Suicide gene therapy of glioblastoma with lentiviral vectors
Results of the particular research project can be subdivided into two parts— 1) We have shown that the lentiviral vector-mediated suicide gene therapy is not toxic for normal brain cells. The therapy does not cause the elimination of neural cells and no significant therapy-related immune inflammation was observed. The research animals (rat) also expressed normal behavior during the therapy. As a result, the therapy is warranted to be followed up by clinical trials. (PMID 27490042) 2) We have also observed a significant improvement of the suicide gene therapy by using a prolonged regimen of prodrug administration. The manuscript of this study is being prepared and will soon be submitted for publication following some additional experiments.
Lentiviral HSV-Tk mediated suicide gene therapy is not toxic for normal brain cells
A major goal for suicide gene therapies of brain tumors is the specific targeting of tumor cells sparing normal brain cells. However, it is not known whether lentiviral vector mediated suicide gene therapy is harmful to normal brain cells in a non-tumor setting. We showed that HSV-Tk mediated suicide gene therapy is not toxic to normal brain cells.
We have studied and showed that lentiviral vector mediated transduction of the suicide gene HSV-tk into normal brain cells in vivo with subsequent Ganciclovir treatment is not toxic. Our results are important in the context of suicide gene therapies for brain tumors as previous viral vectors were chosen based on their ability to transduce brain tumor cells and to spare neural tissue. This is in particular the case for retroviral vectors which were one of the first viral vectors used to deliver HSV-tk to brain tumors. Efficient transduction of normal brain cells with Tk.007 was confirmed by immunostaining. The tropism of VSV-G pseudotyped lentiviral vectors has been analyzed previously and we have shown transduction of different types of brain cells. Here, we showed transduction of neurons, astrocytes and oligodendrocyte precursor cells in both Tk-only and Tk+GCV groups. We also observed that, neurons and oligodendrocyte precursor cells were more abundantly transduced than astrocytes. Importantly the total number of transduced cells did not vary between groups, which indicated that Ganciclovir treatment does not eliminate normal brain cellsThe specific killing of tumor cells and sparing of normal brain cells can be explained by the difference in proliferation activity of those cell populations. In a longer time-window, tumor cells are actively proliferating whereas the majority of normal brain cells are in a post-mitotic state. Phosphorylated GCV is incorporated as a chain terminator into replicating DNA when cells are dividing and thus will affect only actively dividing cells. For treatment of brain tumor patients with lentiviral vectors, it may appear likely to have some extent of off-target transduction, especially if vector injection is executed in the resection cavity. Our results have an important impact on the translation of lentiviral suicide gene therapies into the clinic, as the vector specificity for tumor cells is not anymore mandatory when using HSV-Tk/GCV therapy. The article is prepared as a manuscript and will be submitted to Moleculat Threapy within several weeks.
Enhanced distribution of LCMV-pesudotyped bicistronic lentiviral vector for Glioblastoma treatment
By introducing EGFR, a gene that enhances invasion, in addition to the suicide gene HSV-tk into our vector system, we want to improve the distribution of the therapeutic gene targeting against Glioblastoma multiforme which is one of most difficult-to-cure cancer.
Malignant gliomas, the largest group of primary intracerebral tumours, are one of the most difficult-to-cure cancers. The outcome has not improved significantly in the past years, despite considerable advances in our understanding of the molecular pathogenesis and the improvement of surgical techniques, radio- and chemotherapy. For glioblastoma multiforme (GBM), the most malignant form of glioma, the median survival time is generally less than one year. Invasive glioma cells escape current therapies and are initiating recurrent tumors. Although complete remission of experimental GBM on MRI was observed, recurrent tumors came up which were frequently observed at different sites compared to the primary tumor . Thus invasive cells migrated to thes areas and initiated recurrent tumors.The distribution of the therapeutic gene needs to be enhanced in order to target the invasive glioma cells and thereby improve the therapeutic effect and prolong the recurrence-free time window. By introducing EGFR, a gene that enhances invasion, in addition to the suicide gene HSV-tk into our vector system, we want to improve the distribution of the therapeutic gene and also track invasive tumor cells. Key findings: Expression of EGFR initiates an invasive program in transduced glioblastoma cells in vitro. EGFR mediated infiltration of glioblastoma cells is retained in vivo in a rat xenograft model. [figure3 a &b] The bicistronic lentiviral vector containing HSV-TK and EGFR [figure4] shows satisfactory cytotoxic profile in vitro. [figure5a & b] Conclusion: Expression of EGFR can enhance infiltrative nature of glioblastoma cells both in vitro and in vivo. We are currently taking preparations for an in vivo therapeutic study of our bicistronic lentiviral construct containing a recombinant HSV-TK and EGFR in a clinically relevant GBM rat model.
Lentiviral HSV-Tk.007-mediated suicide gene therapy is not toxic for normal brain cells.
J Gene Med 2016 Sep;18(9):234-43.
PMID: 27490042 - Inngår i doktorgradsavhandlingen
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