312 In Vitro and In Vivo Functional Characterization of Recombinant SV40 Derived CFTR Vectors Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S121[.]
LUNG AND RESPIRATORY DISEASE Lung and Respiratory Disease 310 Attenuation of Monocrotaline-Induced Pulmonary Hypertension in Rats by Luminal Delivery of AAV Prostacyclin Synthase Gene Transfer Vectors Sara R Martin,1 Amanda K Pangle,1 Gabor Molnar,1 Ruth S Everett,1 Larry G Johnson.1 Pulmonary and Critical Care Medicine, University of Arkansas for Medical Sciences, Little Rock, AR Idiopathic pulmonary arterial hypertension (iPAH) is a devastating disease with a high morbidity and mortality untreated We hypothesized that luminal delivery of the human prostacyclin synthase (hPGIS) cDNA with a long-term expression vector could attenuate PAH The hPGIS cDNA was isolated by RT-PCR from human mRNA and cloned into an adeno-associated virus (AAV) serotype vector ( R Jude Samulski, Ph.D., University of North Carolina at Chapel Hill) AAV5 and AAV9 capsids were obtained from the Penn Vector Core (James M Wilson, M.D.) The chicken β-actin promoter with the cytomegalovirus enhancer (CB promoter) was also obtained from the Penn Vector Core AAV5 and AAV9CBhPGIS vectors were produced by calcium phosphate co-transfection of vector, helper, and capsid plasmids, purified by CsCl gradient centrifugation and dialysis, then titered by dot blot assay AAV5-CBPGIS vector or AAV9-CBhPGIS vector (2 x1010 vector genomes), or saline (vehicle) was instilled into airways of Fisher 344 rats (150 g) injected intraperitoneally days later with monocrotaline (MCT, 60 mg/kg) or saline Biochemical, hemodynamic, and morphologic assessments were performed when the rats developed clinical symptoms (∼3-4 weeks after MCT) or at six weeks First, we measured levels of the nonenzymatic hydrolysis production of prostacyclin, 6-keto-PGF1α, in serum and in lung, heart, and liver homogenates of euthanized rats with an enzyme immunoassay kit Subsequently, the heart was removed, the right ventricle (RV) was dissected from the left ventricle (LV) and septum (S), and each portion weighed Then, the right ventricular systolic pressures (RVSP) of anesthetized rats were measured with a 2F pressure transducer (Millar) catheter interfaced to a computerized data capture system (AD Instruments Inc.) Finally, the lungs of euthanized rats were inflation fixed with 4% paraformaldehyde, embedded in paraffin, and sections stained with hematoxylin and eosin or an elastic stain Intraluminal administration of AAV5 and AAV9 vectors (MCT-AAV5 and MCT-AAV9 rats) increased PGF1α levels by three-fold and ten-fold, respectively, as compared to MCTsaline controls, and resembled levels measured in rats not treated with MCT (saline-saline) A significant increase in PGF1α levels was also detected in lung and heart homogenates from MCT-AAV9 rats as compared to MCT-saline or saline-saline rats, but not in MCTAAV5 rats RV/LV+S ratios were greater in MCT-saline rats than in saline-saline rats, whereas the ratios in MCT- AAV5 and MCT AAV9 rats were similar to the ratios in saline-saline rats, consistent with a reduction in MCT-induced cardiac remodeling Similarly, RVSP was two-fold greater in MCT-saline rats than in saline-saline rats, whereas RVSP in MCT-AAV5 and MCT-AAV9 rats was not different from saline-saline controls AAV-mediated gene transfer of hPGIS also attenuated MCT-induced remodeling of small pulmonary arteries Thus, we have demonstrated that luminal delivery of AAV5 and AAV9CBhPGIS vectors attenuated MCT-induced PAH These data suggest a potential role for luminal gene transfer of hPGIS in the treatment of human PAH Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 311 Mini-uPA Gene Therapy for Pulmonary Injury Xiang Gao,1 Jiang Li,1 Annette Wilson,1 Song Li.1 Center for Pharmacogenetics, Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA The normal balance between coagulation and fibrinolysis, both intravascularly and extravascularly, is significantly altered during pulmonary injuries The coagulation pathway is activated that is accompanied by an inhibition of fibrinolytic activity Upregulation of fibrinolytic activity via pulmonary delivery of urokinase plasminogen activator (uPA) (protein- or gene-based therapeutics) has been shown to be not only important for the improvement of hemodynamics and prevention of pulmonary damage at the early stages of pulmonary injury but also beneficial for the prevention of late-stage fibrosis One potential concern with uPA therapy is a proinflammatory activity that is attributed to the growth factor-like and kringle linker domains We have developed an EBV-based mini-uPA expression plasmid that retains the proteolytic domain but lacks the proinflammatory portions In vitro transfection with the mini-uPA plasmid resulted in an increased uPA activity that was similar to that with wildtype uPA plasmid A single intravenous injection of plasmid-LPEI complexes effectively transfected pulmonary endothelial cells with minimal side effects Pulmonary delivery of mini-uPA transgene two days after bleomycin challenge successfully prevented animals from developing pulmonary fibrosis as shown by histology analysis and collagen content assay Furthermore, mini-uPA gene transfer significantly prolonged the survival of bleomycin-treated mice Our studies suggest that pulmonary delivery of mini-uPA transgene via non-viral vectors may represent a new and safe strategy for the management of lung injuries 312 In Vitro and In Vivo Functional Characterization of Recombinant SV40-Derived CFTR Vectors Christian Mueller,1 Sofia Braag,2 Jeff Sirininger,3 Marlene Strayer,1 Fransisco Branco,2 Jean-Pierre Louboutin,2 Terrence R Flotte,1 David S Strayer.2 Pediatrics and Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA; 2Pathology, Jefferson Medical College, Philadelphia, PA; 3Veterinary