RESUMEN
Ocular gene transfer may provide a means for arresting the retinal degeneration characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). Previously, we have shown in immunodeficient animals that recombinant adeno-associated virus (rAAV) mediates transduction of photoreceptors as well as the retinal pigment epithelium (RPE) following subretinal injection. In this study we extend these observations and show that highly purified recombinant AAV vectors encoding the reporter gene LacZ transduce photoreceptors in an immunocompetent mouse strain following subretinal injection and efficiently transduce ganglion cells after intravitreal injection. Levels of transduction increase over time. Sublethal gamma-irradiation is shown to facilitate this process.
Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Degeneración Retiniana/genética , Animales , Rayos gamma , Vectores Genéticos/genética , Operón Lac/genética , Ratones , Ratones Endogámicos BALB C , Retina/patología , Retina/efectos de la radiaciónRESUMEN
Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.
Asunto(s)
Dependovirus/genética , Amplificación de Genes , Vectores Genéticos , Simplexvirus/genética , Proteínas del Envoltorio Viral/genética , Animales , Línea Celular , Chlorocebus aethiops , Cricetinae , Dependovirus/fisiología , Eliminación de Gen , Ratones , Ratones Endogámicos BALB C , Plásmidos , Recombinación Genética , Células Vero , Replicación ViralRESUMEN
We report the development of a relatively quick and simple method for the assessment of X inactivation status for carrier determination in families affected by X-linked agammaglobulinemia (XLA). This method utilises an immunomagnetic separation technique for B cell purification and a polymerase chain reaction (PCR) based assay for the determination of methylation status at the androgen receptor (AR) gene locus to assess whether X inactivation is random or non-random at this locus. We report the results we have obtained using this assay to investigate females known to be carriers of various X-linked immunodeficiency disorders. In addition, we investigated four females from different families affected by XLA, two of whom were of unknown carrier status, and we discuss the results obtained with this and other X-inactivation assays. A similar assay has recently been described by Allen et al. (1992) and applied to members of one family affected by XLA.
Asunto(s)
Agammaglobulinemia/genética , Compensación de Dosificación (Genética) , Tamización de Portadores Genéticos/métodos , Agammaglobulinemia/diagnóstico , Linfocitos B/metabolismo , Secuencia de Bases , Separación Celular , ADN/química , ADN/genética , Cartilla de ADN/genética , Femenino , Ligamiento Genético , Humanos , Metilación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores Androgénicos/genética , Cromosoma XRESUMEN
A major limitation of adeno-associated virus (AAV) based vectors for clinical applications to date is the production of high-titer recombinant AAV vector stocks. Despite recent improvements, the amount of recombinant adeno-associated virus vectors (rAAV) particles produced per cell continues to be significantly lower than that of wild-type AAV. In this study, an HSV-based system for rAAV production was used to examine the influence of different parameters including transfection conditions (vector-to-packaging plasmid ratio, amount of total transfected DNA, cell confluency) and multiplicity of infection of herpes helper virus on the resulting titre of rAAV stocks. For herpes helper virus, time-course experiments were carried out to analyse the effect on rAAV yields up to 72 h postinfection and to determine the ideal harvesting time. Taken together, the optimized production scheme consistently yields more than 3x10(3) transducing units per producer cell.
Asunto(s)
Dependovirus/genética , Dependovirus/fisiología , Vectores Genéticos , Virus Helper/genética , Simplexvirus/genética , Animales , Línea Celular , ADN Viral , Terapia Genética/métodos , Células HeLa , Virus Helper/fisiología , Humanos , Plásmidos/genética , Recombinación Genética/genética , Simplexvirus/patogenicidad , Simplexvirus/fisiología , Transducción Genética , Transfección , Cultivo de Virus , Replicación ViralRESUMEN
PURPOSE: We examined the efficacy and cytopathogenicity of adeno-associated (AAV) and herpes simplex viruses (HSV) as vectors for gene transfer to corneal endothelial cells (CECs). METHODS: Recombinant AAV and HSV were examined for their ability to deliver a lacZ histochemical marker gene to whole-thickness rabbit and human corneas ex vivo. Transgene expression was detected with histochemistry and quantified by a colorimetric assay. RESULTS: Rabbit and human corneas transduced with AAV showed increasing numbers of cells expressing marker gene over a 3- to 4-week period. Using 2.5 x 10(6) or 1.5 x 10(7) infective units for rabbit and human corneal specimens, respectively, approximately 2% of CECs expressed the reporter gene. HSV (10(6) plaque-forming units/specimen) transduced approximately 5% of rabbit and human CECs but showed cytotoxicity. In contrast to the duration of recombinant AAV-mediated lacZ expression, recombinant HSV expression was maximal at day 1 and declined to low levels at day 7. CONCLUSION: AAV is a promising vector, but its usefulness for corneal transduction is currently limited by the technical difficulties preparing high titres. The HSV vector examined is efficient but needs further genetic modification to prolong transgene expression and reduce its toxicity.
