High-Capacity Adenovirus Vector -mediated transfer of conditional cytotoxic (herpes simplex type 1-thymidine kinase [HSV1-TK] and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L] transgenes elicits immune-mediated long-term survival in a synge

PUNTEL; MUHAMMAD; KROEGER; CANDOLFI; YAGIZ; LIU; SALEM; GOZO; BAKER; SHI; FOULAD; XIONG; PALMER; NG; LOWENSTEIN; CASTRO

San Diego, California, USA

Congreso; American Society of Gene Therapy; 2009

High-Capacity Adenovirus Vector -mediated transfer of conditional cytotoxic (herpes simplex type 1-thymidine kinase [HSV1-TK] and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L] transgenes elicits immune-mediated long-term survival in a syngeneic intracranial GBM model.M. Puntel1, A.K.M. Muhammad1, K.M. Kroeger1, M. Candolfi1, K. Yagiz1, C. Liu1, A. Salem1, M. Gozo1, G. Baker1, Y. Shi1, D. Foulad1, W. Xiong1, D. Palmer2, P. Ng2, P. R. Lowenstein1and M. G. Castro1.1Board of Governors Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, and Departments of Medicine, and Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA90048, USA. 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA. Email: castromg@cshs.orgGlioblastoma multiforme (GBM) is a malignant primary brain tumor associated with 5% survival at 5 years post-diagnosis. Adenoviral (Ad)-mediated transfer of conditional cytotoxic, HSV1-TK and immunostimulatory Flt3L transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. Helper dependent, high-capacity Ad vectors (HC-Ads) have a significantly favorable immunological profile due to the lack of Ad encoding sequences. Even in the presence of a preexisting systemic anti-Ad immune response, transgene expression from HC-Ads remains stable for up to 1 year.In order to test efficacy, transgene expression, distribution of vector genomes, and potential toxicity of the high capacity vectors for the treatment of GBM we performed intratumoral delivery of the combined therapy using HC-Ad-TK and HC-Ad-TetON-Flt3L co-administered with gancyclovir and doxicycline. Three doses of the HC-Ads were administered intra-tumorally and analyzed at 5 days, 30 days and 6 months after injection. Neuropathology studies were conducted by immunocytochemistry of brain sections; distribution of HC-Ads genomes were analyzed by qPCR as genome copy numbers within 13 different tissues including brain injection side and contra-lateral side, spleen, and liver; FLt3L expression in brain tissue and plasma. Approximately 70% of the rats treated survived for long term when treated with the combined therapy. At 5 days after treatment Flt3L was detected in brain tissue and serum; strong infiltration of immune cells into the tumor mass was observed; an average of 7.4x105, 1x106, and 7.2 x106 total vector genomes were detected for the doses 5x108, 1x109 and 5x109 vp, respectively; the vector genomes were exclusively restricted to the brain injection side for all the doses tested. At 30 days after treatment, Flt3L was not detected in neither brain tissue nor serum; infiltration of immune cells into the tumor mass was observed limited to the scar region within the striatum; vector genomes were not detected at the doses tested.Also, no toxic effects were found as consequence of the treatment. Our data indicate that the combined administration of HC-Ad vectors encoding TK and Flt3L was efficacious and safe for the treatment of glioma in a syngeneic, intracranial rodent model. These results will form the basis for further developments towards the implementation of HC-Ad mediated combined gene therapy for the treatment of GBM in human patients.Funded by NINDS, R01-NS054193-01A, R01-NS061107-01A1, The Drown Foundation and Bram & Elaine Goldsmith Chair in Gene Therapeutics to P.R.L.; NINDS U01-NS052465-01, R01NS057711-01A2, R21-NS05414031A2 to MGC; The Linda Tallen & David Paul Kane Foundation and the Board of Governors at CSMS.