238 Enhancing Immunotherapy with a Replicating Vaccinia Virus Expressing CXCL11 to Chemoattract T Cells into Tumors Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society[.]
GENE AND CELL THERAPY FOR PULMONARY DISEASES differentiation of airway epithelia with small molecule treatments The resulting drugs identified were tested for their abilities to safely improve target knockdown Interestingly, treatment of welldifferentiated cells with several different drug classes resulted in modest to robust knockdown of the target when delivered along with a DsiRNA oligo These results support that well-differentiated airway epithelia, normally resistant to siRNA delivery, can be pretreated with small molecules to improve oligo uptake and RNAi responses 236 Gene Therapy of Cystic Fibrosis: Impact of Plasmid DNA on In Vivo Lipid-Based Transfection Efficacy Mattias Lindberg,1 Tony Le Gall,1,2 Nathalie Carmoy,1,2 Stephen C Hyde,3,4 Deborah R Gill,3,4 Mathieu Berchel,2,5 Aurore Fraix,2,5 Paul-Alain Jaffrès,2,5 Pierre Lehn,1 Tristan Montier.1,2 Gene and Cell Therapy Group, INSERM U1078, University of Western Brittany, Brest, France; 2Platform IbiSA SyNanoVect, SFR 148, University of Western Brittany, Brest, France; 3Gene Medicine Reasearch Group, University of Oxford, United Kingdom; 4UK Cystic Fibrosis Gene Therapy Consortium, Oxford, London, Edinburgh, United Kingdom; 5UMR CNRS 6521, University of Western Brittany, Brest, France Synthetic carriers, such as cationic lipids, have been developed for gene therapy of cystic fibrosis (CF) as an alternative to viralbased systems that are still associated with immunogenic and oncogenic side effects Our objective was to evaluate the impact of two plasmid constructions encoding the luciferase on monocationic lipophosphoramidates in vivo transfection efficiencies, depending on the administration route Two plasmids were tested in combination with the original cationic arsenolipids BSV4 and KLN47: pTG11033 (9,6 Kb; Transgene) and pGM144 (3,7 Kb; UK CF Gene Therapy Consortium), the last one being completely devoid of any CG dinucleotides After systemic delivery of BSV4-based lipoplexes to naive mice, the bioluminescent expression was exclusively confined to the lungs and was observed up to days with the pTG11033 whereas pGM144 was expressed during more than 20 days With this latter, several organs also possessed luc activities (heart, kidneys, ) Moreover, high levels of luciferase were detected in the serum in the first days after administration, suggesting its excretion by the transfected cells Once the expression has decreased under the detection threshold, a second administration of BSV4/pGM144 leads to a similar gene expression than the first one, contrary to BSV4/ pTG11033 where a very short-term re-expression was observed only when the two administrations were spaced for a sufficient period of time Equivalent results were obtained with KLN47 A transient hepatotoxicity was observed during the first days, independently of the plasmid used We then aimed at developing a lipidic combination permitting to transfer genes into the lungs after intratracheal deposit A first positive result has been obtained with KLN47/pGM144 leading to a bioluminescent signal in the lungs during more than 100 days Furthermore, immunohistochemical stainings demonstrated that bronchial epithelial cells were specifically transfected, urging us to go forward by transferring the gene of interest cftr 237 Lentiviral Vector Gene Transfer to Porcine Sinus and Pulmonary Airways Patrick L Sinn,1 Ashley Cooney,1 Mayumi Oakland,1 Eugene Chang,1 Paul B McCray,Jr.1 The University of Iowa, Iowa City The development of gene therapy as a treatment for cystic fibrosis (CF) lung disease has been limited in part by the lack of an animal model that presents a similar disease progression as humans In anticipation of future preclinical disease correction studies in CF pigs, we investigate lentiviral vector development and transduction Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) in vitro and wild-type pigs in vivo Using a feline immunodeficiency virus (FIV)-based vector, we previously reported that the envelope glycoproteins from baculovirus (GP64), retrovirus (JSRV), coronavirus (SARS), and