Asunto(s)
Adenoviridae/genética , Endotelio Corneal/enzimología , Técnicas de Transferencia de Gen , Vectores Genéticos , Herpesvirus Humano 1/genética , Operón Lac/genética , beta-Galactosidasa/metabolismo , Animales , Endotelio Corneal/virología , Expresión Génica , Histocitoquímica , Humanos , ConejosRESUMEN
In two trials, 68 cattle and buffalo calves, four to 10 months of age, were vaccinated with a vaccine containing a live streptomycin dependent mutant of Pasteurella multocida type B, isolated in Sri Lanka. Immunity was assessed using the passive protection test in mice with cattle and buffalo sera before and two or three weeks after vaccination. A single dose of vaccine conferred immunity in 66.6 to 83.3 per cent of cattle and 100 per cent of buffalo calves. A booster dose given three weeks later enhanced the immunity in cattle. There was no significant difference in the response, whether the vaccine was administered by the subcutaneous or intramuscular route. No adverse reactions were observed in any of the vaccinated animals.
Asunto(s)
Vacunas Bacterianas , Búfalos , Enfermedades de los Bovinos/inmunología , Septicemia Hemorrágica/veterinaria , Infecciones por Pasteurella/veterinaria , Pasteurella/inmunología , Estreptomicina/farmacología , Animales , Anticuerpos Antibacterianos/análisis , Bovinos , Septicemia Hemorrágica/inmunología , Mutación , Pasteurella/efectos de los fármacosRESUMEN
An international effort (sponsored by the Australian Centre for International Agricultural Research) is being made to develop oral vaccines that will protect village chickens against Newcastle disease. The vaccines being used are derivatives of the avirulent Australian V4 strain that have been selected for enhanced heat resistance. The present study, undertaken in Sri Lanka, used local processed (parboiled) rice as a vehicle for the vaccine. Chickens receiving two doses of vaccine on cooked, parboiled rice were completely protected against contact challenge with the virulent SL 88/1 Sri Lankan strain of Newcastle disease virus Chickens kept in contact with these vaccinated chickens were similarly protected. Lower levels of protection were achieved with vaccine given on uncooked parboiled rice. V4 vaccine administered intranasally also gave complete protection. Serums from vaccinated chickens that survived challenge were tested for haemagglutination-inhibition antibodies, using both vaccine virus and challenge virus as antigens. Titres were higher against vaccine virus.
Asunto(s)
Pollos , Enfermedad de Newcastle/prevención & control , Virus de la Enfermedad de Newcastle/inmunología , Vacunas Virales/administración & dosificación , Administración Oral , Animales , Anticuerpos Antivirales/biosíntesis , Manipulación de Alimentos , Masculino , Oryza , Vacunas Virales/inmunologíaAsunto(s)
Búfalos , Septicemia Hemorrágica/veterinaria , Pasteurella multocida , Animales , Antígenos Bacterianos/análisis , Septicemia Hemorrágica/patología , Técnicas para Inmunoenzimas , Macrófagos Alveolares/microbiología , Macrófagos Alveolares/patología , Pasteurella multocida/aislamiento & purificación , Pleura/microbiología , Pleura/patología , Alveolos Pulmonares/microbiología , Alveolos Pulmonares/patologíaAsunto(s)
Infecciones por Citomegalovirus/complicaciones , Citomegalovirus/aislamiento & purificación , Histiocitosis de Células no Langerhans/diagnóstico , Insuficiencia Multiorgánica/diagnóstico , Terapia Combinada , Infecciones por Citomegalovirus/terapia , Femenino , Estudios de Seguimiento , Histiocitosis de Células no Langerhans/etiología , Histiocitosis de Células no Langerhans/terapia , Humanos , Persona de Mediana Edad , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/terapia , Medición de RiesgoRESUMEN
Data on the mortality of cattle and buffaloes in 62 epizootics of haemorrhagic septicaemia (HS) in the HS enzootic and non-enzootic regions of Sri Lanka was collected and subjected to statistical analysis. It was found that the overall mortality for buffaloes was higher than for cattle (45.2 and 15.8% respectively, P less than 0.001). For buffaloes in enzootic areas only the overall mortality was 29% whilst in non-enzootic areas it was 64.5% (P less than 0.05). In the enzootic areas deaths were mainly confined to young animals whereas in the non-enzootic areas when sporadic outbreaks of HS occurred deaths were scattered over all age groups. The vaccines used, vaccination schedules adopted and the coverage of vaccination in these herds did not appear to influence the mortality among buffaloes in enzootic areas to a statistically significant degree.
