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311. Mini-uPA Gene Therapy for Pulmonary Injury
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 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 2 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 2 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. 1
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 1 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
Sara R. Martin,1 Amanda K. Pangle,1 Gabor Molnar,1 Ruth S. Everett,1 Larry G. Johnson.1 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 2 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 2 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. 1
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 1 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