3c; and see Supplementary Fig. Vector genomes were restricted to the injection site. Serum chemistry did not uncover adverse systemic side effects at any of the doses tested. Taken together, our data show that doses of up to 1109 VP of each HC-Ad can be safely administered into the normal brain. This comprehensive toxicity and biodistribution study will lay the foundations for implementation of a phase 1 clinical trial for GBM using HC-Ads. Introduction Despite improvements in neurosurgical techniques, radiotherapy, and chemotherapy, the prognosis in glioblastoma multiforme (GBM) remains poor, with median survival of 14.6C19.6 months (Stupp characterization of the HC-Ad vectors were previously published (Candolfi (modified LabDiet laboratory rodent diet 5001 with 2000?ppm doxycycline; PMI Nutrition International/Purina Mills, Richmond, IN). Groups of rats were injected unilaterally via the right striatum with any of the three escalating doses of HC-Ad-TK and HC-Ad-TetON-Flt3L MI-773 (1108, 1109, or 11010 VP of each vector). The vectors were first mixed together and resuspended in a final volume of 3?l of saline. Using a 10-l Hamilton syringe fitted with a 26-gauge needle, the vector combination was administered at the following stereotactic coordinates: 1?mm anterior MI-773 and 3.2?mm lateral to the bregma and the injection volume of 3?l was delivered in three locations (1?l each) at depths of ?5.5, ?5.0, and ?4.5?mm from your dura. Twenty-four hours after treatment, the rats received ganciclovir (GCV, 25?mg/kg, intraperitoneal; Roche Laboratories, Nutley, NJ), twice daily for up to 10 consecutive days. The control group of rats MI-773 (naive) received 3?l of saline at the same stereotactic coordinates as the treatment groups. Groups of rats were evaluated 5 days, 1 month, 6 months, and 1 year after vector delivery to determine biodistribution of HC-Ad vector genomes, neurotoxicity, peripheral blood cell counts and serum biochemistry, and circulating levels of anti-adenovirus neutralizing antibodies and anti-TK antibodies. In addition, the 1-month, 6-month, and 1-12 months groups were evaluated for HC-Ad vector-induced behavioral deficiencies. All animal procedures were carried out in accordance MI-773 with the NIH and approved by the Cedars-Sinai Medical Center Institutional Animal Care and Use Committee. Serum biochemistry and Rabbit Polyclonal to RRAGB hematology Five days, 1 month, 6 months, or 1 year posttreatment, blood was collected from each animal during euthanasia. The samples were sent to the laboratory of ANTECH Diagnostics (Irvine, CA) for analysis of routine hematological and biochemical parameters. The median, minimal, and maximal values for each parameter are shown in the Results section and supplementary furniture (supplementary data are available online at http://www.liebertpub.com/hgtb). Circulating neutralizing anti-adenovirus antibodies The level of adenovirus-specific neutralizing antibodies was assessed as explained previously (Puntel values less than 0.05 were considered as significant. Results Dose escalation of HC-Ad-TetOn-Flt3L plus HC-Ad-TK delivered into naive rat brain followed by neuropathological analysis To examine the effects of HC-Ad-TetOn-Flt3L and HC-Ad-TK on normal brain architecture and inflammation, we performed a comprehensive neuropathological analysis 5 days, 1 month, 6 months, and 1 year after intrastriatal delivery of HC-Ads. Naive adult Lewis rats received a combination of either 1108, 1109, or 11010 VP of each HC-Ad vector or saline (Fig. 1a). GCV was administered twice daily for up to 10 days. The rats were fed doxycycline-containing chow for up to 4 weeks because this routine was found to be optimal for therapeutic efficacy (Muhammad for up to 4 weeks starting 2 days before treatment. Groups of rats were evaluated at 5 days (short-term), 30 days (medium-term), 180 days or 365 days (long-term) for biodistribution of vector genomes, neuropathology, peripheral blood cell counts and serum biochemistry, circulating levels of anti-adenovirus neutralizing antibodies, and anti-TK antibodies. In addition, the 30-day, 180-day, and 365-day posttreatment groups underwent neurobehavioral screening. The rats were killed at experiment end points and organs were harvested for processing. Neuropathological analysis was performed with the brains of rats at 5 days (B) and at 1, 6, and 12 months (C). Neuropathology results at 1 and 6 months are shown in Supplementary Fig. S1. The brains were processed for Nissl staining to show gross morphology, and immunocytochemistry with main antibodies against myelin basic protein (MBP) to label oligodendrocytes and myelin sheaths, MI-773 tyrosine hydroxylase (TH) to label.