Asunto(s)
Búfalos , Enfermedades de los Bovinos/mortalidad , Brotes de Enfermedades/veterinaria , Septicemia Hemorrágica/veterinaria , Infecciones por Pasteurella/veterinaria , Animales , Bovinos , Septicemia Hemorrágica/mortalidad , Estaciones del Año , Especificidad de la Especie , Sri Lanka , Vacunación/veterinariaRESUMEN
Fifty-seven young buffaloes were experimentally infected or naturally exposed to haemorrhagic septicaemia (HS). Of these animals 32 became immune carriers. They were observed in groups for periods up to 360 days to monitor the appearance of pasteurellae in the nasopharynx and antibody status. In most animals pasteurellae appeared in the nasopharynx for a short period initially and disappeared. The organism reappeared intermittently and the longest observed period of reappearance was 215 days after exposure. All animals showed rising antibody titres with a peak lasting for 150 to 180 days and declining thereafter. Pasteurellae persisted in the tonsils and were isolated from 20 out of 27 carriers after slaughter. The longest period when isolation was made after slaughter was 229 days. The organism lodged in the tonsils was unaffected by antibacterial therapy using drugs to which the organism displayed in vitro sensitivity. It is concluded that in HS, carrier animals exist in an active as well as a latent state, the former appearing for short intermittent periods between long latent periods, when pasteurellae continue to remain in the tonsils which constitute a long-term reservoir.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Búfalos/inmunología , Portador Sano/veterinaria , Septicemia Hemorrágica/veterinaria , Pasteurella/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Búfalos/sangre , Portador Sano/sangre , Portador Sano/inmunología , Cloranfenicol/uso terapéutico , Septicemia Hemorrágica/sangre , Septicemia Hemorrágica/etiología , Septicemia Hemorrágica/inmunología , Nasofaringe/microbiología , Oxitetraciclina/uso terapéutico , Pasteurella/aislamiento & purificación , Factores de TiempoRESUMEN
A large number of streptomycin dependent mutants were produced from bovine haemorrhagic septicaemia strains of Pasteurella multocida. The mutants required a minimum concentration of 25-50 microgram/mL streptomycin for growth and tolerated a concentration of 200 mg/mL. These mutants were avirulent to mice, when inoculated alone, but some mutants killed mice when inoculated with streptomycin. Biochemically all mutants were uniform and similar to the wild type. Most mutants were stable, but a few produced streptomycin independent revertants. The rate of reversion varied with each mutant. Most revertants were highly virulent for mice, some totally avirulant and a few relatively avirulent.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Septicemia Hemorrágica/veterinaria , Mutación , Infecciones por Pasteurella/veterinaria , Pasteurella/metabolismo , Estreptomicina/metabolismo , Animales , Bovinos , Septicemia Hemorrágica/microbiología , Ratones , Pasteurella/aislamiento & purificación , Pasteurella/patogenicidadRESUMEN
We describe a family affected by X linked severe combined immunodeficiency (SCIDX1) in which genetic prediction of carrier status was made using X chromosome inactivation studies together with limited genetic linkage analysis. Linkage studies in this family showed a confusing pattern of inheritance for the X chromosome. A female with a random pattern of X chromosome inactivation in her T cells appeared to have inherited an X chromosome with four recombinations within 10 cM. The odds of this happening in a single meiotic event make this an unlikely explanation. Data obtained from studying the X chromosomes of her two unaffected sons showed that this could be explained simply on the basis of her having inherited three alleles each of the relevant polymorphic DNA loci. We used fluorescent in situ hybridisation (FISH) to confirm that this person had inherited three complete X chromosomes. Thus, although the results from X chromosome inactivation analysis indicated that this subject was not a carrier of the affected chromosome, FISH and genetic linkage analysis showed clearly that the affected chromosome had been inherited. The implications of this finding for diagnosis of carrier status in this family and for other families with X linked inherited immunodeficiencies is discussed.
