- Email: [email protected]
185. Targeted Correction and Restored Function of CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells
Cardiovascular and Pulmonary Diseases However, the role of this process in renal fibrosis is unknown. Thus, we hereby discuss the correlations of NRF-1 and renal interstitial fibrosis. Materials and Methods: NRK-49F (Normal Rat kidney fibroblast) cells were used. In vitro fibrosis model was established by treatment with transforming growth factor-b1 (TGF-b1). The fibrogenic marker (e.q. fibronectin) and fibrogenic signal proteins (e.q. pSmad2/3,Smad2/3,Smad7) were investigated. Here, we used that two distinct mechanisms regulate NRF1 activition and degradation of NRF1. NRF-1 was transfect by NRF-1 overexpression gene to evaluate NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by NRF-1 shRNA to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Western blot was used to examine the intracellular fibrogenic signal proteins (e.q. pSmad2/3,Smad2/3,Smad7). Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin). Results: Our study demonstrated that expression of NRF-1 is significantly decreased in renal fibrosis. However, transfection with pcDNA-NRF-1 (2mg/mL) expression vector did dramatically reverse TGF-b1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin) and fibrogenic signal protein. Intriguingly, NRF-1 shRNA dramatically increased TGF-b1-induced cellular fibrosis. Collectively, these finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells.
Cardiovascular and Pulmonary Diseases 185. Targeted Correction and Restored Function of CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells
Brian R. Davis,1 Ana M. Crane,1 Philipp Kramer,1 Jacquelin H. Bui,1 Wook Joon Chung,2 Xuan Shirley Li,1 Manuel L. GonzalezGaray,3 Finn Hawkins,4 Wei Liao,1 Daniela Mora,1 Jianbin Wang,5 Helena C. Sun,5 David E. Paschon,5 Dmitry Y. Guschin,5 Philip D. Gregory,5 Darrell N. Kotton,4 Michael C. Holmes,5 Eric J. Sorscher.2 1 Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center, Houston, TX; 2Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama, Birmingham, AL; 3Center for Molecular Imaging, University of Texas Health Science Center, Houston, TX; 4Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA; 5Sangamo BioSciences, Inc., Richmond, CA.
Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources – potentially useful for the development of new therapeutic approaches. The primary defect in Cystic Fibrosis (CF), an autosomal recessive disorder, is the regulation of epithelial chloride transport by a chloride channel protein encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recurrent pulmonary infections are responsible for 80 to 90% of the deaths in CF patients. Starting with skin fibroblasts from patients diagnosed with CF, we have derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patientderived lines via homology directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele vs. the other. Differentiation for a total of 19 days in a protocol designed to derive anterior foregut endoderm, subsequently up-regulated expression of NKX2-1, SOX9, TP63, FOXP2, FOXA2, and CFTR, suggesting commitment of at least a sub-population of cells within the endodermal culture to a lung epithelial cell fate. S74
Once differentiated, mutant CF iPSCs yielded neither mature CFTR protein nor CFTR-specific chloride channel activity (as assayed in Ussing chamber experiments) – whereas corrected CF iPSCs and the normal control WA09 hESCs yielded mature CFTR protein and CFTR-specific chloride channel activity. In vitro differentiation of the mutant CF iPSCs into lung epithelial cells and tissue, controlled for by the parallel differentiation of the otherwise isogenic corrected CF iPSCs, may provide a valuable tool for drug screening and examining the functional consequence of mutant CFTR expression. Furthermore, corrected CF iPSCs present a potential source of patient-specific cells capable, in vitro, of differentiation into various lung stem/progenitor cells -- either for transplantation of autologous lung cells or for seeding de-vitalized lung scaffolds ex vivo to generate autologous lungs.
186. Long-Term Results of pCMV-vegf165 Intramuscular Gene Transfer in Patients With Chronic Lower Limb Ischemia
Ilia Y. Bozo,1,2 Roman V. Deev,1 Igor L. Plaksa,1 Nina D. Mzhavanadze,3 Yuriy V. Chervyakov,4 Ilia N. Staroverov,4 Dmitriy A. Voronov,5 Kalinin E. Kalinin,3 Artur A. Isaev.1 1 Human Stem Cells Institute, Moscow, Russian Federation; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russian Federation; 3Ryazan State I.P. Pavlov Medical University, Ryazan, Russian Federation; 4Yaroslavl State Medical Academy, Yaroslavl, Russian Federation; 5Russian National Research Center of Surgery, Moscow, Russian Federation. Restriction of blood flow due to atherosclerosis leads to chronic lower limb ischemia (CLI) affected 3-8% of the general population. Current pharmacological therapies have limited effects. Therefore, developing new treatments is pivotal. Gene therapy is targeted at inducing angiogenesis in ischemic extremities via increased endogenous expression of vascular growth factors. Among many, plasmid DNA vectors play a significant role in gene therapy and are safe with low immunogenicity and no risk of insertional mutagenesis. A novel drug “Neovasculgen” became the first gene therapy for patients with CLI approved by regulatory authorities in Russia in 2011. It contains a plasmid DNA encoding vascular endothelial growth factor VEGF165 (pCMV-vegf165). There was conducted a multicentre randomised controlled clinical trial of the intramuscular transfer of this new gene product in 100 patients with CLI, stage 2a-3 disease according to Fontaine. The trial lasted 6 months. It was concluded that pCMV-vegf165 transfer significantly improved painfree walking distance (PWD) and caused no adverse effects. Efficacy and safety of this therapy were evaluated and translated into practice. Most of patients enrolled in the trial gave their consent to participate in the long-term follow-up study. The primary endpoint was PWD, the secondary – ankle-brachial index (ABI) and transcutaneous oxygen tension (TcPO2). PWD in patients who received pCMV-vegf165 increased throughout the 3-year follow-up period: by the end of the 1st year the parameter increased by 167.2% as compared to the 135.3 m baseline (p<0.05). The tendency remained positive throughout the following two years of monitoring (Fig. 1). PWD decreased by 27% within a 3-year follow-up period in control patients who received conventional therapy alone (p<0.05).
