RESUMO
In hepatocellular carcinoma (HCC), tumor specificity of gene therapy is of utmost importance to preserve liver function. MicroRNAs (miRNAs) are powerful negative regulators of gene expression and many are downregulated in human HCC. We identified seven miRNAs that are also downregulated in tumors in a rat hepatoma model (P<0.05) and attempted to improve tumor specificity by constructing a panel of luciferase-expressing vectors containing binding sites for these miRNAs. Attenuation of luciferase expression by the corresponding miRNAs was confirmed across various cell lines and in mouse liver. We then tested our vectors in tumor-bearing rats and identified two miRNAs, miR-26a and miR-122, that significantly decreased expression in liver compared with the control vector (6.40 and 0.26%, respectively; P<0.05). In tumor, miR-122 had a nonsignificant trend towards decreased (â¼50%) expression, whereas miR-26 had no significant effect on tumor expression. To our knowledge, this is the first work using differentially expressed miRNAs to de-target transgene expression in an orthotopic hepatoma model and to identify miR-26a, in addition to miR-122, for de-targeting liver. Considering the heterogeneity of miRNA expression in human HCC, this information will be important in guiding development of more personalized vectors for the treatment of this devastating disease.
Assuntos
Carcinoma Hepatocelular/genética , Vetores Genéticos , Neoplasias Hepáticas/genética , MicroRNAs/genética , Animais , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Células HEK293 , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas Experimentais , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Ratos , Ratos Endogâmicos BUF , TransgenesRESUMO
Ideal cancer gene therapies should have high tumor specificity and efficacy, and allow systemic administration to target metastases. We recently developed a bi-directional, two-step transcriptional amplification (TSTA) system driven by the tumor-specific Survivin promoter (pSurv) to amplify the correlated expression of both the reporter gene firefly luciferase (FL) and therapeutic gene tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we compare the specificity and potency of an adenovirus carrying this system (Ad-pSurv-TSTA-TRAIL-FL) to a nonspecific vector (Ad-pCMV-FL) in an orthotopic hepatocellular carcinoma (HCC) rat model after systemic administration. At 24 h after injection of Ad-pCMV-FL, bioluminescence imaging revealed a trend (P=0.30) towards greater FL expression in liver versus tumor. In striking contrast, Ad-pSurv-TSTA-TRAIL-FL showed increased FL activity within the tumor compared with the liver (P<0.01), a strong trend towards reduced liver expression compared with Ad-pCMV-FL (P=0.07), and importantly, similar FL levels within tumor compared with Ad-pCMV-FL (P=0.32). Hence, this vector shows potent, tumor-specific transgene expression even after extensive liver transduction and may be of significant value in avoiding hepatotoxicity in HCC patients. Future studies will explore the benefits of tumor-specific TRAIL expression in this model, the potential to target metastases and the extension of this vector for the treatment of other Survivin-positive tumors is warranted.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas Associadas aos Microtúbulos/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Adenoviridae/genética , Animais , Expressão Gênica , Marcação de Genes , Genes Reporter , Vetores Genéticos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Regiões Promotoras Genéticas , Ratos , Sensibilidade e Especificidade , Survivina , TransgenesRESUMO
There is growing interest in delivering cellular agents to infarcted myocardium to prevent postinfarction left ventricular remodeling. MRI can be effectively used to differentiate infarcted from healthy myocardium. MR-guided delivery of cellular agents/therapeutics is appealing because the therapeutics can be precisely targeted to the desired location within the infarct. In this study, a steerable intramyocardial injection catheter that can be actively tracked under MRI was developed and tested. The components of the catheter were arranged to form a loopless RF antenna receiver coil that enabled active tracking. Feasibility studies were performed in canine and porcine myocardial infarction models. Myocardial delayed-enhancement (MDE) imaging identified the infarcted myocardium, and real-time MRI was used to guide left ventricular catheterization from a carotid artery approach. The distal 35 cm of the catheter was seen under MRI with a bright signal at the distal tip of the catheter. The catheter was steered into position, the distal tip was apposed against the infarct, the needle was advanced, and a bolus of MR contrast agent and tissue marker dye was injected intramyocardially, as confirmed by imaging and postmortem histology. A pilot study involving intramyocardial delivery of magnetically labeled stem cells demonstrated the utility of the active injection catheter system.
Assuntos
Cateterismo Cardíaco , Injeções Intralesionais , Imageamento por Ressonância Magnética , Transplante de Células-Tronco Mesenquimais , Infarto do Miocárdio/terapia , Miocárdio , Animais , Cateterismo Cardíaco/instrumentação , Cateterismo , Meios de Contraste , Dextranos , Cães , Desenho de Equipamento , Óxido Ferroso-Férrico , Ferro , Nanopartículas de Magnetita , Infarto do Miocárdio/patologia , Miocárdio/patologia , Óxidos , Imagens de Fantasmas , SuínosRESUMO
PURPOSE: Technetium-99m-labeled sulfur colloid lymphoscintigraphy is useful to evaluate lower extremity lymphatic circulation in cases of possible lymphedema and to reveal abnormal lymphatic collections. Groin lymphatic fistulas and lymphoceles are known complications of peripheral vascular surgical procedures. The authors describe a patient with ascites that developed into right lower extremity swelling after surgical repair of a femoral artery injury. Even after surgical ligation of multiple lymphatic channels, the patient continued to have lymphorrhea. It was unclear whether this was attributable to a persistent lymphatic leak or an ascitic leak from a postsurgical defect resulting in an abnormal connection with the peritoneal cavity. METHODS: Lymphoscintigraphy of the lower extremities was performed using Tc-99m sulfur colloid. Images were obtained at several intervals after injection of the radiotracer. Images were also acquired after the wound packing was removed. RESULTS: The images revealed an accumulation of radiotracer in the right groin, confirming the lower extremity lymphatic origin of the collection. CONCLUSIONS: Lymphoscintigraphy is useful to evaluate the origin of serous collections in the groin, a region in which lymphatic complications of vascular surgery are not uncommon.