RESUMO
OBJECT: The authors had previously reported on a replication-competent retrovirus (RCR) that has been demonstrated to be stable, capable of effective transduction, and able to prolong survival in an intracranial tumor model in nude mice. The purpose of this study was further investigation of this gene therapy option. METHODS: The transduction efficiency of RCR in RG2, an immunocompetent intracranial tumor model, was tested in Fischer 344 rats. The immune response to the RCR vector was expressed by the quantification of CD4, CD8, and CD11/b in tumors. The pharmaceutical efficacy of the suicide gene CD in converting prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) was measured using fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy. Animal survival data were plotted on Kaplan-Meier survival curves. Finally, the biodistribution of RCR was determined using quantitative real-time polymerase chain reaction (RT-PCR) for the detection of retroviral env gene. There was no evidence of viral transduction in normal brain cells. Neither severe inflammation nor immunoreaction occurred after intracranial injection of RCR-green fluorescent protein compared with phosphate-buffered saline (PBS). The 19F-NMR spectroscopy studies demonstrated that RCR-CD was able to convert 5-FC to 5-FU effectively in vitro. The infection of RG2 brain tumors with RCR-CD and their subsequent treatment with 5-FC significantly prolonged survival compared with that in animals with RG2 transduced tumors treated with PBS. In contrast to the nude mouse model, evidence of virus dissemination to the systemic organs after intracranial injection was not detected using RT-PCR. CONCLUSIONS: The RCR-mediated suicide gene therapy described in this paper effectively transduced malignant gliomas in an immunocompetent in vivo rodent model, prolonging survival, without evidence of severe intracranial inflammation, and without local transduction of normal brain cells or systemic organs.
Assuntos
Neoplasias Encefálicas/terapia , Genes Transgênicos Suicidas/genética , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Glioma/terapia , Terapia Viral Oncolítica/métodos , Retroviridae/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , DNA Viral/análise , DNA Viral/genética , Modelos Animais de Doenças , Encefalite/fisiopatologia , Encefalite/prevenção & controle , Flucitosina/metabolismo , Terapia Genética/tendências , Vetores Genéticos/genética , Glioma/genética , Glioma/metabolismo , Humanos , Hospedeiro Imunocomprometido/genética , Masculino , Terapia Viral Oncolítica/tendências , Ratos , Ratos Endogâmicos F344 , Taxa de Sobrevida , Transdução Genética/métodos , Transdução Genética/tendências , Resultado do Tratamento , Replicação Viral/genéticaRESUMO
Amphotropic retroviruses with modified envelope displaying single-chain antibody fragment (scFv) directed against the c-Met receptor were recently generated and found to efficiently and selectively deliver genes into hepatocarcinoma cells. A large proportion of human gliomas also frequently overexpresses c-Met. We therefore explored the possibility of infecting glioma cells using such retroviruses bearing an scFv directed against c-Met. In one construct, a urokinase (uPA) cleavage site was inserted between the scFv and the envelope. We assessed the transduction by these chimeric viruses of a panel of seven human glioma cell lines that we characterized for their c-Met and uPA levels. We found that abundance of the c-Met receptor and viral infection were inversely correlated if we used the retrovirus displaying scFv directed against c-Met, suggesting that the chimeric virus binds preferentially to the c-Met receptor, resulting in virus sequestration. Addition of the uPA site between the scFv moiety and the envelope restored the infectivity of the virus, consistent with a "two-step" infection process: (1) virus binding to the c-Met receptor, (2) cleavage of the scFv moiety by uPA, enabling the virus to dissociate from c-Met and entry into the cells via the Pit-2 receptor. Our study has significant implications for the design of targeting strategies for gliomas expressing high levels of c-Met.
Assuntos
Marcação de Genes , Glioma/virologia , Fragmentos de Imunoglobulinas/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Retroviridae/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Anticorpos/genética , Anticorpos/imunologia , Linhagem Celular Tumoral , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glioma/genética , Glioma/metabolismo , HumanosRESUMO
BACKGROUND: Recently, several cancer gene therapy studies have shown that replication-competent retroviral vectors represent a major improvement over replication-defective ones in terms of transgene propagation efficiency. However, this positive effect is somewhat spoiled by the increased risk of dissemination and oncogenesis that replication-competent retroviral vectors entail. To enhance both their integral safety and their transgene capacity, we developed a semi-replication-competent retroviral vector system. METHODS: The semi-replication-competent retroviral vector system is based on two transcomplementing replication-defective retroviral vectors termed gag-pol vector (GPv) and env vector (Ev). Vector propagation was monitored in vitro and in solid tumors in vivo, using different reporter transgenes for GPv and Ev. Systemic vector dissemination and leukemogenesis was assessed by direct intravenous vector injection and subsequent bone marrow transplantation, in MLV-sensitive mice. RESULTS: In vitro and in vivo the semi-replication-competent retroviral vectors propagate transgenes almost as efficiently as replication-competent ones. The semi-replication-competent retroviral vector system does not lead to detectable dissemination or leukemogenesis as does the replication-competent vector or the parental virus. Additionally, the vector duo allows co-propagation of different transgenes as well as mobilization of a third replication-defective vector. CONCLUSIONS: This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumors in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in in vitro studies and allows envisioning its further development for cancer gene therapy.
Assuntos
Replicação do DNA , Técnicas de Transferência de Genes , Vetores Genéticos , Retroviridae/genética , Animais , Transplante de Medula Óssea , Linhagem Celular , Genes Reporter , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos , Retroviridae/metabolismo , Transdução Genética , TransgenesRESUMO
Poor efficiency of gene transfer into cancer cells constitutes the major bottleneck of current cancer gene therapy. We reasoned that because tumors are masses of rapidly dividing cells, they would be most efficiently transduced with vector systems allowing transgene propagation. We thus designed two replicative retrovirus-derived vector systems: one inherently replicative vector, and one defective vector propagated by a helper retrovirus. In vitro, both systems achieved very efficient transgene propagation. In immunocompetent mice, replicative vectors transduced >85% tumor cells, whereas defective vectors transduced <1% under similar conditions. It is noteworthy that viral propagation could be efficiently blocked by azido-thymidine, in vitro and in vivo. In a model of established brain tumors treated with suicide genes, replicative retroviral vectors (RRVs) were approximately 1000 times more efficient than defective adenoviral vectors. These results demonstrate the advantage and potential of RRVs and strongly support their development for cancer gene therapy.