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
Cardiovascular and Pulmonary Diseases 185. Targeted Correction and Restored Function of CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells
Brian R. Davis,1 Ana M. Crane,1 Philipp Kramer,1 Jacquelin H. Bui,1 Wook Joon Chung,2 Xuan Shirley Li,1 Manuel L. GonzalezGaray,3 Finn Hawkins,4 Wei Liao,1 Daniela Mora,1 Jianbin Wang,5 Helena C. Sun,5 David E. Paschon,5 Dmitry Y. Guschin,5 Philip D. Gregory,5 Darrell N. Kotton,4 Michael C. Holmes,5 Eric J. Sorscher.2 1 Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center, Houston, TX; 2Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama, Birmingham, AL; 3Center for Molecular Imaging, University of Texas Health Science Center, Houston, TX; 4Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA; 5Sangamo BioSciences, Inc., Richmond, CA.
Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources – potentially useful for the development of new therapeutic approaches. The primary defect in Cystic Fibrosis (CF), an autosomal recessive disorder, is the regulation of epithelial chloride transport by a chloride channel protein encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recurrent pulmonary infections are responsible for 80 to 90% of the deaths in CF patients. Starting with skin fibroblasts from patients diagnosed with CF, we have derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patientderived lines via homology directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele vs. the other. Differentiation for a total of 19 days in a protocol designed to derive anterior foregut endoderm, subsequently up-regulated expression of NKX2-1, SOX9, TP63, FOXP2, FOXA2, and CFTR, suggesting commitment of at least a sub-population of cells within the endodermal culture to a lung epithelial cell fate. S74
Once differentiated, mutant CF iPSCs yielded neither mature CFTR protein nor CFTR-specific chloride channel activity (as assayed in Ussing chamber experiments) – whereas corrected CF iPSCs and the normal control WA09 hESCs yielded mature CFTR protein and CFTR-specific chloride channel activity. In vitro differentiation of the mutant CF iPSCs into lung epithelial cells and tissue, controlled for by the parallel differentiation of the otherwise isogenic corrected CF iPSCs, may provide a valuable tool for drug screening and examining the functional consequence of mutant CFTR expression. Furthermore, corrected CF iPSCs present a potential source of patient-specific cells capable, in vitro, of differentiation into various lung stem/progenitor cells -- either for transplantation of autologous lung cells or for seeding de-vitalized lung scaffolds ex vivo to generate autologous lungs.
186. Long-Term Results of pCMV-vegf165 Intramuscular Gene Transfer in Patients With Chronic Lower Limb Ischemia
Ilia Y. Bozo,1,2 Roman V. Deev,1 Igor L. Plaksa,1 Nina D. Mzhavanadze,3 Yuriy V. Chervyakov,4 Ilia N. Staroverov,4 Dmitriy A. Voronov,5 Kalinin E. Kalinin,3 Artur A. Isaev.1 1 Human Stem Cells Institute, Moscow, Russian Federation; 2A.I. Evdokimov Moscow State University of Medicine and Dentistry, Moscow, Russian Federation; 3Ryazan State I.P. Pavlov Medical University, Ryazan, Russian Federation; 4Yaroslavl State Medical Academy, Yaroslavl, Russian Federation; 5Russian National Research Center of Surgery, Moscow, Russian Federation. Restriction of blood flow due to atherosclerosis leads to chronic lower limb ischemia (CLI) affected 3-8% of the general population. Current pharmacological therapies have limited effects. Therefore, developing new treatments is pivotal. Gene therapy is targeted at inducing angiogenesis in ischemic extremities via increased endogenous expression of vascular growth factors. Among many, plasmid DNA vectors play a significant role in gene therapy and are safe with low immunogenicity and no risk of insertional mutagenesis. A novel drug “Neovasculgen” became the first gene therapy for patients with CLI approved by regulatory authorities in Russia in 2011. It contains a plasmid DNA encoding vascular endothelial growth factor VEGF165 (pCMV-vegf165). There was conducted a multicentre randomised controlled clinical trial of the intramuscular transfer of this new gene product in 100 patients with CLI, stage 2a-3 disease according to Fontaine. The trial lasted 6 months. It was concluded that pCMV-vegf165 transfer significantly improved painfree walking distance (PWD) and caused no adverse effects. Efficacy and safety of this therapy were evaluated and translated into practice. Most of patients enrolled in the trial gave their consent to participate in the long-term follow-up study. The primary endpoint was PWD, the secondary – ankle-brachial index (ABI) and transcutaneous oxygen tension (TcPO2). PWD in patients who received pCMV-vegf165 increased throughout the 3-year follow-up period: by the end of the 1st year the parameter increased by 167.2% as compared to the 135.3 m baseline (p<0.05). The tendency remained positive throughout the following two years of monitoring (Fig. 1). PWD decreased by 27% within a 3-year follow-up period in control patients who received conventional therapy alone (p<0.05).
Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy