latter is upregulated in hypertrophied and failing heart, allowing "induction-by-disease" gene therapy. METHODS AND RESULTS: Part of the PL cDNA was cloned in antisense and sense directions into adenovectors under the control of either a CMV (Ad5CMVPLas and Ad5CMVPLs, respectively) or ANF (Ad5ANFPLas and Ad5ANFPLs, respectively) promoter. Infection of cultured rat neonatal cardiomyocytes with Ad5CMVPLas reduced PL mRNA to 30+/-7% of baseline and PL protein to 24+/-3% within 48 and 72 hours, respectively. The effects were vector dose dependent. Ad5CMVPLas increased the Ca(2+) sensitivity of SERCA2 and reduced the time to 50% recovery of the Ca(2+) transient. A decrease of PL protein was also achieved by infection with Ad5ANFPLas, and the presence of the hypertrophic stimulus, endothelin-1, led to enhanced downregulation of PL. The adenovectors expressing PL sense RNA had no effect on any of the tested parameters. CONCLUSIONS: Vector-mediated PL antisense RNA expression may become a feasible approach to modulate myocyte Ca(2+) homeostasis in the failing heart. The inducible ANF promoter for the first time offers the perspective for induction-by-disease gene therapy, ie, selective expression of therapeutic genes in hypertrophied and failing cardiomyocytes.
Asunto(s)
Proteínas de Unión al Calcio/genética , Regulación de la Expresión Génica/fisiología , Corazón/fisiología , Contracción Miocárdica/fisiología , Oligonucleótidos Antisentido/genética , Adenoviridae , Animales , Proteínas de Unión al Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/genética , Células Cultivadas , Endotelina-1/genética , Terapia Genética , Vectores Genéticos , Oligonucleótidos Antisentido/administración & dosificación , Regiones Promotoras Genéticas/genética , RatasRESUMEN
During the past few years great efforts have been made to construct and to test human factor VIII (hFVIII) and IX (hFIX) vectors suitable for haemophilia gene therapy in vivo. However, little is known about the molecular mechanisms of persistence and shut-off of transgene expression in the target organs after gene transfer using recombinant adenoviral vectors. To evaluate low transgene mRNA levels in different tissues, especially at long times after the gene transfer, the common northern blot method is often not sensitive enough. For this reason we developed a new, highly sensitive and species-specific method for hFIX mRNA quantification and employed it in mice treated with an adenoviral vector (Ad5CMVFIX) expressing human FIX. In addition to its very high sensitivity (lowest detection level=1 fg RNA), the method was shown to be strictly species-specific, since hFIX mRNA signals were never detected in untreated mice. In a long-term study of 18 vector-treated mice we compared the human FIX:Ag levels in the mouse plasma, the human FIX mRNA levels and human FIX vector DNA concentrations in the mouse liver. We found that a slow but continuous decrease of hFIX:Ag levels in mouse plasma was associated with a corresponding decrease of hFIX mRNA levels in the liver. However, the Ad5CMVFIX vector DNA levels did not decrease to a comparable degree, suggesting that the decrease of human FIX:Ag levels in mouse plasma is, to a significant extent, also caused by CMV promotor shut-off and only to a minor degree by loss of vector DNA.
Asunto(s)
Vectores Genéticos/análisis , Transgenes/genética , Adenoviridae/genética , Animales , Factor IX/genética , Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
Nitric oxide (NO) produced in endothelial cells has been implicated in the regulation of blood pressure, regional blood flow, inhibition of platelet aggregation, and endothelial and vascular smooth muscle cell proliferation. In a variety of cardiovascular disease states, such as atherosclerosis, arterial hypertension, and restenosis, expression of endothelial NO synthase (NOS-III) and endothelial NO production appear to be altered. Thus, NOS-III is an attractive target for cardiovascular gene therapy for which adenoviral vectors are one of the most effective vector systems. Therefore, a recombinant adenoviral vector expressing NOS-III (adenovirus type 5 [Ad5] cytomegalovirus [CMV] NOSIII) was constructed and biochemically and pharmacologically characterized both in vitro and in intact cells. Ad5CMVNOSIII-derived recombinant NOS-III was successfully expressed, as shown by immunoprecipitation and immunocytochemistry, and biologically active, as shown by functional assays in human primary umbilical vein and EA.hy926 endothelial cells, as well as 293 human embryonic kidney and Chinese hamster ovary cells. The Km values for NADPH and L-arginine and the Ka for tetrahydrobiopterin as well as the enzyme's dependency on other cofactors were similar to recombinant reference enzyme and literature values. NOS-III expression levels correlated linearly with the multiplicity of infection with Ad5CMVNOSIII and lasted for at least 8 days. NOS-III transfection inhibited endothelial cell proliferation. In conclusion, adenovirus-mediated gene transfer of Ad5CMVNOSIII to vascular and nonvascular cells resulted in the dose-dependent expression of intact, physiologically regulated, and functionally active NOS-III.
Asunto(s)
Adenoviridae/genética , Endotelio Vascular/enzimología , Técnicas de Transferencia de Gen , Óxido Nítrico Sintasa/genética , Óxido Nítrico/metabolismo , Adenoviridae/fisiología , Animales , Células CHO , Células Cultivadas , Clonación Molecular/métodos , Cricetinae , Endotelio Vascular/metabolismo , Vectores Genéticos , Humanos , Cinética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III , Replicación ViralRESUMEN
High transgene stabilities of 1 year and more have been reported in immunodeficient hosts after adenovirus-mediated gene transfer. Transgene persistence of this duration could be due to inherently high stability of the episomal viral vector DNA. An alternative explanation would be limited 'autoreplication' of transgenic vector DNA, just sufficient to counteract slow but continuous degradation within the host cells. Autoreplication could occur in the absence of any production of infectious virus particles, based on residual activity of the adenoviral DNA replication system only. To test this hypothesis, a series of DNA metabolic labeling studies in non-permissive cells cultures transfected with different vectors was conducted. Due to extensive E1 region deletions none of the vectors was able to produce viral progeny in non-permissive cells. Vectors fell into two categories, however, with respect to their autoreplication potential. Neosynthesis of vector DNA in non-permissive vector-transfected cells was readily detectable in 'type A', but not in 'type B' vectors. In addition to their different transgene expression cassettes, vector DNA sequencing showed a less extensive E1 deletion in type A (nucleotides 453-3333 of wild-type virus) as compared to type B vectors (nucleotides 325-3523). Autoreplication was also associated with high transcriptional activity of several viral genes (E1B-14k, adenoviral DNA polymerase, single-strand DNA-binding protein, E4-25k), in contrast to type B vectors. In addition to these 'wild-type' transcripts, 'irregular' recombinant transcripts were detected in autoreplication vectors which contained the transgenic cDNA in conjunction with adenoviral vector sequences. Exogenous or cryptic promotors may (under certain conditions) enhance the transcriptional activity of a vector in such a way that autoreplication occurs. Conditions determining the level of transcriptional enhancement (extent of E1 deletion, type of promoter and transgene, etc) need to be further defined before rational design of adenovectors with high autoreplication capacity becomes possible. In summary, we have shown autoreplication to be a novel feature of certain E1-deleted adenovectors with likely relevance for their stability in vivo, but also with possibly adverse consequences for target cell function or vector immunogenicity. Full characterization of adenoviral vector systems should therefore include a description of their autoreplication capacity.
Asunto(s)
Adenoviridae/genética , Genes Virales/genética , Replicación Viral/genética , Células Cultivadas , Eliminación de Gen , Vectores Genéticos/genética , Genoma Viral , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Secuencia de ADNRESUMEN
Deficiency of the serine proteinase inhibitor (serpin) alpha 1-antitrypsin (alpha 1AT) is the most common autosomal recessive genetic disorder in Northern Europe. alpha 1AT is the physiological regulator of the proteolytic enzyme neutrophil elastase and severe deficiency states are associated with an increased risk of developing chronic obstructive pulmonary disease (COPD) as a consequence of chronic proteolytic damage to the lungs. Among the known mutations of the alpha 1AT gene causing severe alpha 1AT deficiency and COPD a few alleles are also associated with liver disease. When expressed in cell cultures, all these particular alleles cause intracellular alpha 1AT accumulation which appears to be a prerequisite for the development of hepatic injury. Liver disease is seen in only a small fraction of all patients carrying such alleles, however. The reason for this is not completely clear, but there is evidence that PI ZZ individuals 'susceptible' to liver disease carry an additional defect affecting protein degradation in the endoplasmic reticulum (ER). We characterise a newly identified defective alpha 1AT allele PI Mwürzburg (Pro369 [CCC] to Ser [TCC]) associated with a complete intracellular transport block in cell cultures in vitro. The allele PI Mheerlen, a previously described different amino acid substitution in the same position as PI Mwürzburg (Pro369 [CCC] to Leu [CTC]) is shown to cause complete retention of the mutant alpha 1AT in the ER, too, whereas in the recently described mutant allele PI Q0lisbon (Thr68 [ACC] to Ile [ATC]) a significantly reduced alpha 1AT secretion from the cells was observed. Adenovirus-mediated recombinant expression of mutant Mwürzburg and Mheerlen, and of wild-type alpha 1AT in mouse liver in vivo showed that the mutant human proteins were not secreted into the mouse plasma, in contrast with human wild-type alpha 1AT which circulated at high concentrations over several weeks. In summary, all transportation deficient alpha 1ATs analysed have the potential to cause lung disease in the homozygous state or in heterozygous carriers of another deficiency allele, and they may also cause liver disease in certain patients. The mutant PI Mwürzburg and Mheerlen alpha 1ATs are completely retained within synthesising cells, and the molecular defect of transportation in these two alleles may be similar to that in the common PI Z allele. The molecular defect in the PI Q0lisbon allele (Thr68Ile) shows similarity with the immediately neighbouring Mmineral springs mutation (Gly67Glu).
