RESUMEN

Platelet-derived growth factor (PDGF) gene therapy offers promise for tissue engineering of tooth-supporting alveolar bone defects. To date, limited information exists regarding the safety profile and systemic biodistribution of PDGF gene therapy vectors when delivered locally to periodontal osseous defects. The aim of this preclinical study was to determine the safety profile of adenovirus encoding the PDGF-B gene (AdPDGF-B) delivered in a collagen matrix to periodontal lesions. Standardized alveolar bone defects were created in rats, followed by delivery of matrix alone or containing AdPDGF-B at 5.5 x 10(8) or 5.5 x 10(9) plaque-forming units/ml. The regenerative response was confirmed histologically. Gross clinical observations, hematology, and blood chemistries were monitored to evaluate systemic involvement. Bioluminescence and quantitative polymerase chain reaction were used to assess vector biodistribution. No significant histopathological changes were noted during the investigation. Minor alterations in specific hematological and blood chemistries were seen; however, most parameters were within the normal range for all groups. Bioluminescence analysis revealed vector distribution at the axillary lymph nodes during the first 2 weeks with subsequent return to baseline levels. AdPDGF-B was well contained within the localized osseous defect area without viremia or distant organ involvement. These results indicate that AdPDGF-B delivered in a collagen matrix exhibits acceptable safety profiles for possible use in human clinical studies.

Asunto(s)

Pérdida de Hueso Alveolar/terapia , Regeneración Ósea/fisiología , Terapia Genética , Vectores Genéticos/metabolismo , Proteínas Proto-Oncogénicas c-sis/genética , Adenovirus Humanos/genética , Pérdida de Hueso Alveolar/patología , Animales , Regeneración Ósea/genética , Huesos/metabolismo , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Masculino , Proteínas Proto-Oncogénicas c-sis/metabolismo , Ratas , Ratas Sprague-Dawley , Seguridad , Transducción Genética

RESUMEN

OBJECT: Adenovirus transduction in gene therapy is dependent on the expression of the coxsackie virus-adenovirus receptor (CAR) for initial binding and on the integrin receptors (avb3, avb5) for viral internalization. Low and variable expression of CAR may be responsible for the low transduction rates seen with native adenoviral vectors. The goal of this study was to demonstrate increased transduction efficiency by retargeting the adenovirus with a fibroblast growth factor (FGF) ligand, FGF-2. METHODS: The retargeted adenoviruses were used to transduce human glioblastoma multiforme (GBM)-derived ECs (tumor-associated brain endothelial cells [TuBECs]), in which there is minimal CAR expression but a high expression of FGF receptor (FGFR). The results demonstrate that the transduction efficiency of TuBECs can reach as high as 80% when one uses an FGF2-conjugated adenovirus containing green fluorescent protein (FGF2-AdGFP) yet be only 5% when one uses the native adenovirus (AdGFP). The TuBECs were transduced with either a native adenovirus (AdHSV-TK) or a retargeted adenovirus (FGF2-AdHSV-TK), both of which carry the suicide herpes simplex virus-thymidine kinase (HSV-TK) gene. Administered as a cytotoxic prodrug, ganciclovir induced a significant decline in the proliferation rate and increased apoptosis in TuBECs treated with the retargeted adenovirus, compared with its effect on TuBECs treated with the native adenovirus. Increased transduction efficiency was determined by performing GFP-based flow cytometry, and the expression of the TK protein by the retargeted adenovirus was assessed by performing an immunohistochemical analysis focused on HSV-TK. The mechanism of cytotoxicity was determined to be apoptosis by performing a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. CONCLUSIONS: Fibroblast growth factor-2-retargeted adenoviral vectors may be used to increase the transduction of GBM-derived endothelial cells, enabling a new and efficient antiangiogenesis strategy for the treatment of malignant gliomas.

Asunto(s)

Neoplasias Encefálicas/terapia , Factor 2 de Crecimiento de Fibroblastos/farmacología , Terapia Genética/métodos , Vectores Genéticos/efectos de los fármacos , Glioblastoma/terapia , Neovascularización Patológica/terapia , Viroterapia Oncolítica/métodos , Adenoviridae/genética , Inhibidores de la Angiogénesis/genética , Antivirales/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/fisiopatología , Células Cultivadas , Resistencia a Medicamentos/genética , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/virología , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Ganciclovir/farmacología , Genes Transgénicos Suicidas/genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Glioblastoma/irrigación sanguínea , Glioblastoma/fisiopatología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/farmacología , Proteínas Fluorescentes Verdes/uso terapéutico , Humanos , Neovascularización Patológica/genética , Neovascularización Patológica/prevención & control , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Timidina Quinasa/genética , Transducción Genética/métodos

