138. hChonJb#7 Cells (Chondrocytes Expressing TGF-b1) Reduced Pain in a Rat Osteoarthritis Model

PHYSICAL METHODS OF DELIVERY activation (e.g. NANOG, Sox2, Oct4), 2) a specific dosage of Oct4HATHFUN in relation to other applied HATHFUN proteins such as Sox2, NANOG and Klf4 is necessary to achieve optimal activation and 3) when small molecule inhibitors which aid in reprogramming such as AM580, sodium butyrate, PD0325901 and SB431542 are applied that the optimal protein dosages may change. Conclusion: Protein-based reprogramming will offer a viable alternative to the dosage, reproducibility and safety issues inherent in viral-based reprogramming.

138. hChonJb#7 Cells (Chondrocytes Expressing TGF-b1) Reduced Pain in a Rat Osteoarthritis Model

Kyoung Baek Choi,1 Hyeon Youl Lee,1 Dae Wook Kim,1 Su Jeong Kim.1 1 Bio Innovation Center, Kolon Life Science, Geumcheon-gu, Seoul, Korea. Background: TissueGene-C (TG-C) has been developed as a cell and gene therapy for osteoarthritis. It is a mixture of non-transduced human chondrocytes (hChonJ) and genetically modified chondrocytes expressing TGF-b1 (hChonJb#7). Phase 2a and 2b clinical trials have shown that TG-C treatment significantly reduced pain when administered to the osteoarthritic patients. In the present study, we investigated to see if hChonJb#7 cells modulate pain by itself without being combined with hChonJ cells in a rat osteoarthritis model. Methods: Osteoarthritis in rats was induced by injection of monosodium iodoacetate (MIA) into the left knee joint. The injection of MIA resulted in the degeneration of the joints and induced pain. The rats showing pain-related behaviors were selected for the evaluation of hChonJb#7 cells. At two weeks post MIA administration, various doses of hChonJb#7 cells were injected into the osteoarthritic knees. The effect of hChonJb#7 cells on the mechanical allodynia was evaluated by von Frey filament test. After completing the filament tests, the knees were harvested for the histological analysis. Results: The intraarticular injection of hChonJb#7 cells reduced pain in a rat MIA model. The improvement of pain related behavior was shown in a dose-dependent manner from 1 week post hChonJb#7 cell injections. The minimum effective dose of hChonJb#7 was 3 x 10^4 cells. The effect was maintained upto 6 week post hChonJb#7 cell injections. The histological analysis showed that the integrity of cartilage was severely damaged by MIA treatment but hChonJb#7 cell treatment improved the cartilage from the MIA-induced damage when compared with the untreated group. Conclusion: This study showed that hChonJb#7 cell treatment reduced pain in a rat osteoarthritis model, which suggested that hChonJb#7 cells alone can contribute to the effect of relieving pain during the clinical trials. The molecular mechanisms of relieving pain, however, need to be further investigated.

Physical Methods of Delivery 139. Electro-Chemo-Gene Therapy for LongTerm Management of Cancer in Canines

Jeffry Cutrera,1 Xueqing Xia,1 Glenn King,2 Pamela Jones,2 Kristin Kicenuik,2 Shulin Li.1 1 Pediatrics Research, MD Anderson Cancer Center, Houston, TX; 2 Oncology, Gulf Coast Veterinary Specialist, Houston, TX. Previously, the results of a canine clinical trial were presented, and these results demonstrated that only two administrations of electroporation-mediated intratumoral treatments with bleomycin and Interleukin 12 (IL12) plasmid DNA (pDNA) are required to eradicate or inhibit tumor growth in spontaneous acanthomatous ameloblastoma, histiocytic sarcoma, and fibrosarcoma. In this new trial, one important question was examined--is it feasible to manage S56

persistent or recurrent tumors using multiple Electro-Chemo-Gene Therapy (ECGT) treatments in the same canine patient without causing side effects? To address this question, several patients with large (> 2 cm), recurrent tumors were enrolled in this study. Even though to conventional treatments such as surgery, radiation, and chemotherapy, ECGT treatments with IL12 pDNA and bleomycin were capable of slowing the growth of these large tumors; however, the tumors eventually maintained previous growth rates. To stop this growth, ECGT treatments were performed again with IL12 pDNA and either bleomycin or gemcitabine when the tumor volume began to increase. By performing these intermittent, tumor growthbased treatments, the large, recurrent tumors can be maintained and even reduced for extended periods of time. Additionally, by switching the chemotherapeutic agent and incrementally increasing the chemotherapeutic dose, chemo-resistance can be overcome. ECGT with either bleomycin or gemcitabine and IL12 pDNA has been tested in multiple breeds of canines ranging in size from 4 kg to 70 kg, and the number of treatments varied from 5 to14 across a period of 150 to 805 days. Regardless of the number of treatments, size of the patient, or volume of the tumor, no significant treatmentassociated side effects were seen, and all patients safely withstood repeated ECGT treatments. Importantly, this study illustrates that this ECGT procedure can serve as a standard procedure to manage or control recurrent and aggressive tumors and extend survival without necessarily curing the tumor. These results along with the current and completed gene therapy clinical trials and the clinical acceptance of electroporation in the European Union verify the efficacy and safety of ECGT for the treatment of cancer and pave the way for this technology to be applied to human cancer patients.

140. Engineering Novel Transducers for Effective Ultrasound-Mediated Gene Therapy

Misty L. Noble,1 Shuxian Song,1 Ryan R. Sun,1 Kyle P. Morrison,2 George W. Keilman,2 Luping Fan,1 Marla Paun,3 Keith R. Loeb,4 Carol H. Miao.1,5 1 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA; 2Sonic Concepts, Inc., Bothell, WA; 3 Applied Physics Laboratory, University of Washington, Seattle, WA; 4Fred Hutchinson Cancer Research Center, Seattle, WA; 5 Dept. of Pediatrics, University of Washington, Seattle, WA. We have achieved significant progress in enhancing gene delivery into livers of large animals using ultrasound (US)-targeted microbubble (MB) destruction methods. US can induce cavitation of exogenous nuclei, such as MBs, to temporarily permeabilize the vascular endothelium and cell membranes and allow the entrance of naked DNA into cells in the target region. Short US pulses (20 cyc) at low frequency (1.1 MHz) and high pressure amplitudes (>1 MPa) are inductive towards creating cavitation conditions for successful gene transfection. Using an apodized, unfocused, dual-element US transducer (H105, with a 52-mm effective diam), we obtained a 64fold enhancement in luciferase expression in pig livers compared to sham-control when injected with pGL4 plasmid and lipid-based MBs via a portal vein branch and exposed to US for 4 mins at 8400 W, or 2.7 MPa peak negative pressure (PNP). No liver damage was observed by histological analysis or liver transaminase assays. However, the planar, unfocused design of H105 significantly limits the electrical energy that can be put into the transducer. We designed and developed new therapeutic US transducers that are more efficient in converting electrical signals to acoustic energies, and are capable of delivering higher output intensity. These next generation transducers are designed to be spherically (H185A) or cylindrically focused (H185B) that should reduce the electrical power (Pel) requirement. With appropriate scanning over the liver surface, these transducers will produce a homogeneous acoustic field similar to H105. The 41-mm diam. H185A is composed of 19 individual 10-mm planoMolecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy