Preclinical Efficacy and Safety Profile of Allometrically Scaled Doses of Doxycycline Used to Turn "On" Therapeutic Transgene Expression from High-Capacity Adenoviral Vectors in a Glioma Model.

Glioblastoma multiforme (GBM) is the most commonly occurring primary brain cancer in adults, in whom its highly infiltrative cells prevent total surgical resection, often leading to tumor recurrence and patient death. Our group has discovered a gene therapy approach for GBM that utilizes high-capacity "gutless" adenoviral vectors encoding regulatable therapeutic transgenes. The herpes simplex type 1-thymidine kinase (TK) actively kills dividing tumor cells in the brain when in the presence of the prodrug, ganciclovir (GCV), whereas the FMS-like tyrosine kinase 3 ligand (Flt3L) is an immune-stimulatory molecule under tight regulation by a tetracycline-inducible "Tet-On" activation system that induces anti-GBM immunity. As a prelude to a phase I clinical trial, we evaluated the safety and efficacy of Food and Drug Administration (FDA)-approved doses of the tetracycline doxycycline (DOX) allometrically scaled for rats. DOX initiates the expression of Flt3L, which has been shown to recruit dendritic cells to the brain tumor microenvironment-an integral first step in the development of antitumor immunity. The data revealed a highly safe profile surrounding these human-equivalent doses of DOX under an identical therapeutic window as proposed in the clinical trial. This was confirmed through a neuropathological analysis, liver and kidney histopathology, detection of neutralizing antibodies, and systemic toxicities in the blood. Interestingly, we observed a significant survival advantage in rats with GBM receiving the 300 mg/day equivalent dosage of DOX versus the 200 mg/day equivalent. Additionally, rats rejected "recurrent" brain tumor threats implanted 90 days after their primary brain tumors. We also show that DOX detection within the plasma can be an indicator of optimal dosing of DOX to attain therapeutic levels. This work has significant clinical relevance for an ongoing phase I clinical trial in humans with primary GBM and for other therapeutic approaches using Tet-On transactivation system in humans.

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma.

Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

Gene Therapy for the Treatment of Neurological Disorders: Central Nervous System Neoplasms.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a median survival of 16.2-21.2 months post diagnosis (Stupp et al., N Engl J Med 352(10): 987-996, 2005). Because of its location, complete surgical resection is impossible; additionally because GBM is also resistant to chemotherapeutic and radiotherapy approaches, development of novel therapies is urgently needed. In this chapter we describe the development of preclinical animal models and a conditionally cytotoxic and immune-stimulatory gene therapy strategy that successfully causes tumor regression in several rodent GBM models.

In vivo studies of polypyrrole/peptide coated neural probes.

Neural probes are micromachined multichannel electrode arrays that facilitate the functional stimulation and recording of neurons in the peripheral and central nervous system. For long-term implantations, surface modification is necessary for maintaining the stable connection between electrodes and neurons. The conductive polymer polypyrrole (PPy) and synthetic peptide DCDPGYIGSR were co-deposited on the electrode surface by electrochemical polymerization. The stability of PPy/DCDPGYIGSR coatings was tested in soaking experiments. It was found that the peptide was entrapped in the PPy film and did not diffuse away within 7 weeks of soaking in DI water. Coated probes were implanted in guinea pig brain for periods of 1, 2 and 3 weeks. Recording tests were performed and the impedance was monitored. The explanted probes and tissue were examined by immunocytochemical studies. Significantly more neurofilament positive staining was found on the coated electrode which indicated that the coatings had established strong connections with the neuronal structure in vivo. Good recordings were obtained from the coated sites that had neurons attached. First week tissue sections had no significant gliosis. In week 2, a layer of non-neuronal tissue consisting of mostly meningeal fibroblasts and ECM protein including at least fibronectin was formed around the probe tracks of both coated and uncoated probes. Astrocytes started to form a loosely organized layer by the end of the third week.