RESUMEN

OBJECT: Adenovirus vector (AdV)-mediated gene delivery has been recently demonstrated in clinical trials as a novel potential treatment for malignant gliomas. Combined coxsackievirus B and adenovirus receptor (CAR) has been shown to function as an attachment receptor for multiple adenovirus serotypes, whereas the vitronectin integrins (alphavbeta3 and alphavbeta5) are involved in AdV internalization. In resected glioma specimens, the authors demonstrated that malignant gliomas have varying levels of CAR, alphavbeta3, and alphavbeta5 expression. METHODS: A correlation between CAR expression and the transduction efficiency of AdV carrying the green fluorescent protein in various human glioblastoma multiforme (GBM) cell lines and GBM primary cell lines was observed. To increase transgene activity in in vitro glioma cells with low or deficient levels of CAR, the authors used basic fibroblast growth factor (FGF2) as a targeting ligand to redirect adenoviral infection through its cognate receptor, FGF receptor 1 (FGFR1), which was expressed at high levels by all glioma cells. These findings were confirmed by in vivo study data demonstrating enhanced transduction efficiency of FGF2-retargeted AdV in CAR-negative intracranial gliomas compared with AdV alone, without evidence of increased angiogenesis. CONCLUSIONS: Altogether, the results demonstrated that AdV-mediated gene transfer using the FGF2/FGFR system is effective in gliomas with low or deficient levels of CAR and suggested that FGF2-retargeting of AdV may be a promising approach in glioma gene therapy.

Asunto(s)

Adenoviridae/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Marcación de Gen , Terapia Genética , Vectores Genéticos , Glioma/terapia , Línea Celular Tumoral , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Humanos , Integrina alfaVbeta3/metabolismo , Integrinas/metabolismo , Ligandos , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores Virales/metabolismo , Receptores de Vitronectina/metabolismo , Transducción Genética

RESUMEN

OBJECTIVES: The purpose of this study was to investigate whether a novel fibroblast growth factor-2 gene formulation, providing a localized and sustained availability of the adenoviral vector from a collagen-based matrix, in combination with CO 2 transmyocardial laser revascularization would lead to an enhanced angiogenic response and improved myocardial function. METHODS: Fibroblast growth factor-2 gene was delivered by means of an adenoviral vector (adenoviral fibroblast growth factor-2) formulated in a collagen-based matrix. The ischemic areas of 33 animals were then treated. Group 1 was treated with CO 2 transmyocardial laser revascularization; group 2 was treated with intramyocardial injections of adenoviral fibroblast growth factor-2 in a collagen-based matrix; group 3 had a combination treatment of matrix adenoviral fibroblast growth factor-2 and CO 2 transmyocardial laser revascularization; and group 4 received injections with saline-formulated adenoviral fibroblast growth factor-2. Baseline left ventricular function was assessed by echocardiography and cine magnetic resonance imaging. Studies were repeated 6 weeks after treatment. Vascular development was assessed using anti-alpha-actin immunohistochemistry. RESULTS: Matrix adenoviral fibroblast growth factor-2 + transmyocardial laser revascularization-treated areas had a 105% increase in arteriolar development versus either treatment alone ( P < .05) and a 390% increase compared with saline-formulated adenoviral fibroblast growth factor-2 treatment alone ( P < .05). Contractility was significantly improved in matrix adenoviral fibroblast growth factor-2 + transmyocardial laser revascularization-treated areas as measured by myocardial wall thickening. This functional improvement was confirmed by cine magnetic resonance imaging, in which a 90% increase in the contractility of the treated segments was demonstrated after matrix adenoviral fibroblast growth factor-2 + transmyocardial laser revascularation. The other treatments provided significantly less restoration of myocardial function. CONCLUSIONS: The increase in angiogenesis as a result of matrix adenoviral fibroblast growth factor-2 gene therapy in combination with CO 2 transmyocardial laser revascularization is greater than that seen in either therapy alone. A concomitant improvement in myocardial function was seen as a result of this angiogenic response.

