RESUMO
PURPOSE: Lentiviral vectors pseudotyped with glycoproteins of the lymphocytic choriomeningitis virus (LCMV-GP) are promising candidates for gene therapy of malignant glioma, as they specifically and efficiently transduce glioma cells in vitro and in vivo. Here, we evaluated the therapeutic efficacy of LCMV-GP and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors. EXPERIMENTAL DESIGN: Therapeutic efficacy was tested for unmodified (9L) and DsRed-modified (9LDsRed) gliomas using the suicide gene thymidine kinase of the herpes simplex virus type 1 (HSV-1-tk). Positron emission tomography (PET) and magnetic resonance imaging were done to analyze transduction of tumors and monitor therapeutic outcome. RESULTS: LCMV-GP pseudotypes mediated a successful eradication of 9LDsRed tumors with 100% of long-term survivors. Before initiation of ganciclovir treatment, a strong HSV-1-tk expression within the tumor was detected by noninvasive PET using the tracer 9-[4-[(18)F]fluoro-3-(hydroxymethyl)butyl]guanine. Therapeutic outcome was successfully monitored by magnetic resonance imaging and PET imaging and correlated with the histopathologic data. In the 9L model, LCMV-GP and VSV-G pseudotyped lentiviral vectors displayed similar therapeutic efficacy. Further studies revealed that normal brain cells transduced with VSV-G pseudotypes were not eliminated by ganciclovir treatment and contributed significantly to the bystander killing of tumor cells. CONCLUSIONS: Suicide gene transfer using pseudotyped lentiviral vectors was very effective in the treatment of rat glioma and therefore is an attractive therapeutic strategy also in human glioblastoma especially in conjunction with an imaging-guided approach. In addition, high selectivity of gene transfer to tumor cells may not always be desirable for therapeutic genes that exert a clear bystander effect.
Assuntos
Neoplasias Encefálicas/terapia , Genes Transgênicos Suicidas , Terapia Genética , Vetores Genéticos/genética , Glioma/terapia , Lentivirus/genética , Animais , Antivirais/farmacologia , Encéfalo/citologia , Ganciclovir/farmacologia , Glicoproteínas/genética , Herpesvirus Humano 1/genética , Humanos , Vírus da Coriomeningite Linfocítica/genética , Imageamento por Ressonância Magnética , Tomografia por Emissão de Pósitrons , Ratos , Timidina Quinase/genética , Vesiculovirus/genéticaRESUMO
BACKGROUND: Early clinical trials for gene therapy of human gliomas with retroviral packaging cells (PC) have been hampered by low transduction efficacy and lack of dissemination of PC within the tumor. In the current approach, these issues have been addressed by creating a stable packaging cell line for retroviral vectors pseudotyped with glycoproteins of lymphocytic choriomeningitis virus (LCMV) based on tumor-infiltrating progenitor cells. METHODS: Tumor-infiltrating progenitor cells, which had been isolated from adult rat bone marrow (BM-TIC), were modified to stably express Gag-Pol proteins of moloney murine leukemia virus (Mo-MLV) and glycoproteins of LCMV. Packaging of a retroviral vector was measured by titration experiments on human fibroblast cells as well as on mouse and human glioma cell lines. Additionally, gene transfer was tested in a rat glioma model in vivo. RESULTS: The BM-TIC-derived packaging cell line (BM-TIPC) produced retroviral vectors with titers between 2-8 x 10(3) transducing units (TU)/ml. Extended culturing of BM-TIPC over several weeks and freezing/thawing of cells did not affect vector titers. No replication-competent retrovirus was released from BM-TIPC. In a rat glioma model, BM-TIPC infiltrated the tumors extensively and with high specificity. Moreover, BM-TIPC mediated transduction of glioma cells in vivo. CONCLUSION: This proof-of-principle study shows that primary adult progenitor cells with tumor-infiltrating capacity can be genetically modified to stably produce retroviral LCMV pseudotype vectors. These BM-TIPC may be a useful tool to enhance specificity and efficacy of gene transfer to gliomas in patients.
Assuntos
Linhagem Celular , Movimento Celular , Vetores Genéticos , Glioma/terapia , Células-Tronco Mesenquimais/fisiologia , Retroviridae/genética , Animais , Células da Medula Óssea , Células Cultivadas , Terapia Genética/métodos , Humanos , Vírus da Coriomeningite Linfocítica/química , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Vírus da Leucemia Murina de Moloney/química , Ratos , Transdução Genética , Proteínas Virais/genética , Montagem de VírusRESUMO
Malignant gliomas are the most frequent primary brain tumors and have a dismal prognosis due to their infiltrative growth. Gene therapy using viral vectors represents an attractive alternative to conventional cancer therapies. In a previous study, we established lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus (LCMV) glycoproteins (GPs) and demonstrated transduction of human malignant glioma cells in culture. In the current approach, we compared the transduction efficacy of LCMV-GP- and vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral vectors for malignant glioma cells and normal brain cells in vitro and in vivo. LCMV-GP pseudotypes transduced almost exclusively astrocytes, whereas VSV-G pseudotypes infected neurons as well as astrocytes. LCMV-GP pseudotypes showed an efficient transduction of solid glioma parts and specific transduction of infiltrating tumor cells. In contrast, VSV-G-pseudotyped lentiviral vectors transduced only a few tumor cells in solid tumor parts and infected mostly normal brain cells in infiltrating tumor areas. In conclusion, lentiviral vectors pseudotyped with LCMV glycoproteins represent an attractive option for gene therapy of malignant glioma.