Asunto(s)
Alelos , Isoleucina/genética , Leucina/genética , Prolina/genética , Inhibidores de Serina Proteinasa/genética , Serina/genética , Treonina/genética , alfa 1-Antitripsina/genética , Adulto , Animales , Niño , Femenino , Expresión Génica , Humanos , Masculino , Ratones , LinajeRESUMEN
In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.
Asunto(s)
Agonistas de los Canales de Cloruro , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/agonistas , Isoenzimas/metabolismo , Adenoviridae/genética , Animales , Línea Celular , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Técnicas de Transferencia de Gen , Isoenzimas/genética , Técnicas de Placa-Clamp , Fosforilación , RatasRESUMEN
Complex interactions between replication deficient adenoviral vectors (Ad5) and the immune system of the host influence the stability of transgenes in vivo. Vector-infected cells are attacked by diverse cellular immune mechanisms which limit transgene persistence. On the other hand, the products of several E3 region genes of wild-type adenovirus can suppress host immune reactions by interference with the expression of MHC class I molecules and by other mechanisms. We have developed an adenoviral vector for human factor IX (Ad5E3+FIX) which carries the E3 region of wild-type adenovirus, and an E3-deleted vector of otherwise similar structure (ad5 delta E3FIX). Intravenous injection of Ad5E3+FIX in C57BI/6 mice resulted in expression levels up to 6000 ng/ml of recombinant human factor IX in the mouse plasma and in enhanced transgene stability as compared with the vector Ad5 delta E3FIX. Whereas expression from E3-deleted vectors was essentially turned off 8 weeks after the gene transfer, the vector Ad5E3+FIX3+FIX supported transgene expression with therapeutic levels of human factor IX in the mouse plasma for > 4 months. The enhanced stability of the vector Ad5E3+FIX appears to be a consequence of efficient E3 region-mediated suppression of the host's antivector immune response. As an additional approach to improving transgene stability the influence of transient CD4+ T cell depletion of the host was investigated. CD4+ cytotoxic T lymphocytes contribute to the clearance of adenovirus-infected cells and play a pivotal role in the activation of CD8+ cytotoxic T cells and as helper T cells in the formation of human adenovirus neutralizing antibodies (HANA). Transient anti-CD4 treatment of the host limited to the time of vector injection resulted in a significant prolongation of transgene expression from the factor IX vector Ad5E3+FIX and a luciferase vector Ad5Luc. The combination of transient anti-CD4 treatment of the host and integration of a complete E3 region in an adenoviral vector resulted in markedly improved transgene stability after gene transfer to the liver (therapeutic factor IX levels for > 6 months).
Asunto(s)
Adenoviridae , Proteínas E3 de Adenovirus/genética , Anticuerpos Monoclonales/farmacología , Antígenos CD4/inmunología , Factor IX/biosíntesis , Factor IX/genética , Expresión Génica , Vectores Genéticos , Animales , Formación de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Cartilla de ADN , Endotelio Vascular , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Subgrupos de Linfocitos T/inmunología , Transfección , Venas UmbilicalesRESUMEN
Mouse chromosomes possessing multiple Robertsonian rearrangements (Rb chromosomes) have been examined using fluorescence in situ hybridization with the telomeric consensus sequence (TTAGGG)n. No hybridization signals were detected at the primary constriction of Rb chromosomes. This observation leads us to conclude that the formation of Rb chromosomes in the mouse is invariably associated with the loss of telomeric regions. More significantly, a further alteration in regions flanking the primary constrictions was observed after hybridizing with a minor satellite DNA probe to Rb chromosomes. It seems likely that the breakpoints required for a Robertsonian process do not include telomeric sites exclusively but extend to the adjacent pericentromeric regions of the original acrocentric chromosomes. In contrast to previous reports, these observations demonstrate the elimination of substantial amounts of chromosomal DNA during the formation of mouse Rb chromosomes.