Asunto(s)
Ligamiento Genético , Inmunodeficiencia Combinada Grave/genética , Trisomía/genética , Cromosoma X , Mapeo Cromosómico , Femenino , Tamización de Portadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Masculino , Linaje , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Retinal angiogenesis is a central feature of the leading causes of blindness. Current treatments for these conditions are of limited efficacy and cause significant adverse effects. In this study, we evaluated the angiostatic effect of gene transfer of the soluble VEGF receptor sFlt-1 in a mouse model of ischaemia-induced retinal neovascularisation using adenovirus and adeno-associated virus (AAV) vectors. We induced proliferative retinopathy in mice by exposure to 75% oxygen from postnatal day 7 (p7) to p12 and injected intravitreally recombinant viral vectors expressing the reporter green fluorescent protein (GFP) or vectors expressing the VEGF inhibitor sFlt-1. Efficient adenovirus-mediated GFP expression was evident in cells of the corneal endothelium and iris pigment epithelium. AAV-mediated GFP expression was evident in ganglion cells and cells of the inner nuclear layer of the retina. Vector-mediated sFlt-1 expression was confirmed by ELISA of pooled homogenised whole eyes. Injection of either vector expressing sFlt-1 resulted in a reduction in the number of neovascular endothelial cells by 56% and 52% for adenovirus and AAV vectors, respectively (P < 0.05). Local gene transfer of sFlt-1 consistently inhibits experimental retinal neovascularisation by approximately 50% and offers a powerful novel approach to the clinical management of retinal neovascular disorders.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Terapia Genética/métodos , Neovascularización Retiniana/terapia , Adenoviridae/genética , Animales , Dependovirus/genética , Expresión Génica , Vectores Genéticos/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Cadenas Pesadas de Miosina , Miosina Tipo IIB no MuscularRESUMEN
The retinal degeneration slow (rds or Prph2(Rd2/Rd2)) mouse, a model of recessive retinitis pigmentosa, lacks a functional gene encoding peripherin 2. This membrane glycoprotein is required for the formation of photoreceptor outer segment discs. The striking feature of the rds mouse is the complete failure to develop outer segments. We have previously examined the short-term effect of gene replacement therapy using an adeno-associated (AAV) vector and demonstrated induction of outer segments and improvement of photoreceptor function. Here we have extended our analysis and have demonstrated that the potential for ultrastructural improvement is dependent upon the age at which animals are treated, but the effect of a single injection on photoreceptor ultrastructure may be long-term. However, there was no significant effect on photoreceptor cell loss, irrespective of the date of administration, despite the improvements in morphology and function. Our investigation excluded procedure-related damage, vector toxicity and immune responses as major factors which might counteract the benefits of photoreceptor restoration, but suggested that transgene over-expression is of significance. These findings suggest that successful gene therapy in patients with photoreceptor defects may ultimately depend upon intervention in early stages of disease and upon accurate control of transgene expression.
Asunto(s)
Terapia Genética , Proteínas de Filamentos Intermediarios/genética , Glicoproteínas de Membrana , Proteínas del Tejido Nervioso/genética , Degeneración Retiniana/terapia , Animales , Animales Recién Nacidos , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Humanos , Proteínas de Filamentos Intermediarios/administración & dosificación , Proteínas Luminiscentes/administración & dosificación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos CBA , Ratones Mutantes , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Proteínas del Tejido Nervioso/administración & dosificación , Periferinas , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Epitelio Pigmentado Ocular/metabolismo , Epitelio Pigmentado Ocular/patología , Epitelio Pigmentado Ocular/ultraestructura , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Retina/metabolismo , Retina/patología , Retina/ultraestructura , Degeneración Retiniana/genética , Retinitis Pigmentosa/genética , Factores de Tiempo , TransfecciónRESUMEN
Recombinant AAV vectors mediate efficient and sustained transgene expression in retinal tissues and offer a powerful approach to the local, sustained delivery of angiostatic proteins for the treatment of ocular neovascular disorders. The application of such strategies may also require regulated gene expression to minimize the potential for unwanted adverse effects. In this study, we have evaluated the effect of a hypoxia-responsive element (HRE) on the kinetics of recombinant adeno-associated (rAAV)-mediated reporter gene expression in murine models of retinal and choroidal neovascularization. In murine ischaemia-induced retinal neovascularization, intravitreal delivery of rAAV.HRE.GFP results in reporter gene expression specifically at sites of vascular closure during the period of active neovascularization and not after vector delivery in normal controls. In murine laser-induced choroidal neovascularization, subretinal delivery of rAAV.HRE.GFP results in reporter gene expression at sites of active neovascularization but not elsewhere or after vector delivery in normal controls. HRE-driven gene expression offers an attractive strategy for the targeted and regulated delivery of angiostatic proteins to the retina in the management of neovascular disorders.
Asunto(s)
Neovascularización Coroidal/terapia , Regulación de la Expresión Génica , Marcación de Gen/métodos , Terapia Genética/métodos , Neovascularización Retiniana/terapia , Animales , Hipoxia de la Célula/genética , Dependovirus/genética , Técnicas de Transferencia de Gen , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes , Hipoxia , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Elementos de Respuesta/genética , TransgenesRESUMEN
Chronic granulomatous disease (CGD) comprises a heterogeneous group of inherited conditions characterized biochemically by disordered function of a unique multicomponent enzyme system present in phagocytic cells, the NADPH-oxidase. Clinically, it is characterized by recurrent bacterial and fungal infections that are relatively resistant to treatment by conventional means. Curative bone marrow transplantation has been successfully achieved in a small number of cases, but the wider application of this procedure is limited by availability of suitable donor material. Somatic gene therapy would overcome this problem, and several groups have now shown correction of the biochemical defect in hematopoietic cells by retrovirus-mediated gene transfer. However, the failure of the current generation of retroviral vectors to efficiently transduce quiescent cells greatly restricts their potential for gene transfer to pluripotent hematopoietic stem cells. Given these limitations, we have constructed vectors based on adeno-associated virus and used these to transfer a functional copy of the p47phox gene to immortalized B cells derived from patients with p47phox-deficient autosomal recessive CGD. We show stable expression of protein and restoration of NADPH-oxidase function in these cells in the absence of selection. Adeno-associated virus vectors may overcome some of the limitations of retroviral gene delivery systems and may therefore be a useful vehicle for curative gene therapy of CGD and other primary immunodeficiencies.
Asunto(s)
Dependovirus/genética , Terapia Genética , Vectores Genéticos , Enfermedad Granulomatosa Crónica/terapia , NADH NADPH Oxidorreductasas/genética , Linfocitos B/enzimología , Linfocitos B/virología , Southern Blotting , Western Blotting , Línea Celular Transformada , Células Cultivadas , Dependovirus/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Enfermedad Granulomatosa Crónica/enzimología , Enfermedad Granulomatosa Crónica/genética , Humanos , NADH NADPH Oxidorreductasas/biosíntesis , NADH NADPH Oxidorreductasas/deficiencia , NADPH Oxidasas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Estallido Respiratorio , Superóxidos/metabolismo , TransfecciónRESUMEN
The human parvovirus, adeno-associated virus-2 (AAV-2), has many attributes that recommend its use as a gene transfer vehicle, including a broad tissue tropism, the ability to integrate stably into the host genome, and efficient transduction of cells which proliferate slowly. However, application to human gene therapy is currently limited by existing methods for generation of recombinant AAV (rAAV), resulting in relatively low transducing titres. In an attempt to overcome some of these problems, we have developed a defective adenoviral vector which improves the efficiency of rAAV vector delivery to cells in which rAAV is propagated, and from which the rAAV genome can be efficiently rescued. A functional copy of the p47phox gene was successfully transferred to cell lines derived from patients with autosomal recessive chronic granulomatous disease (CGD) by rAAV recovered in this way, and function of the NADPH-oxidase was restored to levels which were stable for at least 8 weeks. This method for generation of rAAV, although still limited by the need for cotransfection of AAV Rep and Cap functions, may permit recovery of higher titre transducing stocks from cell lines in which these genes are stably incorporated, and significantly reduces the risk of contamination with wild-type adenovirus (wtAd).