Pathology, Louisiana State University, Baton Rouge, LA In cystic fibrosis (CF) respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa Despite considerable improvement in patient survival, conventional therapies are mainly supportive Recent progress towards gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy As alternative gene therapy vectors for CF we examined the feasibility of SV40-derived vectors (rSV40s) which may circumvent some of these obstacles To accommodate the large CFTR cDNA, we removed not only SV40 Tag genes, but also all capsid genes We therefore tested whether “gutless” rSV40s could be packaged and were able to express a functional human CFTR cDNA Results from our in vitro analysis determined that rSV40-CFTR was able to successfully result in the expression of CFTR protein which localized to the plasma membrane and restored channel function to CFTR deficient cells Similarly in vivo experiments delivering rSV40-CFTR to the lungs of Cftr-/- mice resulted in a reduction of the pathology associated with intra-tracheal pseudomonas aeruginosa challenge rSV40-CFTR treated mice had had less weight loss when compared to control treated mice as well as demonstrably reduced lung inflammation as evidence by histology S121 LUNG AND RESPIRATORY DISEASE and reduced inflammatory cytokines in the BAL The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted 313 Soluble Receptor Gene Therapy for Allergic Responses Targeting IL-13 and IL-17e Christian Mueller,1 Allison Keeler,1 Sofia Braag,1 Terence R Flotte.1 UMass Gene Therapy Center, UMass Medical School, Worcester, MA Cystic fibrosis (CF) patients have increased levels of proinflammatory cytokines (TNFa, IL-4, IL-8, IL-13 and IL-6) The increase in the pro-inflammatory cytokines leads to a hyperinflammatory response, which causes lung tissue destruction This phenotype has also been documented in CFTR-deficient Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus (Af) antigen to mimic allergic bronchopulmonary aspergillosis (ABPA), an unusual IgE mediated hypersensitivity syndrome seen in up to 15% of CF patients and rarely outside this condition We hypothesized that expression of soluble receptors directed against IL-13 and IL-17e (two cytokine that help orchestrate allergic responses) would prevent the cytokines from engaging the cell bound receptors and therefore help attenuate allergic responses in the ABPA mouse model To achieve this we constructed IgG Fc fusions of the extracellular domains of the IL-17e and IL-13 receptors to be expressed from a rAAV constructs CFTR-/- mice injected with rAAV1 intra-muscularly expressing soluble receptors to IL-17E (IL-17ER-fc) or IL-13 (IL-13R-fc) showed steady systemic levels of the receptors at weeks post injection Total IgE levels 48 hours after the last airway challenge with Af, in mice receiving the IL-17ER-Fc therapy where 25% lower and about 35% lower in the IL-13-Fc group than in the control rAAV-GFP group Interestingly AFspecific IgE levels were undetectable in both the mice receiving the IL-17ER-Fc and IL-13-Fc therapies Further flow cytometry analysis of intracellular gene expression in CD4+ and Cd11b+ cells showed a divergence between therapies In the IL-17-Fc group the cytokine levels for IL-17, IL-17e, Il13 and IL-4 were reduced in both CD4 and Cd11b cells Intracellular cytokine levels in the IL-13-Fc group remained unchanged as compared to controls in CD4 and Cd11b cells This suggest that blocking IL-17e may be interfering with signaling upstream of CD4 and Cd11b cells and may be reducing IgE levels by affecting signaling at these cell populations In contrast it appears that Il-13 blockade acts downstream of this to reduce IgE levels probably by directly affecting B cell maturation Further experiments are need to resolve this difference, in addition this suggest that delivery of both therapies may have a synergistic effect 314 Six Months Lentiviral Correction of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Defect in Cystic Fibrosis Mice Patricia L Cmielewski,1,3 Don S Anson,2 David W Parsons.1,3,4,5 Respiratory and Sleep Medicine, CY&WHS, Adelaide, SA, Australia; 2Gene Technology Unit, CY&WHS, Adelaide, SA, Australia; 3Paediatrics, University of Adelaide, Adelaide, SA, Australia; 4WCHRI, CY&WHS, Adelaide, SA, Australia; 5Centre for Stem Cell Research, University of Adelaide, Adelaide, SA, Australia The assessment of functional genetic correction of the bioelectric defect in cystic fibrosis (CF) mice nasal airway via transepithelial potential difference (TPD) measurement is a technically challenging technique Unreliable cannula placement and importantly, insufficient sample sizes for long term studies with CF transgenic mice can prevent studies having sufficient statistical power to discern the S122 required improvements in TPD measurements We examined the effectiveness of our lentiviral (LV) gene therapy protocols over more than months using a repeated-measures experimental design in CF mice Methods: Male and female CF tm1Unc mice were instilled nasally using either PBS (control) or 0.3% lysophosphatidylcholine (LPC) one hour prior to delivery of an LV vector containing the functional cystic fibrosis transmembrane conductance regulator (CFTR) gene An additional group of CF mice received LPC with an empty LV vector (LV-MT) To date nasal TPD measurements (under domitor:ketamine aneasthesia) have been performed at week and 1, and months post LV instillation using standard basal, basal+amiloride (10-4) and low chloride+amiloride Krebs ringer solutions The ∆PD was calculated as the low chloride minus the basal TPD response under amiloride conditions Results: At and months there was a significant response towards correction of the ∆PD towards normal in CF mice that received LPC pretreatment and LV-CFTR compared to either PBS/LV-CFTR or LPC/LV-MT control treatments (p