RESUMEN
Modern cancer therapy combines recombinant viruses with traditional chemotherapeutic agents that are metabolized by hepatic cytochrome P450 3A4 (CYP3A4). A single dose of recombinant adenovirus (Ad) expressing beta-galactosidase (AdlacZ) significantly alters CYP3A2, the correlate of CYP3A4, in rats for 14 days. Recombinant adenovirus expressing human p53 (Adp53) also suppresses CYP3A2. Plasma clearance of docetaxel (DTX) in animals given AdlacZ (3.38+/-0.22 l h(-1) kg(-1)) was significantly lower than that of those given DTX alone (7.35+/-1.22 l h(-1) kg(-1), PAsunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo
, Inhibidores Enzimáticos del Citocromo P-450
, Vectores Genéticos/genética
, Hígado/metabolismo
, Proteínas de la Membrana/metabolismo
, Taxoides/farmacocinética
, Adenoviridae/genética
, Adenoviridae/metabolismo
, Animales
, Antineoplásicos Fitogénicos/farmacocinética
, Citocromo P-450 CYP3A
, Modelos Animales de Enfermedad
, Docetaxel
, Regulación Enzimológica de la Expresión Génica/efectos de los fármacos
, Hígado/efectos de los fármacos
, Hígado/virología
, Masculino
, Ratas
, Ratas Sprague-Dawley
, Transaminasas/sangre
RESUMEN
Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos/inmunología , Polietilenglicoles , Animales , Fenómenos Químicos , Química Física , Citocinas/biosíntesis , Portadores de Fármacos , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/toxicidad , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Recuento de Plaquetas , Propiedades de Superficie , Transaminasas/sangre , Transducción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
This study summarizes our initial efforts to address an issue that is critical to the success of any multicenter gene therapy clinical trial - maintenance of vector viability during shipping and storage at remote test sites. We have identified formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and storage conditions. Adenovirus and adeno-associated virus expressing E. coli beta-galactosidase (lacZ) were suspended in blends of complex carbohydrates, cyclodextrins and various surfactants. X-gal stains of 293 and 84-31 cells were used to determine infectious titer of all preparations. Potassium phosphate-buffered preparations consistently maintained high viral titers after storage at -20 and 4 degrees C. Blends of sucrose, mannitol, and surfactant showed negligible loss of titer for 35 days at 4 degrees C. Formulations of sucrose and cyclodextrin were stable for 2 years at -20 degrees C. Negligible loss in titer was observed in unit-dose viral preparations lyophilized in sucrose and stored at 4 degrees C for 1 year after an initial loss of 0.5 log due to processing. Studies with lyophilized sucrose/mannitol blends have shown that viral recovery after processing is directly related to the final moisture content of the dried product. Virus concentration also plays a significant role in recovery after processing with highly concentrated preparations showing minimal loss in titer after lyophilization. In summary, lyophilized preparations that can be shipped and stored at 25 degrees C offer a solution to the current problem of distribution of viral vectors for clinical trials.
Asunto(s)
Adenoviridae , Dependovirus , Terapia Genética/métodos , Vectores Genéticos , Ingeniería Genética , Humanos , Transducción GenéticaRESUMEN
Despite remarkable progress in the development of both viral and non-viral gene delivery vectors for cystic fibrosis therapy, low efficiency of gene transfer to the airway epithelium is a major obstacle to clinical application. Here we develop formulations that enhance cellular absorption of adenoviral vectors. We selected excipients from a panel of pharmaceutically acceptable com-pounds known to enhance drug absorption. Transduction efficiency of the virus in the presence of each ingredient was assessed in vitro and in vivo. Mannitol and chitosan substantially enhanced transduction efficiency in vitro and augmented expression in vivo by 4 and 8 log units, respectively. The most successful formulation (a blend of sucrose, mannitol, and Pluronic F68) transduced 100% of an A549 cell population in vitro and produced areas of intense gene expression in both large and small airways in vivo with minimal toxicity. Dose response studies also indicate that when placed in this formulation, the viral dose can be lowered by 1/2 log while maintaining superior levels of transgene expression. This formulation also enhanced the physical stability of the virus. No significant loss in titer was detected from a lyophilized formulation after storage at 25 degrees C for 30 days.