Asunto(s)

Modelos Animales de Enfermedad , Terapia Genética/métodos , Terapia por Láser/métodos , Contracción Miocárdica , Isquemia Miocárdica/terapia , Revascularización Miocárdica/métodos , Adenoviridae , Animales , Arteriolas/crecimiento & desarrollo , Química Farmacéutica , Enfermedad Crónica , Terapia Combinada , Ecocardiografía , Prueba de Esfuerzo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/uso terapéutico , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Inmunohistoquímica , Imagen por Resonancia Cinemagnética , Isquemia Miocárdica/diagnóstico , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/fisiopatología , Neovascularización Fisiológica , Distribución Aleatoria , Porcinos , Resultado del Tratamiento , Función Ventricular Izquierda

RESUMEN

We have developed a therapeutic approach to wound repair involving immobilization of gene transfer vectors within biocompatible matrices (gene-activated matrix, or GAM). The matrix also serves as a scaffold for cellular in-growth and subsequent gene uptake and expression. An adenoviral vector encoding human platelet-derived growth factor-B delivered in collagen (AdPDGF-B/GAM) has demonstrated efficacy in models of wound repair. The safety, biodistribution, and immunogenicity profiles of AdPDGF-B/GAM were examined using a rabbit dermal wound model. Four weekly doses at 1 x 10(10) and 1 x 10(11) viral particles/cm2 of wound surface stimulated dose-related increases in granulation tissue formation and cell proliferation. In situ hybridization and immunostaining demonstrated concordant expression of human PDGF-B mRNA and protein. No treatment-related changes in hematology, serum chemistry, or histopathology were observed. Although AdPDGF-B DNA and PDGF-B mRNA were detected in wounds and axillary lymph nodes of treated animals, no AdPDGF-B was detected in blood or other organs. No immunologic responses against collagen were observed; however, as expected, IgG responses to AdPDGF-B and human PDGF-BB protein were detected. In adenovirus-preimmunized rats, attenuation of the wound healing response was modest (approximately 16%). Collectively, these observations indicate that repeat doses of AdPDGF-B/GAM are well tolerated and lead to robust, localized tissue repair.

Asunto(s)

Adenoviridae/genética , Colágeno/química , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Proteínas Proto-Oncogénicas c-sis/genética , Cicatrización de Heridas , Animales , Formación de Anticuerpos , Modelos Animales de Enfermedad , Expresión Génica , Terapia Genética/efectos adversos , Tejido de Granulación/citología , Tejido de Granulación/crecimiento & desarrollo , Ganglios Linfáticos/metabolismo , Reacción en Cadena de la Polimerasa , Alcohol Polivinílico/química , Proteínas Proto-Oncogénicas c-sis/análisis , Proteínas Proto-Oncogénicas c-sis/inmunología , ARN Mensajero/análisis , Conejos , Piel/patología

RESUMEN

HYPOTHESIS: Tissue flaps are commonly used for surgical reconstruction, especially to cover difficult wounds and in breast reconstruction following mastectomy. Complications due to inadequate flap perfusion are a source of morbidity and, in the lower extremity, can result in amputation. SETTING: Laboratory. INTERVENTIONS: We evaluated the ability of platelet-derived growth factor (PDGF) B and fibroblast growth factor 2 plasmid DNA, formulated in a type I collagen matrix, to promote tissue survival in a rat transverse rectus abdominis muscle flap model based on the inferior deep epigastric vascular supply. In the absence of any therapeutic agent, only about 24% of flap tissue survives in this model. The DNA/matrix formulations were delivered subcutaneously into the skin paddles 7 days before flap elevation, and tissues were harvested 7 days later. RESULTS: Our studies reveal dramatic increases in overall vascularity after treatment with PDGF-B and fibroblast growth factor 2 plasmid DNA; however, only PDGF-B increased flap survival (130% increase at 228 micro g/cm(2) of plasmid DNA vs controls; P<.01). Transdermal spectral imaging demonstrated an increase in patent vessels supporting blood flow in flaps treated with PDGF-B plasmid DNA vs the fibroblast growth factor 2 transgene. CONCLUSION: Matrix-enabled gene therapy may provide an effective nonsurgical approach for promoting flap survival and is well suited for surgical applications in which transient therapeutic transgene expression is desired.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/farmacología , Isquemia/prevención & control , Neovascularización Fisiológica/efectos de los fármacos , Plásmidos/farmacología , Proteínas Proto-Oncogénicas c-sis/farmacología , Colgajos Quirúrgicos/irrigación sanguínea , Animales , ADN , Modelos Animales de Enfermedad , Femenino , Rechazo de Injerto , Supervivencia de Injerto , Inmunohistoquímica , Masculino , Neovascularización Fisiológica/fisiología , Probabilidad , Ratas , Ratas Sprague-Dawley , Recto del Abdomen/patología , Recto del Abdomen/cirugía , Factores de Riesgo , Sensibilidad y Especificidad

