Glioblastoma multiforme (GBM) is a highly invasive and aggressive primary brain tumor. The median survival (~12 months after diagnosis) has remained unchanged for the past several decades despite advances in surgery, chemotherapy, and radiotherapy.1–6
Surgical success is often limited by a poorly defined tumor border because of the highly infiltrative nature of GBM and its close proximity to vital brain structures. Chemotherapy plus radiation are unable to eliminate all residual GBM cells. Residual glioma cells, often become resistant to chemotherapeutic agents, and give rise to recurrent GBM tumors, a hallmark of the disease.6
Gene therapy has been proposed as a new therapeutic approach to treat this devastating disease.2–4,6–8
Therapeutic approaches include the use of oncolytic, conditionally replicating viral vectors,7,9,10
replication-defective viral vectors to transfer conditionally cytotoxic genes such as herpes simplex virus type 1 thymidine kinase (HSV-1-TK),8,11,12
death receptor ligand interactions using TRAIL or FasL,13–15
and immunostimulatory genes such as interleukin-2 (IL-2), IL-12, interferon-β, tumor necrosis factor-α, and granulocyte–macrophage colony-stimulating factor.6,16–18
Several gene therapy strategies have been implemented in clinical trials to treat GBM, using adenoviral, adeno-associated viral (AAV), HSV-1, and retroviral vectors.6
Most of the clinical trials using gene therapy approaches to treat GBM have been Phase I safety trials, and therefore efficacy data cannot be extracted from these trials. One large Phase III trial using retroviral vectors expressing HSV1-TK failed to extend patients’ survival.19
To date, two gene therapy clinical trials have shown statistically significant prolongation of life expectancy (~2 month increase), by using adenoviral vectors to deliver HSV1-TK.8,12
In a Phase IIa trial Sandmair et al. compared delivery of TK into the tumor cavity with either replication-defective adenoviral vectors or retroviral vector-producing cells.12
Patients treated with an adenoviral vector expressing TK showed prolonged survival. In a Phase IIb clinical trial conducted by Immonen et al
., 36 patients were randomized and treated with either current standard of care consisting of radical tumor resection followed by radiotherapy, or with standard of care plus intra-operative injections of an adenoviral vector expressing HSV1-TK into the margins of the tumor cavity following tumor resection. The mean survival time for patients treated with adenoviral vectors was 70.6 weeks as compared to 39 weeks in those who received standard of care alone.8
This approach has been recently tested in a large multicenter Phase III trial.20
We have shown earlier that treatment of microscopic tumors with Ad-TK plus ganciclovir (GCV) effectively inhibits tumor progression of syngeneic intracranial GBM in 100% of the animals.21
In order to model the characteristics of human GBM more closely, we developed a large, macroscopic, syngeneic rat model of GBM. In this stringent GBM animal model Ad-TK plus GCV resulted in the survival of only ~20% of the animals, while other single therapies tested in this model failed.22
We therefore used this model to test the efficacy of novel, combination gene therapies, and demonstrated that the efficacy of Ad-TK is greatly enhanced when combined with gene transfer of Fms-like tyrosine kinase 3 ligand (Flt3L), rescuing ~70% of the treated, tumor-bearing animals.22,23
We have also shown that treatment with Ad-Flt3L and Ad-TK is effective in a multifocal model of intracranial GBM, in which only one GBM lesion was treated.24
Striatal lesions induced by using the neurotoxin 6-hydroxydopamine are used to generate a model of Parkinson’s disease in rats, resulting in rapid reduction in the tyrosine hydroxylase (TH) immunoreactive cells in the striatum25
and elicits behavioral deficits such as asymmetry in limb use and abnormalities in amphetamine-induced turning behavior and sensorimotor reactivity.26–29
These behavioral abnormalities can be quantitated and used as response variables for testing the efficacy of Parkinson’s disease therapies.26–29
Because the growth of the GBM in the striatum induces the displacement of normal striatal tissue, seen as a displacement of TH-immunoreactive axons in the striatum, we sought to use the same tests to assess the behavioral impact of the combined Ad-Flt3L/Ad-TK-mediated tumor progression/regression in this model.
Because the GBM tumor cells were implanted into the striatum we hypothesized that tumor growth would cause behavioral abnormalities in the experimental animals. Further, we also hypothesized that because the combined gene therapy approach using Flt3L with TK plus GCV was effective at inducing tumor regression and long-term survival of the treated animals, the treatment would also correct the behavioral abnormalities. We therefore assessed the behavior and neuropathology of tumor-bearing animals treated with our combined gene therapy at three time points: 3 days after treatment, in long-term survivors (60 days after tumor implantation), and in long-term survivors that had been rechallenged with a second GBM tumor in the contralateral brain hemisphere (200 days after the initial tumor implantation).
Our results demonstrate that the large, syngeneic, intracranial GBM displaces the normal striatal tissue and induces behavioral deficits. We show a complete resolution of tumor-induced behavioral deficits as a result of gene therapy-mediated tumor regression in long-term survivors. In addition, behavioral abnormalities were not observed in long-term survivors that had been rechallenged with a second GBM tumor. Neuropathological analysis of long-term survivors after tumor rechallenge revealed an overall recovery of normal architecture of the injected striatum. The lack of long-term behavioral deficits and limited neuropathological abnormalities demonstrate the efficacy and good safety profile of the combined Flt3L and TK adenoviral vector-mediated gene therapy for GBM. These results may form the basis for further developments, leading toward the implementation of a Phase I clinical trial for GBM using this combined gene therapy strategy.