Sibiril1, Paul-Alain Jaffrès3, Tristan Montier1. 1Université Bretagne Occidentale, INSERM U1078, Brest, France,. 2INSERM U1078, Brest, France, 3Université ...
Non-Viral Gene Transfer and Therapy II levels of F.IX transgene expression to naïve animals treated with conventional AAV8 vector. At the highest IVIg dose tested (8mg/ mouse), residual levels of F.IX transgene expression were about 30% of naïve animals treated with conventional AAV8 vectors. We next evaluated the efficiency of exo-AAV8 vs. AAV8 vectors in naïve animals. In male mice, no statistically significant difference in F.IX transgene expression levels was observed between the two vector types; also indicated by the similar pattern of vector genome biodistribution. Interestingly, female mice (in which the efficiency of liver transduction with AAV is extremely low compared with male animals) treated with exo-AAV8 showed a dramatic increase in transgene expression, comparable to that of male mice receiving conventional AAV8 vectors. In conclusion, exo-AAV8 vectors present an enhanced liver transduction profile compared with conventionally purified AAV vectors, both in naïve female animals and in animals carrying anti-capsid neutralizing antibodies.
596. Effect of Chemokine/Cytokine Delivered by Nanoparticles on Tumor Migration of Neuroblastoma Targeting Chimeric Antigen Receptor T Cells
Mai T. Huynh Center for Cell and Gene Tharapy, Baylor College of Medicine, Houston, TX INTRODUCTION: Neuroblastoma is the most common extracranial solid tumor in children and morbidity and mortality of high risk disease remains significant. The ganglioside GD2 is overexpressed on neuroblastoma and T cells expressing chimeric antigen receptors (CARs) specific for GD2 (GD2.CAR T cells) have been shown to induce complete tumor remissions without toxicity in a clinical trial. However, insufficient migration of CAR T cells to solid tumors remains a major problem. XCL1 (lymphotactin) is a chemokine that attracts T cells and natural killer cells, however, like other chemokines, diffuses quickly when injected at the tumor site. Calcium Alginate Nanoparticles (CANs) have been used clinically to safely deliver vaccines and chemokines. We are now evaluating the use of CANs to deliver chemokines to the tumor microenvironment and improve tumor homing of GD2.CAR T cells. METHODS/ RESULTS: In our in vitro experiments, XCL1 was released more gradually, with a change from 798 pg/µL detected after 1 hour to 1233 pg/ µL at 50 hours when loaded with CANs compared to a decrease from 1434 pg/ µL to 398 pg/ µL at 50 hours without CANs as measured by ELISA and was able to attract CAR T cells. For in vivo experiments, T cells obtained from fresh peripheral blood mononuclear cells were activated on CD3 and CD28 coated plates and transduced 3 days later with firefly luciferase and GD2. CD28-OX40z or GD2. 41BBz retroviral supernatants with 70-95% transduction rate. Immunodeficient NSG mice were subcutaneously injected with GD2 expressing LAN-1 tumor cells and divided into different treatment groups which received IL-2 intraperitoneally twice weekly (n=5). Mice that received CANs loaded with XCL1 showed a 6.6-fold increase in CAR T cell signal on day 10 while peritumorally injected XCL1 alone or empty nanoparticles had no such impact on T cell migration. We subsequently incorporated IL15 in our experiments based on its reported enhancement of T cell expansion in vivo. Without other exogenous cytokines, mice receiving the combination of IL-15 and XCL1 with CANs on days 0, 7, 12, and 19 showed remarkable improvement in T cell signal at the tumor site: 8.19 and 4.62-fold increase compared to groups treated with CANs only and XCL1 loaded with CANs on day 7, respectively. As early as 6 days after T cell injection and the first CANs injections, mice in the control CANs had increased tumor burden, nearly double that of IL-15/XCL1/CANs group with intermediate tumor burden in the CANs/XCL1 group. The combination of both chemokine and cytokine S236
delivered by CANs also had a positive impact on survival, which was lengthened to >45 days compared to 26 days in the CANs only control group. CONCLUSION: Calcium Alginate Nanoparticles loaded with a combination of both XCL1 and IL-15 achieved superior T cell expansion, tumor control and better survival outcome compared to controls. These findings indicate that CANs are a promising delivery vehicle for chemokines in combination with adoptive T cell therapies. In the future we will attempt to optimize dosing and plan to transduce T cells with selected phenotypes to further enhance T cell persistence.
597. Development of Non-Viral Gene Delivery Systems with Antibacterial Effects Deliverable via Aerosol for Lungs Gene Therapy
Angélique Mottais1, Tony Le Gall2, Mathieu Berchel3, Yann Sibiril1, Paul-Alain Jaffrès3, Tristan Montier1 1 Université Bretagne Occidentale, INSERM U1078, Brest, France, 2 INSERM U1078, Brest, France, 3Université Bretagne Occidentale, CNRS UMR 6521, Brest, France Objective: Cystic Fibrosis (CF) is a genetic disorder caused by the absence or malfunction of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CF gene therapy would consist in delivering a normal copy of the CFTR gene in the nucleus of respiratory epithelial cells. The presence of bacteria in the pulmonary tract can alter the transported DNA, the transfection efficiency as well as targeted cells. At present, the leading method to target the respiratory epithelium being the nebulisation, the goal of this research project is to develop a formulation that can be delivered as an aerosol exhibiting both a transfection activity and an antibacterial activity. Methods: The formulated complexe was composed of 50% (molar percentage) of cationic lipid (an arseno-phospholipid whose transfection efficiency and antibacterial activity against Gram-positive bacteria have already been demonstrated) and 50% of a silver salt (active against Gram-negative bacteria). The transfection activity was evaluated according to the expression of a luciferase reporter gene into human bronchial epithelial cells grown in liquid culture or at air-liquid interface. The antibacterial activity was determined according to the difference of growth in agar plate between a protected area and an exposed area to the aerosol. Results: Contrary to the aerosolization of the classical lipoplexe, the formulation developed containing a silver salt possesses an antibacterial activity against the Gram-negative bacteria. The transfection activity is maintained post-aerosolization with some similar levels of expression to those observed with the cationic lipid used alone, and this both in liquid culture and at air-liquid interface. Discussion and Conclusion: This study shows that an equimolar combination of two compounds: an arseno-phospholipid and a silver salt (i) is simultaneously effective on bacteria frequently isolated from patients, (ii) has a very low human bronchial epithelial cells toxicity, and (iii) exhibits a transfection efficacy under clinically relevant conditions. Unlike a gene transfer system devoid of an antibacterial activity, such a combination may therefore participate in the eradication of bacteria, thus reducing the stress on the bronchial epithelial cells while promoting the transfection process and finally, the expression of the transgene.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