filovirus (Ebola) confer apical entry into differentiated primary cultures of human airway epithelia whereas VSV-G confers basolateral entry In addition, we evaluated FIV vector pseudotyped with the influenza envelope glycoproteins (HA and NA) derived from swine influenza H3N2 and human influenza H1N1 We screened the apical and basolateral transduction efficiency of pseudotyped FIV on well-differentiated PAE, and noted gene transfer efficiency and polarity similar to that observed for human airway epithelia Retroviral restriction factors are species specific and may present barriers against lentiviral gene transfer FIV-based vectors reproducibly transduced immortalized pig cells as well as pig primary cells with greater efficacy than HIV-1 based vectors PAE express porcine TRIM5-alpha We contrasted the restrictive properties of porcine TRIM5-alpha against both FIV- and HIV-based vectors using gain and loss of function approaches We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5-alpha overexpression or knockdown Both vectors were similarly inhibited by bovine or rhesus TRIM5-alpha overexpression To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we bronchoscopically delivered GP64-FIV expressing mCherry vector to the right upper lobe of the lung or the ethmoid sinus of four-week old non-CF pigs One week later, cells expressing mCherry were readily detected in the epithelium Our findings indicate that pseudotyped FIV lentiviral vectors confer similar tropisms in porcine epithelia as observed in human primary airway epithelial cultures and provide further support for the selection of GP64 as an appropriate envelope pseudotype for preclinical gene therapy studies in the porcine model 238 Enhancing Immunotherapy with a Replicating Vaccinia Virus Expressing CXCL11 to Chemoattract T-Cells into Tumors Liang-Chuan S Wang,1 Rachel C Lynn,1 Veena Kapoor,1 Stephen H Thorne,2 Steven M Albelda.1 Thoracic Oncology Research Laboratory, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA; Immunology and Surgery, University of Pittsburgh, PA Rationale: Insufficient trafficking of activated, antigen-specific T-cells is a limitation to cancer immunotherapy A plausible explanation is that chemokine receptors expressed on activated T-cells not “match” with the intratumoral chemokine milieu In our previous study with vaccinia virus (VV) expressing IFNβ, we showed that VV.IFNβ could enhance the efficacy of cancer vaccine by inducing IFNβ and the IFN-related chemokines (IP-10 and CXCL11) to attract activated antigen-specific T cells into tumors Herein, we evaluated the potential of a VV expressing CXCL11 to enhance the efficacy of immunotherapy in our mouse lung cancer models Methods: Replication of VV vectors in vitro was examined by infecting mouse TC-1 lung cancer (expressing HPV-E7) cells with VV at MOI of The cell lysates and supernatants were collected at various time points (6, 24, 48 and 72 hours) and titered with a standard plaque protocol For in vivo viral replication, TC-1 tumors were harvested at various timepoints following intravenous injection of VV-CXCL11 Tumor lysates were then titered on BSC-1 cells CXCL11 levels in culture supernatants or tumor lysates were measured using ELISA For the combination studies with the Ad.E7 cancer vaccine, mice bearing TC1 tumors (approximately 150 mm^3 in size) were vaccinated subcutaneously twice, days apart, with 10^9 pfu of Ad.E7 vector Two days later, VV-Luciferase (VV-Luc) and VV-CXCL11 were injected into corresponding mice intravenously via tail vein Tumors were monitored times weekly until they reached S93 GENE AND CELL THERAPY FOR PULMONARY DISEASES large size Infiltration of T cells into the tumor was determined by RTPCR and flow cytometry Results: VV.CXCL11 replicated well and generated over 10 ng/ml of CXCL11 in mouse TC-1 lung cancer cells in vitro over two days The control vector VV.Luc, in comparison, replicated well in the same tumor cell line, but did not generate any significant level of CXCL11 in culture Similar finding was also seen in mice, in that both VV.Luc and VV.CXCL11 replicated well in TC-1 tumors after intravenous injection, but only VV.CXCL11 induced significant levels of CXCL11 This chemokine production was associated