Adenoviral-Mediated Gene Transferinto the Canine Brain in Vivo

Abstract

NIH Public Access Author ManuscriptNeurosurgery. Author manuscript; available in PMC 2007 November 27. Published in final edited form as:Neurosurgery. 2007 January ; 60(1): 167-178. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding β-galactosidase (βGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODS:J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding βGal (Ad-βGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, βGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration.RESULTS: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and βGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm 2 area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSION:We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials. KeywordsAdenovirus; Dog; Flt3L; Ganciclovir; Gene therapy; Glioblastoma; HSV1-TK 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)(22)(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 r...

Advances in Diagnostic and Treatment Modalities for Intracranial Tumors

“…Common therapeutic strategies include suicide gene therapy, oncolytic treatment, immunomodultion, gene replacement, proapoptotic treatment, and antiangiogenesis 175, 203. Successful delivery or efficacy of gene therapy approaches using viral vectors and plasmid DNA has been shown experimentally in canine brain tumor cells and brain using adenoviral,204, 205, 206, 207, 208, 209 retroviral201, 202, 210 herpes,211 and adeno‐associated viral212, 213, 214 delivery. Few viral therapies have progressed to phase III clinical trials, and none have been shown to have significant efficacy in high‐grade brain tumors in phase III trials to date 175…”

Gene Therapy for Brain Cancer: Combination Therapies Provide Enhanced Efficacy and Safety

“…This allows performing preclinical trials that will mimic more closely the clinical scenario, in which new therapies are applied in patients that simultaneously undergo traditional treatment. We and others have previously demonstrated the feasibility of delivering therapeutic transgenes to dog GBM cells in vitro and dog brain cells in vivo upon intracranial injection of gene therapy vectors, such as type 5 adenoviral vectors [69-71], adeno-associated viral vectors [72], plasmid DNA/polyethylenimine (PEI) complexes [71], which suggests that dogs bearing spontaneous GBM would be a suitable model to test novel gene therapy approaches. Importantly, the availability of canine GBM J3T [70, 73] and W&W [74] cell lines allows in vitro screening of novel therapeutic agents before moving to preclinical trials in dogs bearing spontaneous GBM.…”

“…Also, considering the requirement of exogenous nucleotide analogs to induce cell death, the withdrawal of the pro-drug could stop the development of putative adverse side effects. Our previous results indicate that administration of Ad-TK in the naïve brain of rats [20] and dogs [69-71] does not significantly alter the structure of the normal brain and induces only a mild, transient local inflammation. Importantly, it has been demonstrated that injection of adenoviral vectors encoding HSV1-TK into the margins of the tumor cavity after GBM surgical resection followed by GCV administration was well tolerated in over 70 patients in 6 early clinical trials [2].…”

Flt3L and TK gene therapy eradicate multifocal glioma in a syngeneic glioblastoma model

“…Ad-expressed transgenes were detected utilizing custom-generated rabbit antibodies specific to TK 35,44,45 or human soluble Flt3L 19,30,31,44 (developed in rabbit; 1:1,000) combined with the mouse antivimentin antibody. Alexa 488 directly conjugated fluorescent secondary antibodies (1:1,000) were used to detect transgenes (Invitrogen, Carlsbad, CA, USA).…”