ADENOVIRAL-MEDIATED GENE TRANSFERINTO THE CANINE BRAIN IN VIVO

Glioblastoma multiforme (GBM), the most common type of primary brain tumor in adults, is an aggressive and locally invasive tumor. Survival of patients affected by GBM has remained virtually unchanged during the past decades (6−12 mo after diagnosis) despite advances in surgery, radiation, and chemotherapy (8, 12, 16, 24, 27, 37). Considering the poor prognosis of patients with high-grade glioma, novel therapeutic approaches are needed. Gene therapies are currently being developed and implemented in rodent preclinical models and in clinical trials (2, 13, 21–23, 26, 38). However, the lack of a large animal model of GBM makes it difficult to predict the outcome of preclinical therapies when translated directly from small rodent brains to those of human patients. Promising results in preclinical rodent experiments have led to human clinical trials, some of which showed therapeutic efficacy (13, 26, 40, 50). Nevertheless, the lack of a preclinical model that closely mimics the human condition and neuroanatomy, in addition to host immune responses to the gene therapy vectors, has hampered the clinical implementation of novel therapies. The small size of the rodent brain does not allow accurate optimization of volumes and doses that will be suitable for human GBM patients. It is likely that the different sizes of rat (2 g) and human (1300−1500 g) brains may contribute to discrepancies in preclinical efficacy studies and results obtained in human trials. Our laboratory has shown efficacy in eradicating GBM using an immunostimulatory therapy in large, intracranial syngenic GBM model in Lewis rats in which the tumor occupies most of the volume of the striatum in one hemisphere by using adenoviral-mediated gene transfer of herpes simplex virus (HSV1) 1-derived thymidine kinase (TK) and Fms-like tyrosine kinase 3 ligand (Flt3L) (1, 2). Because one of our aims is to translate this approach to human patients, we implemented it in dogs with spontaneous GBM, which may better model human disease parameters. Dogs have large brains (72 g in beagles) compared with rodents. This makes them an attractive model to test gene delivery efficiency, evaluate vector and transgene biodistribution, and optimize treatment protocols before clinical implementation in humans. Dogs have already proven to be an excellent model of human Duchenne muscular dystrophy (9, 11). Furthermore, brachycephalic breeds, such as Boston terriers and boxers (20, 25, 29), are predisposed to develop spontaneous glioblastomas that closely resemble the human disease (44, 45). Clinical signs, imaging, histopathology, treatment, and prognosis of human and canine GBMs are similar. In addition, there is a high correlation of neuroimaging features observed with magnetic resonance imaging scans in canine and human GBM, which is used as a diagnostic tool for canine GBM. Peritumoral edema is a consistent finding in both human and canine GBM (30, 35). Canine GBMs are histopathologically indistinguishable from human GBMs (30). Both are highly infiltrative, contain areas of intratumoral necrosis, pseudopalisading, hemorrhage, cellular pleomorphism, nuclear atypia, abnormal mitosis, and endothelial proliferation (34). Thus, dogs bearing spontaneous GBM constitute an excellent large animal model to test efficacy and toxicity of novel glioma therapies. In this article, we demonstrate that adenoviral vectors (Ads) Type 5 can transduce canine glioma cells in vitro and the dog brain in vivo. We tested the transduction efficacy in dog J3T dog glioma cells infected with adenoviral-encoding therapeutic transgenes, such as HSV1-TK and Flt3L, or the reporter gene, β-galactosidase (βGal). Our immunotherapeutic approach combines the cytotoxic properties of HSV1-TK, which kills glioma cells in the presence of ganciclovir (GCV) (5, 32) and causes the concomitant release of tumor antigens, with the immunostimulatory effects of Flt3L (15), which attracts antigen-presenting cells to the brain and the tumor mass (1, 2, 15). This combined approach is efficient in eliminating large, intracranial GBM in the syngenic Lewis rat model, in which all other therapies fail (2). We administered Ads in vivo in the brain of dogs and determined transgene expression in neurons and astrocytes, as well as limited infiltration of immune cells to the injection site. We demonstrate effective adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and βGal in dog glioma cells in vitro and into the normal dog brain in vivo. Importantly, no adverse clinical or neuropathological side effects were observed. This report constitutes an original description of adenoviral-mediated transgene expression into the brain of dogs in vivo and supports the use of these vectors to develop an efficacy trial of gene therapy for canine GBM. The results generated will be used as a prelude to implementing this novel therapeutic approach to treat human GBM patients.