Asunto(s)
Química Farmacéutica , Excipientes/farmacología , Vectores Genéticos/administración & dosificación , Adenoviridae/genética , Animales , Materiales Biocompatibles , Biopolímeros , Quitina/análogos & derivados , Quitina/metabolismo , Quitosano , Relación Dosis-Respuesta a Droga , Expresión Génica , Terapia Genética , Vectores Genéticos/metabolismo , Humanos , Manitol/metabolismo , Ratones , Ratones Endogámicos BALB C , Transducción GenéticaRESUMEN
Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy.
Asunto(s)
Adenovirus Humanos/inmunología , Expresión Génica , Vectores Genéticos/inmunología , Polietilenglicoles , Succinimidas/inmunología , Sulfonas/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Línea Celular , Genes Reporteros , Humanos , Inmunidad Celular , Inmunocompetencia , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Pulmón/inmunología , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , beta-Galactosidasa/genéticaRESUMEN
PEGylation is the covalent attachment of activated monomethoxy poly(ethylene) glycols (MPEGs) to free lysine groups of therapeutic proteins. This technology has enhanced the physical stability of proteins and ablated humoral immune responses generated against them. In this study, adenoviral vectors were modified with MPEGs activated by cyanuric chloride, succinimidyl succinate, and tresyl chloride. Under proper buffering conditions, reactions were complete within 2 hr. Transduction efficiency of PEGylated adenoviruses was not compromised by neutralizing antibodies to native adenovirus in vitro. These preparations retained titers that were significantly greater than those of the unconjugated virus after storage at 42, 25, 4, and -20 degrees C. Stability profiles of PEGylated preparations at -20 degrees C suggest that glycerol could be eliminated from formulations without significant loss of viral titer. PEGylated adenoviruses produced a two- to threefold increase in transduction in the lung when administered by intratracheal injection and a fivefold increase in transduction in the liver when administered intravenously.
Asunto(s)
Adenoviridae/química , Cápside/química , Técnicas de Transferencia de Gen , Lisina/química , Polietilenglicoles/química , Adenoviridae/genética , Adenoviridae/crecimiento & desarrollo , Animales , Tampones (Química) , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Vectores Genéticos/química , Ratones , Ratones Endogámicos C57BL , Pruebas de Neutralización , Succinatos , Sulfonas , Transfección , TriazinasRESUMEN
In order to identify suitable adenoviral vectors for efficient delivery of transgenic proteins and peptides to the intestine, the ability of adenovirus types 5 and 41 (an enterotropic serotype) to bind to and enter undifferentiated and differentiated enterocytes was assessed. FACS analysis showed no significant difference between the virions in their ability to bind to undifferentiated Caco-2 cells as 81.6% of the cellular population bound adenovirus 5 (Ad 5) and 79.8% bound Ad 41. Both virions were also efficiently internalized in this cell type as 99.6% of the cells took up Ad 5, while 95.9% took up Ad 41. In studies with differentiated enterocytes, probable targets for oral gene delivery but rather resistant to adenovirus-mediated gene transfer, 28.4% of the population internalized the Ad 5 vector and less than 10% bound the virus. Adenovirus 41 was efficiently internalized in differentiated enterocytes as 89.6% of the cellular population took up the virus while 37.4% bound the virus. These results were consistent with those observed in vivo in rat jejunum. Thus, molecularly engineered Ad 41-based recombinants could be highly efficient vectors for delivery of transgenic proteins to differentiated enterocytes.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos , Intestinos/virología , Adenoviridae/fisiología , Animales , Células CACO-2 , Diferenciación Celular , Línea Celular Transformada , Separación Celular , Citometría de Flujo , Colorantes Fluorescentes , Humanos , Mucosa Intestinal/virología , Yeyuno/ultraestructura , Yeyuno/virología , Microscopía Electrónica , Ratas , Ratas Sprague-Dawley , Rodaminas , Células Tumorales CultivadasRESUMEN
PURPOSE: In general, the intestinal epithelium is quite refractory to viral and non-viral methods of gene transfer. In this report, various cyclodextrin formulations were tested for their ability to enhance adenoviral transduction efficiency in two models of the intestinal epithelium: differentiated Caco-2 cells and rat jejunum. METHODS: Transduction efficiency of replication-deficient adenovirus type 5 vectors encoded with either the E. coli beta-galactosidase or the jellyfish green fluorescent protein gene was assessed by X-gal staining or visualization of fluorescence 48 hours after infection. In vivo experiments were performed using an intestinal loop ligation technique. RESULTS: Several formulations of neutral and positively charged beta cyclodextrins significantly enhanced adenoviral-mediated gene transfer in the selected models. The cyclodextrin formulations studied increased adenoviral transduction in the intestine by enhancing both viral binding and internalization. Viral binding was significantly increased on cell membranes treated with positively charged cyclodextrins, as seen with confocal microscopy and rhodamine-labeled virus. Permeability studies and TEER readings revealed that the most successful formulations gently disrupt cell membranes. This enhances internalization of viral particles and results in increased levels of gene expression. CONCLUSIONS: These formulations can be of value in gene transfer to cells and tissues in which adenoviral infection is limited due to a lack of fiber and alpha(v) integrin receptors. They are simple to prepare and do not affect the ability of the virus to transduce target cells.