RESUMEN

Tissue repair is driven by migratory macrophages and fibroblasts that infiltrate injury sites and secrete growth factors. We now report the enhancement of skeletal muscle repair by targeting transgene delivery to these repair cells using matrix-immobilized gene vectors. Plasmid and adenovirus vectors immobilized in collagen-gelatin admixtures were delivered to excisional muscle wounds, and when encoding either fibroblast growth factor-2 (FGF2) or FGF6 transgenes, produced early angiogenic responses that subsequently remodeled into arteriogenesis. FGF2 gene delivery enhanced the number of CD31(+) endothelial cells present at treatment sites > 6-fold by day 14, and muscular arteriole density up to 11-fold by day 21 (P<0.0001). Muscle repair was also enhanced, as FGF gene-treated wounds filled with regenerating myotubes expressing the marker CD56 (an average 20-fold increase in CD56 expression versus controls, P<0.0001). These responses required transfection of a threshold level of repair cells, achievable only in injured muscles, and were transgene-driven, as neither platelet-derived growth factor-B (PDGFB) gene nor FGF2 protein delivery produced equivalent responses. In conclusion, using biomatrices to direct gene delivery to repair cells allows for relatively complex regenerative processes such as arteriogenesis and myogenesis, and therefore represents a promising approach to treating injured and ischemic muscle.

Asunto(s)

Factor 2 de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/genética , Desarrollo de Músculos/fisiología , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/fisiología , Neovascularización Fisiológica/fisiología , Proteínas Proto-Oncogénicas/genética , Cicatrización de Heridas/fisiología , Animales , Arteriolas/fisiología , Antígeno CD56/metabolismo , Colágeno/metabolismo , Factor 6 de Crecimiento de Fibroblastos , Citometría de Flujo , Marcación de Gen , Vectores Genéticos , Humanos , Luciferasas/metabolismo , Músculo Esquelético/lesiones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Proto-Oncogénicas c-sis/genética , Ratas , Ratas Sprague-Dawley

RESUMEN

Pancreatic ductal adenocarcinomas (PDACs) overexpress various cell-surface tyrosine kinase receptors, including the type I high-affinity fibroblast growth factor receptor (FGFR-1). The purpose of this study was to determine whether FGFR-targeted gene therapy is feasible in this disorder. Accordingly, the effects of a conjugate consisting of fibroblast growth factor (FGF)-2 linked to a Fab' fragment against the adenovirus knob region were evaluated in human pancreatic cancer cell lines treated with an adenoviral vector containing the herpes simplex virus thymidine kinase (AdTK) gene. An adenoviral vector containing the firefly luciferase reporter gene (AdLuc) served to assess infection efficiency, and was initially tested in L6 rat myoblasts. In parental L6 cells that express exceedingly low levels of high-affinity FGFRs, transduction with AdLuc was enhanced 7- to 10-fold with the FGF2-Fab' conjugate, whereas in L6 cells transfected to express FGFR-1, it was enhanced 39- to 52-fold. The pancreatic cancer cell lines expressed variable levels of the four high-affinity FGF receptors, and exhibited 2- to 34-fold increases in gene transduction in the presence of the FGF2-Fab' conjugate. In the absence of FGF2-Fab' there was no correlation between surface binding of FGF2 and AdLuc transduction efficiency, whereas in the presence of FGF2-Fab', enhanced AdLuc transduction efficiency correlated with greater surface binding of FGF2. In the absence of AdTK, all the cell lines were insensitive to ganciclovir, whereas after AdTK transduction, only ASPC-1 and PANC-1 cells were resistant to ganciclovir even in the presence of FGF2-Fab'. Ganciclovir-mediated inhibition was dependent on the conjugate in CAPAN-1 and COLO-357 cells, but was independent of the conjugate in T3M4 and MIA-PaCa-2 cells. Real-time quantitative PCR of laser-captured cancer cells revealed high levels of various FGFR mRNA species in six of seven PDAC tumor samples. These findings indicate that transduction efficiency with FGF2-Fab' in pancreatic cancer cells is independent of native adenoviral transduction efficiency and is greatest in cells that exhibit concomitant expression of various high-affinity FGFRs. In view of the overexpression of high-affinity FGFRs in the cancer cells in PDAC, our findings also suggest that the combined use of AdTK, ganciclovir, and FGF2-Fab' may ultimately be a promising therapeutic approach in a subgroup of patients with PDAC.

Asunto(s)

Adenocarcinoma/terapia , Técnicas de Transferencia de Gen , Neoplasias Pancreáticas/terapia , Receptores de Factores de Crecimiento de Fibroblastos/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antivirales/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Ganciclovir/farmacología , Genes Reporteros , Humanos , Luciferasas/metabolismo , Masculino , Microscopía Confocal , Persona de Mediana Edad , Unión Proteica , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismo , Células Tumorales Cultivadas