880 Repeat Aerosol Delivery of Concentrated PEI/pDNA to the Sheep Lung Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S338 LUNG AND RESPIRA[.]
LUNG AND RESPIRATORY DISEASE GENE & CELL THERAPY 878 The T1Į Promoter Mediates Nuclear Import of Plasmid DNA into Alveolar Epithelial Type I Cells Lynn F Gottfried, David A Dean Pediatrics, University of Rochester Medical Center, Rochester, NY Nonviral gene delivery to the lung is a promising approach for the treatment of a various number of devastating diseases However, its use has been limited, in part, by the lack of cell specic targeting of DNA The alveolar epithelial type I (ATI) cell, in particular, is an attractive cell type to target, as it comprises 95% of the internal surface area of the lung However, ATI-specific gene delivery studies have been limited due to a lack of understanding of the true transcriptional properties of these cells and difculty in isolation of primary cell cultures To overcome this, we characterized two models of ATI cells, the R3/1 cell line and primary rat alveolar type II cells maintained on plastic for days, to be used for initial gene delivery studies Immunouorescence analysis has identied these cell types as good models for ATI cells, as evidenced by their strong expression of the ATI markers, T1α and Aquaporin-5, and absent expression of ATII markers, Surfactant Protein B and Lamellar Body Membrane 180 Using these models, we have developed a method for ATI-specic targeting of plasmid DNA through utilization of cellspecic DNA nuclear import sequences We have previously shown that nuclear import of plasmids in non-dividing cells is mediated by specic sequences of DNA that bind to transcription factors in the cytoplasm These transcription factors contain nuclear localization signals that can be presented to the importin nuclear import machinery to promote nuclear import If bound to the transcription factors, the DNA can be carried into the nucleus To date, we have identied DNA sequences that act in all cell types as well as specic cell types, due to the transcription factors bound We screened the promoters from several genes previously identied in the literature to be specic for or enriched in ATI cells for the ability to mediate DNA nuclear import in this specic cell type after cytoplasmic microinjection The promoters of these genes were inserted into a plasmid lacking any other DNA nuclear targeting sequence, and the plasmids were labeled with a triplex-forming Cy3-PNA Of the sequences examined, we demonstrate that only a DNA sequence within the rat T1α promoter was able to mediate plasmid DNA nuclear import in R3/1 and day primary rat alveolar epithelial cells When microinjected into other cell types, the T1α promoter displayed no DNA nuclear import activity, suggesting that this sequence acts in a type I cell-specic manner This highlights a new property of the T1α promoter as well as the ability to directly target the ATI cell over other cell types of the lung 879 Effects of LPC on Lung Mechanics in NonHuman Primates P Hiatt, R McConnell, N Grove, D Dang, N Brunetti-Pierri, D Palmer, A Beaudet, P Ng Depts of Pulmonary Pediatrics and Molecular Human Genetics, Baylor College of Medicine, Houston, TX Efcient transduction of the airway epithelium and long-term expression of the transgene are essential elements for successful Cystic Fibrosis (CF) gene therapy Parsons et al (2008) showed that lysophosphatidylcholine (LPC) opens tight junctions between cells allowing improved access to viral receptors on the baso-lateral surface of the airway epithelium in mice We have previously shown that targeted lobar aerosolization of a mixture of LPC and Helper Dependent Adenovirus (HDAd) into nonhuman primate lungs results in uniform, high level pulmonary transduction Our goal is to use LPC as a carrier in the delivery of HDAd for gene therapy in CF patients In this study we investigated the effects of LPC on lung mechanics of non-human primates Methods: Baseline pulmonary function (PF) in baboons was measured using the Flexivent respiratory mechanics platform (Scireq) Animals were intubated with a cuffed endotracheal Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy tube, placed under general anesthesia, and their lungs were inated to 30cm of H2O prior to each of the PF measurements Following baseline measurements, LPC was aerosolized in two different concentrations which were previously shown to yield signicant pulmonary transduction by HDAd Two ml/lobe of 0.1% LPC was delivered into each of the six major lung lobes of baboons using an intracorporeal nebulizing catheter (AeroProbe) inserted into a bronchoscope PF measurements were taken after LPC administration and every 15 minutes thereafter up to 2.5 hours to determine changes in lung mechanics A nal PF measurement was taken at 24 hours post LPC administration for those animals with lung functions that did not return to baseline at the end of the 2.5 hour testing time Three weeks after administration of 0.1% LPC, 0.05% LPC was administered to the same animals and PF measurements were obtained as previously described Results: Maximal PF changes occurred 15 minutes following LPC administration for both 0.05% and 0.1%LPC Mean respiratory resistance increased 53%+14%(SE) and 44%+14% above baseline and compliance decreased 25%+3.9% and 27%+7% below baseline after the delivery of 0.05% LPC(n=6) and 0.05%LPC+vector(n=3), respectively Respiratory resistance in these animals returned to normal in less than hours When 0.1% LPC(n=5) was administered to the same baboons, their mean respiratory resistance increased 127%+32% over baseline and compliance decreased 35%+4.1% from baseline When 0.1%LPC+vector was administered to baboons, their mean respiratory resistance increased 338%+108% over baseline and compliance decreased 63%+4.2% from baseline Pulmonary function changes returned to baseline within 24 hours of LPC+vector administration No significant changes in FiO2 or SpO2 were observed following administration of either concentrations of LPC Conclusion: LPC (0.05% and 0.1%) is associated with increased respiratory resistance and decreased compliance measurements when aerosolized to the lung Maximal changes in PF occur within 15 minutes of delivery These results indicate that LPC delivered by aerosol to the airways of the lung produce signicant but temporary pulmonary function changes in non-human primates in a dose-dependent manner 880 Repeat Aerosol Delivery of Concentrated PEI/pDNA to the Sheep Lung Gerry McLachlan,1 Lee A Davies,2 Cath M Gordon,1 Christina Vrettou,1 Eilidh Baker,1 Peter Tennant,1 Alison Baker,1 Rebecca L Coles,2 Dominique McCormick,2 Stephanie G Sumner-Jones,2 Stephen C Hyde,2 Deborah R Gill,2 D David S Collie.2 The Roslin Institute & R(D)SVS, University of Edinburgh, Edinburgh, United Kingdom; 2Gene Medicine Group, NDCLS, University of Oxford, Oxford, United Kingdom Considerable advances have been made in lung-directed gene therapy, however it is unlikely that clinical benet to patients with chronic lung disease can be achieved without repeat administration For viral vectors, the ability to deliver multiple doses is normally limited by the host immune response, which results in attenuated expression following consecutive exposures Importantly, aerosol delivery of non-viral gene transfer agents to the lung is not generally associated with the same problem We have previously demonstrated that aerosol delivery of plasmid DNA (pDNA) complexed with polyethylenimine (PEI) to sheep, either at the standard (low) concentration or with a novel concentrated formulation (cPEI; Davies et al, 2008, Mol Ther 16:1283) induces a mild transient inammatory response characterised by increased neutrophils in bronchoalveolar lavage (BAL) uid Here we have examined the effect of repeat dosing with cPEI Two groups of sheep (n=6) were each administered a single dose of cPEI/pCIKLux (32mg DNA at 1.6mg/ml) Expression of luciferase (lux) protein and mRNA were measured either at day (Group 1) or day 15 (Group 2) Mean lux protein expression at day was 19.4 +/- 6.5 RLU/mg lung protein, which fell to background S339 OLIGONUCLEOTIDE & RNAI THERAPEUTICS III levels at day 15 A third group was given two doses 14 days apart and gene expression assessed at day 15 Mean Lux expression after a repeat dose was 15.7 +/- 3.7 RLU/mg and was not signicantly different to day level from a single dose (Group 1) A similar pattern of expression was observed when vector-derived lux mRNA was quantied in these animals The mean value from a single dose at day was 62.9 % vector mRNA/endogenous ovCFTR mRNA (%v/e), falling to 0.4 %v/e at day 15 mRNA expression following two doses was somewhat lower than the level obtained with a single dose at day (11.8 %v/e; p=0.03) Consistent with previous studies, the level of neutrophils in BAL uid in response to a single dose was 49% on day 1, which decreased to 5% (within normal range) by day 15 Unexpectedly the BAL neutrophil response observed at day following repeat dosing was signicantly lower than after a single dose (∼23%; p=0.02) These results indicate that a second aerosol administration of the cPEI/pDNA formulation to the whole lung of a large animal directs repeated transgene expression and induces a reduced BAL neutrophil response compared with the rst dose, suggesting that multiple administrations may be effective and well tolerated These data provide the rst evidence that repeated aerosol delivery of a non-viral formulation to the sheep lung is feasible and support the potential use of cPEI formulations in clinical applications where repeated administration to the lung is required UK Cystic Fibrosis Gene Therapy Consortium 881 Pseudotype Switching as a Strategy To Permit Efcient Re-Administration of Lentiviral Vectors to Mouse Airways John C Olsen, Angela Giddings, Manij Patel Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, NC A possible complication of using gene transfer as a repetitive therapy is the generation of a neutralizing immune response against the gene transfer vector This could affect the therapeutic efcacy by inhibiting the efciency of subsequent vector delivery To test for inhibition of repeat lentivirus-mediated airway gene transfer, mice were dosed by nasal inhalation of an inuenza hemagglutinin (HA) pseudotyped EIAV-based lentivirus vector encoding lacZ Three weeks later, these mice or parallel naive mice were challenged by nasal administration of an EIAV vector encoding a different transgene (luciferase) and pseudotyped with HA from the same H7 subtype Imaging of nasal luciferase activity indicated that mice previously dosed with the HA pseudotyped vector had a 4-7 fold lower expression compared to parallel naive mice Sera from mice previously dosed with vector had signicant neutralizing activity towards H7-HA pseudotyped lentiviral vectors, but did not inhibit infectivity of VSV-G pseudotyped vectors This result prompted us to investigate strategies to alter the vector to avoid inhibition of gene transfer during re-dosing For inuenza virus, the HA glycoprotein is the major target of the neutralizing response During inuenza infection or vaccination, the inuenza HA elicits neutralizing antibody that can protect against subsequent challenge of inuenza virus from the same subtype, but has weaker protection against other inuenza virus serotypes Thus, administration of an H7 pseudotype would be predicted to protect against subsequent delivery by another H7 pseudotype, but would be predicted to have a much weaker ability to protect against other inuenza subtypes In order to test whether pseudotyping with a second inuenza HA subtype would permit re-dosing, we optimized lentiviral vector pseudotyping using the HA from an inuenza H3 subtype Pseudotyping with H3 HA was found to be as efcient as pseudotyping with H7 HA As an initial test to determine if changing inuenza subtypes could provide a strategy for overcoming the block to vector re-administration, an in vitro neutralization assay was carried out to determine if sera from mice with high titer serum neutralization activity against H7 HA S340 would inhibit H3 HA pseudotypes It was found that sera from mice previously dosed with H7 pseudotyped vectors or a commercial source of anti-H7 were not effective at inhibiting infectivity of an H3 HA pseudotype This result suggests that subtype switching might be an effective strategy for overcoming the presence of preexisting neutralizing antibody These results also conrm that HA is the major neutralizing determinant of inuenza HA pseudotyped lentiviruses since it was the only variable in the production of the two pseudotypes In vivo challenge experiments to test the efcacy of pseudotype switching are in progress Oligonucleotide & RNAi Therapeutics III 882 Antinociceptive Potentiation and Attenuation of Tolerance by Intrathecal ȕ-Arrestin siRNA in Rats Chung-Ren Lin,1 Kuan-Hung Chen,1 Chih-Hsien Wu,1 Yi-Sen Chen.1 Anesthesiology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung County, Taiwan We tested whether intrathecal administration of siRNA against β-arrestin would reduce the tolerance to chronic morphine use and the severity of precipitated morphine withdrawal Intrathecal β-arrestin (2 ug siRNA/10 ul/rat) were injected onece daily for days Rats then received intrathecal catheter implantation and a continuous intrathecal infusion of morphine (2 nmol/h) or saline for days Daily tail-ick and intrathecal morphine challenge tests were performed to assess the effect of intrathecal β-arrestin siRNA on antinociception and tolerance to morphine Naloxone withdrawal (2 mg/kg) was performed to assess morphine dependence The antinociceptive effect of intrathecal morphine was increased by β-arrestin inhibition The magnitude of tolerance was decreased in the rats receiving the nmol/h infusion with β-arrestin siRNA compared with the control group (morphine nmol/h alone) (AD50, 25.3 vs 143.7 nmol) The severity of naloxone-induced withdrawal was less in the rats receiving β-arrestin siRNA Intrathecal β-arrestin siRNA thus enhances analgesia and attenuates naloxone-induced withdrawal symptoms in rats receiving chronic intrathecal morphine infusion This method may merit further investigation in the context of the long-term use of intrathecal opioids for controlling chronic pain IMPLICATIONS: Control of chronic pain is a major health problem We show here that intrathecal β-arrestin siRNA in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain 883 siRNA Cationic Delivery Systems for Therapeutic Applications Anne-Laure D Bolcato-Bellemin,1 Marie-Elise Bonnet,1 Gaëlle Deglane,1 Nathalie Lenne,1 Patrick Neuberg,1 Clément Paris,1 JeanPaul Behr,2 Patrick Erbacher.1 Polyplus-transfection, Illkirch, France; 2Laboratoire de Chimie Génétique, Université de Strasbourg, Illkirch, France Numerous delivery systems have been described in order to develop the therapeutic potential of siRNAs However, to date, delivery still remains a major obstacle for nucleic acid-based drugs to be a success Anionic oligonucleotides, such as synthetic siRNA, are unable to cross the cell membrane siRNA can efciently enter cells when formulated into cationic complexes The purpose of our work is to develop and improve cationic systems for in vivo delivery of siRNA Given that complexes formed between siRNA and cationic polymers used as carriers, are much weaker than those formed with genes, we have designed sticky siRNAs (ssiRNAs) to increase the Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... provide the rst evidence that repeated aerosol delivery of a non-viral formulation to the sheep lung is feasible and support the potential use of cPEI formulations in clinical applications where repeated... following repeat dosing was signicantly lower than after a single dose (∼23%; p=0.02) These results indicate that a second aerosol administration of the cPEI/pDNA formulation to the whole lung of a... the gene transfer vector This could affect the therapeutic efcacy by inhibiting the efciency of subsequent vector delivery To test for inhibition of repeat lentivirus-mediated airway gene transfer,