Asunto(s)
Adenoviridae/genética , Ciclodextrinas/farmacología , Técnicas de Transferencia de Gen , Vectores Genéticos , Mucosa Intestinal/metabolismo , Animales , Células CACO-2 , Secuencia de Carbohidratos , Diferenciación Celular , Impedancia Eléctrica , Humanos , Mucosa Intestinal/efectos de los fármacos , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Datos de Secuencia Molecular , Concentración Osmolar , Ratas , Ratas Sprague-DawleyRESUMEN
Recently, replication-deficient adenoviruses have received increasing attention as vector for gene delivery and as potential vaccine carriers. With the increased use of the vector in vivo and in clinical trails, the demand for a safe, rapid, and cost effective purification process has been heightened. In this report, a simple and efficient method for the purification of large quantities of live adenoviral vectors was developed. The process involved the replacement of cesium chloride (CsCl) gradients with sucrose gradients. Ultracentrifugation times were reduced and the desalting step eliminated, decreasing total preparation time by 15 hr. A 20-80% linear sucrose gradient provided optimal recovery of infectious viral particles and positioning of the viral band in the gradient. Purification with this gradient system produced a preparation containing 1.39 x 10(14) lac-forming units (lfu)/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the process also removed all associated cellular proteins from the preparation. Studies have shown that direct lyophilization of the vector in sucrose after purification produces a product containing 1.4 x 10(12) lfu/ml. Minimal degradation was seen in the lyophilized preparation. A viral concentration of 6 x 10(11) lfu/ml was detected in the product after 150 days in storage at -20 degrees C. This approach will not only simplify the preparation of adenoviral vectors for in vivo studies and clinical trials, but will facilitate production of stable adenoviral formulations for oral gene delivery.
Asunto(s)
Adenoviridae/aislamiento & purificación , Técnicas de Transferencia de Gen , Vectores Genéticos , Adenoviridae/genética , Administración Oral , Centrifugación por Gradiente de Densidad , Recombinación Genética , Sacarosa/administración & dosificaciónRESUMEN
This report identifies formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and cooling rates. Adenovirus type 5 containing the lacZ marker gene was suspended in combinations of trehalose, sorbitol, sucrose, mannitol, glycine, CaCl2, and gelatin. X-gal stains of 293 cells were used to determine the lac-forming units (lfu)/ml of each preparation before and after treatments. Phosphate-buffered solutions (except those containing sucrose or trehalose) demonstrated a drop of 3 pH units upon freezing regardless of cryoprotectant used. Tris-buffered solutions demonstrated a variation in pH which was dependent upon chosen cryoprotectant, with 1 M trehalose exhibiting no change and a 5% mannitol/10 mM CaCl2 combination showing a 3-unit drop in pH. 4-[2-Hydroxyethyl]-1-piperazine ethanesulfonic acid (HEPES)-buffered solutions showed little change in initial pH when frozen regardless of cryoprotectant chosen. In solution, adenovirus was not affected by incubation for 24 hr in buffers ranging from pH 4 to 8. However, when the solutions were frozen, the number of remaining infectious virions was dependent upon the final pH of the suspending medium. Cryoprotectant solutions that significantly maintained viral stability during a single freeze--thaw cycle were 0.5 M sucrose, 0.5 M trehalose, and 10% sorbitol/0.4% gelatin. Long-term stability studies were performed at 4 degrees C with lyophilized sorbital/gelatin and sucrose preparations. Both formulations provided adequate stability for the adenovirus, with 2.6 and 5.6 x 10(11) lfu/ml detected 150 days after drying, respectively.