with increased numbers of tumor infiltrating CD8 T cells in the VV.CXCL11-treated group versus VV.Luc group When we gave these two VV vectors following the Ad.E7 cancer vaccine, VV.CXCL11, not VV.Luc, enhanced anti-tumor efficacy of Ad.E7; 40% of mice treated with both VV.CXCL11 and Ad.E7 were cured, and maintained disease-free for over two months No cures were seen in any other treatment group, and the survival curves for single agents and VV.Luc plus Ad.E7 looked similar Conclusion: Our data indicated that use of VV.CXCL11 is a promising approach to augment the efficacy of immunotherapy by inducing more T-cell infiltration to the tumor site Similar studies using adoptive T cell transfer using mouse T cells expressing a chimeric antigen receptor are currently underway 239 High Speed X-Ray Imaging Reveals Dosed Fluid Dynamics and Fate in Mouse Nasal and Lung Airways David Parsons,1,5,6 Siu Karen,2,3,4 Morgan Kaye,2 Martin Donnelley.1,5,6 Respiratory & Sleep Medicine, Women’s & Children’s Hospital, WCHN, Nth Adelaide, SA, Australia; 2School of Physics, Monash University, Clayton, VIC, Australia; 3Monash Biomedical Imaging, Monash University, Clayton, VIC, Australia; 4Australian Synchrotron, Clayton, VIC, Australia; 5Paediatrics & Reproductive Health, University of Adelaide, SA, Australia; 6Centre for Stem Cell Research, University of Adelaide, SA, Australia In the lung the spreading of dose fronts along conducting airways and into distal alveolar spaces was easily followed For some deliveries respiratory motion halted temporarily, and cases of subsequent fluid clearance (i,e potential loss) also occurred The 40 μl dose produced coughing that cleared some of the dose from the lungs Dose progress into the lung tree was patchy and not uniform across animals Figure shows a 15 μl dose with the full movie at http://goo.gl/dmMx8 Introduction: Mice are commonly used for developing gene therapy treatments for CF airway disease, but there is little knowledge of how fluid doses distribute after delivery into the airways We have developed high spatial and temporal resolution X-ray imaging methods to help determine the destination and the behaviour of (surrogate) fluid dose deliveries Methods: Nembutal-anaesthetised (intubated and ventilated for lung studies) C57Bl/6 mice were imaged at the SPring-8 synchrotron, Japan Doses of 4, 10 or 20 μl (nose study) or 15, 30, or 40 μl (lung study) of an iodine-based contrast fluid mixture were delivered to mimic standard gene transfer dose deliveries Images were acquired at 6.7 frames per second for minutes following delivery initiation Background subtraction, frame difference and pseudo-colouring techniques revealed the progress of fluid along the airways over time Results: Fluid distributions were dose-dependent In the nose a μl dose typically remained in the anterior and olfactory regions Higher doses saw fluid ‘overflow’ via the nasaopharyngeal airway towards the lung and could affect contralateral nasal spaces Furthermore, residual dose lodging in the distal nasopharynx often refluxed proximally Figure shows a 20 μl dose with the full movie at http://goo.gl/lBsHh Conclusion: These novel non-invasive imaging techniques reveal for the first time the complex real-time dynamics of airway dosing in mice and could also be applied to understanding dose delivery in other animal models The variability noted suggests therapeutic dosing outcomes may be influenced by heterogeneous dose distributions that naturally occur in anatomically complex nasal and lung airways S94 Molecular Therapy Volume 20, Supplement 1, May 2012 Copyright © The American Society of Gene & Cell Therapy ... Biomedical Imaging, Monash University, Clayton, VIC, Australia; 4Australian Synchrotron, Clayton, VIC, Australia; 5Paediatrics & Reproductive Health, University of Adelaide, SA, Australia; 6Centre... Higher doses saw fluid ‘overflow’ via the nasaopharyngeal airway towards the lung and could affect contralateral nasal spaces Furthermore, residual dose lodging in the distal nasopharynx often refluxed... therapy treatments for CF airway disease, but there is little knowledge of how fluid doses distribute after delivery into the airways We have developed high spatial and temporal resolution X-ray