Suicide gene therapy of glioblastoma with lentiviral vectors
Prosjekt
- Prosjektnummer
- 911805
- Ansvarlig person
- Hrvoje Miletic
- Institusjon
- Helse Bergen HF
- Prosjektkategori
- Forskningsprosjekt
- Helsekategori
- Cancer, Neurological
- Forskningsaktivitet
- 5. Treatment Developement
Rapporter
We have developed a suicide gene therapy based on lentiviral vectors for treatment of glioblastoma, the most frequent and most aggressive primary brain tumor. We have shown in different animal models that this treatment strategy is very effective. In this project we want to optimize the treatment efficacy and evaluate the potential toxicity.1. Toxicity:
We have finalized the toxicity study and shown that the treatment with lentiviral vectors delivering the suicide gene HSV-TK.007 is not toxic for normal brain cells. In this study, we have included a detailed analysis of the expression of HSV-TK.007 in different brain cells. In addition, we have quantified different brain cells and the infiltration of immune cells. In collaboration with Janne Grønli and Jelena Mrdalj from the oncology department, we have analysed the behavior of rats under treatment using an open field test. The results have now been published in the "Journal of Gene Medicine".
2. Treatment optimization
A. We have tested another prodrug termed Valganciclovir in a brain tumor animal model and compared the treatment efficacy to Ganciclovir, which is the usual prodrug used for suicide gene therapy. By giving this Valganciclovir over prolonged time periods (3 months), which is possible due to a low toxicity profile, we could show that the animals have a survival benefit. However, recurrent tumors also developed under this treatment. We have analyzed the recurrent tumors in detail and found that they escape therapy by upregulating tyrosine kinase receptors. In particular, we have found that the tyrosine kinase receptor EGFR is highly significantly upregulated after long-term treatment with Valganciclovir. Therefore we aim at inhibiting this receptor by using the small molecule inhibitor Erlotinib, which is used in the clinic to treat colon and lung cancer patients, in combination with our suicide gene therapy.
B. Initially, we wanted to enhance the distribution of the suicide gene within the tumor by co-deliviering the Epidermal growth factor receptor (EGFR) gene together with the suicide gene to the tumor cells. We have previously shown that EGFR enhances the invasion of tumor cells. Thus enhanced invasion would lead to a better distribution of the suicide gene, especially at the tumor border. We have already constructed a lentiviral vector with both the suicide gene and EGFR and shown in vitro that EGFR enhances the invasion of tumor cells. In vivo, we have also demonstrated that EGFR enhances the invasion of tumor cells compared to EGFR negative cells. However, due to our new results described under 2.A, EGFR seems to be important in therapy resistance of our gene therapy. Therefore we think that it would be contra-productive to overexpress EGFR in glioblastoma cells. We have adopted now our therapeutic strategy as described under 2A and aim at performing a double treatment consisting of the suicide gene therapy and the small molecule inhibitor erlotinib, targeting EGFR instead of overexpressing it.
We have developed a suicide gene therapy based on lentiviral vectors for treatment of glioblastoma, the most frequent and most aggressive primary brain tumor. We have shown in different animal models that this treatment strategy is very effective. In this project we want to optimize the treatment efficacy and evaluate the potential toxicity.1. Toxicity:
We have performed a toxicity study by injecting the lentiviral vectors into the brain of healthy, immunocompetent rats. We analyzed extensively the behaviour of the rats during treatment with the prodrug Ganciclovir in collaboration with Jelena Mrdalj and Janne Grønli from the Department of Biological and Medical Psychology. The rats did not show any abnormal behaviour due to the treatment. Furthermore we examined the brains of the rats after treatment. We observed no toxic effect on neurons or other brain cells. Furthermore we could not detect any inflammation in the brains of the treated rats. Thus, our treatment does not show toxic effects on normal brain cells. Our study has a big impact on the translation of suicide gene therapies into the clinic to treat brain tumor patients. The study will soon be submitted for publication
2. Treatment optimization
A. We have tested another prodrug in a brain tumor animal model and compared the treatment efficacy to Ganciclovir, which is the usual prodrug used for suicide gene therapy. Due to patent issues we cannot declare the name of the prodrug. By giving this new prodrug over prolonged time periods, which is possible due to a low toxicity profile, we could show that the animals have a survival benefit.
B. We want to enhance the distribution of the suicide gene within the tumor by co-deliviering the Epidermal growth factor receptor (EGFR) gene together with the suicide gene to the tumor cells. We have previously shown that EGFR enhances the invasion of tumor cells. Thus enhanced invasion would lead to a better distribution of the suicide gene, especially at the tumor border. We have already constructed a lentiviral vector with both the suicide gene and EGFR and shown in vitro that EGFR enhances the invasion of tumor cells. In vivo, we have also demonstrated that EGFR enhances the invasion of tumor cells compared to EGFR negative cells. Our final experiment will be the treatment experiment which will be performed in the course of 2016.
The purpose of the study is:
1. the clinical translation of a lentiviral suicide gene therapy to treat brain tumor patients
2. optimization of this therapy using a preclinical animal model
3. to increase the distribution of the suicide gene by increasing migration of tumor cells that carry this gene within the tumor1. We have finished a small scale toxicity study where we treated healthy rats with injections of lentiviral vectors into the brain followed by Ganciclovir treatment. All rats survived the toxicity study. We did behavioral studies showing that the treatment was well tolerated by the rats without side effects. Initial analysis of the brains does not show toxicity to neurons or inflammation. Detailed analysis of the brains is in progress. Furthermore, we are in contact with two companies that will produce the lentiviral vectors and will perform a large scale pharmacology toxicology study, which is necessary before starting a clinical trial in patients.
2. We have generated a new lentiviral vector which contains the suicide gene and in addition the Epidermal Growth factor receptor (EGFR). This gene can promote invasion of cells. First we have tested the functionality of this new vector and shown that EGFR is expressed by cells infected with the lentiviral vector. This new vector will in the future be used in a therapeutic preclinical experiment to verify whether an increased distribution of the suicide gene also will lead to a stronger therapeutic effect.
3. We have shown that tumor cells transduced with EGFR are more migratory than cells without EGFR in vivo in a rat model. We have labelled these two cell populations with two different fluorescent markers and injected them into the brain of rats. EGFR+ cells migrated significantly longer distances compared to control cells without EGFR, which we observed on brain sections under a fluorescence microscope. The migratory capacity was quantified and showed statistically significant differences between the two groups.
We have developed a novel gene therapy for human glioblastoma based on lentiviral vectors delivering the suicide gene HSV-tk/Ganciclovir. Using two different animal models, we have shown a highly significant survival benefit for animals treated with this novel therapy. Currently, we are translating this therapeutic strategy into the clinic.In the current project, we will deliver the suicide gene HSV-tk-GFP together with EGFR by lentiviral vectors into tumors in vivo. The hypothesis is, that the infected tumor cells will then through upregulation of EGFR infiltrate the tumor mass and even track invading tumor cells. This will lead to a very efficient direct and bystander killing of tumor cells upon systemic application of the prodrug Ganciclovir. Our second goal is to prolong the therapeutic effect by giving multiple cycles of Ganciclovir as we have observed in a previous study that there is a fraction of tumor cells surviving a single cycle of Ganciclovir application.
The first task was to clone EGFR together with HSV-tk into the lentiviral vector. For this purpose we used a 2A sequence in order to get a similar expression level of both proteins. For the clonig we used a PCR strategy, which was in the end successful. The construct has been sequenced and is now ready for quality control. Currently, the vector is tested by producing viral supernatants and infecting cells in culture. In the first experiment we could detect upregulation of EGFR and expression of GFP which indicates that both HSV-tk and EGFR are expressed and that the construct works.
In preliminary in vitro experiments, we have transduced EGFR- glioma cells with EGFR and mixed these cells with the original cells in a collagen invasion assay, developed in our group. To distinguish both cell populations we transduced them with the fluorescating proteins RFP or GFP, respectively. By using fluorescence microscopy, we could show that the EGFR+ cells have a higher migratory capacity compared EGFR- cells, which confirms our hypothesis that EGFR increases invasion. We will now conduct the same experiment in vivo to further prove the hypothesis and to show that our system works, which we want to use for the final therapeutic experiment in vivo.
Vitenskapelige artikler
Hossain JA, Ystaas LR, Mrdalj J, Välk K, Riecken K, Fehse B, Bjerkvig R, Grønli J, Miletic H
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
Doktorgrader
Jubayer Al Hossain
Lentiviral Vector Mediated Suicide Gene Therapy for Glioblastoma Multiforme
- Disputert:
- desember 2016
- Hovedveileder:
- Hrvoje Miletic
Deltagere
- Hrvoje Miletic Prosjektleder
- Lars Andreas Rømo Ystaas Prosjektdeltaker
- Jubayer Al Hossain Ph.d.-kandidat
- Jubayer Hossain Ph.d.-kandidat
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