Asunto(s)
Adenoviridae/genética , Terapia Genética , Línea Celular Transformada , Liofilización , Congelación , Humanos , Concentración de Iones de Hidrógeno , Recombinación GenéticaRESUMEN
Adenoviral vectors are being developed for oral delivery of therapeutic genes to the intestine. Initial studies in the rat using mucolytics and direct application of adenovirus encoded with the interleukin-1 receptor antagonist gene to the jejunum produced limited gene expression. The goal of this study was to determine the role of integrins in adenovirus-mediated gene delivery to the intestinal epithelium. Integrins are involved in cellular differentiation and tight junction formation and mediate adenoviral internalization. Results from Caco-2 and IEC-18 cells suggest that, as enterocytes differentiate, cell-surface integrin expression decreases. Pretreatment of Caco-2 cells with RGD peptides reduced adenoviral transduction efficiency by 80% in undifferentiated cells and 20% in differentiated cells. Both differentiated and undifferentiated IEC-18 cells showed a 70% drop in transduction when pretreated with the peptide. Infection inhibition studies with monoclonal antibodies further suggest that alpha(v)beta3 and alpha6beta1 integrins play significant roles in adenoviral internalization in the intestine. Expression of integrins in cell culture models of the intestine correlated with in vivo expression in intestinal segments. These results indicate that the ileum is a prime target for efficient adenovirus-mediated gene transfer in the rat. To enhance transduction in differentiated enterocytes (probable targets for oral gene delivery), Caco-2 cells were treated with interleukin-1beta (a cytokine known to increase integrin expression) prior to administration of the virus. Transduction efficiency increased four-fold.
Asunto(s)
Adenovirus Humanos/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Integrinas/fisiología , Mucosa Intestinal , Animales , Células CACO-2 , Diferenciación Celular , Línea Celular , Impedancia Eléctrica , Femenino , Humanos , Íleon , Integrinas/análisis , Interleucina-1/farmacología , Mucosa Intestinal/citología , Yeyuno , Oligopéptidos/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/análisisRESUMEN
PURPOSE: Adenoviral (Ad) vectors have been used as efficient tools for gene therapy in various tissues, whereas in some differentiated epithelium transduction efficiency is almost abolished. METHODS: Caco-2 cell monolayers were chosen as an in vitro model for the differentiated intestinal epithelium. Fluorescence-labeled adenoviral particles were used for binding studies to cell surfaces. Internalization receptors for adenoviral uptake were detected by a fluorescence-labeled vitronectin antibody. Gene expression was studied by using the beta-galactosidase reporter gene. All experiments were done on undifferentiated and differentiated Caco-2 cells. Furthermore, adenoviral particles were allowed to bind to differentiated Caco-2 monolayers followed by a trypsinization step that disintegrates the monolayers and result in a cell suspension. Gene expression was tested after reseeding the cells into dishes. RESULTS: The results from adenoviral binding studies, vitronectin immunofluorescence detection and gene expression are in good agreement and indicate that virion binding as well as the expression of internalization receptors almost disappear in fully differentiated cells. Nonetheless, adenoviral binding to differentiated monolayers seems to be sufficient to cause up to 53% gene expression, but only if internalization of the vector can be induced by disintegrating the monolayers and releasing free vitronectin receptors. CONCLUSIONS: These findings indicate that gene transfer to the intestinal epithelium utilizing adenoviral vectors is poor and ineffective, because of the lack of sufficient internalization receptors. If these receptors can be exposed in differentiated epithelium, transduction can be made more efficient. Alternatively, a viral vector must be developed whose uptake mechanism is independent of integrin receptor expression like the enteral virus Ad40, or Ad5 could be conjugated to ligands that trigger viral internalization by receptor-mediated endocytosis.