709 Phase 1 2 Clinical Trial in Patients With Decompensated Liver Cirrhosis Treated With Bone Marrow Derived Endotelial Progenitor Cells Preliminary Safety and Efficacy Analysis Molecular Therapy Volu[.]
SOMATIC STEM CELLS dmLT To determine the frequency of gene transfer; DC progenitor cells, iDC or mDC were transduced with retroviral or lentiviral vectors expressing GFP The CD34+ cells expanded 10-20 fold during 2-3 week in vitro culture When GM-CSF/IL-4 was added, proliferation ceased, but cells acquired DC phenotypic and functional characteristics including cell morphology, phenotype; phagocytosis, chemotaxis to lymph node chemokines, and T cell stimulation properties The retroviral vector LZRS-GFP transduced more than 40% CD34+ cells and iDC Lentiviral vectors transduced iDC most efficiently with >80% GFP+ Cultured DC were able to stimulate viral-specific IFN-γ ELIspot forming units from autologous PBMC In conclusion, a cytokine-based in vitro DC-generation method was established, which started from rhesus CD34+ BM progenitor cells and consisted of 3-steps: DC progenitor cell expansion, iDC differentiation, and mDC activation Gene transfer into these populations generated DC capable of stimulating antigen-specific IFN-γ ELIspot Future studies are necessary to characterize the DC function in vivo to maximize the clinical benefit of the therapy 707 Designer T Cells Redirected To Target HIV Env+ Cells Stephen E Braun,1 Edith Walker,2 Gautam Sahu,2 Gail Skowron,2 Preston A Marx,1 Dorothee von Laer,3 Andrew MacLean,1 Richard P Junghans.2 Tulane National Primate Research Center, Tulane University School of Medicine, Covington, LA; 2Infectious Diseases/Surgery, Roger Williams Medical Center, Providence, RI; 3Virology Section, Innsbruck Medical University, Innsbruck, Austria Introduction: Infection with HIV-1 results in CD4+ T cell depletion and the subsequent loss of immune function results in AIDS Although HAART lowers plasma viremia, it requires life-long drug therapy However, some patients control viral replication without HAART To redirect CTL activity against HIV, the first-generation CD4-chimeric antigen receptors (CARs) used the CD4 extracellular domain to target Envelope and the T-cell receptor zeta (TCRζ) intracellular signaling domain to activate T cells These MLV-vectors targeted infected cells in vitro, but failed to control viremia in clinical trials Previously, we demonstrated that the membrane-associated C46 (maC46) fusion inhibitor, when tethered to susceptible cells, binds HIV and blocks viral replication – demonstrating protection and a strong selective advantage in transduced cells To increase the immunosurveillance of HIV; we genetically modified autologous T cells to bolster and redirect the “designer T cells” (dTc) towards an HIV-specific target and measured CTL activity Methods: To improve the CD4-CAR vectors, we added the intracellular CD28 signaling domain to CD4-TCRz vector CD4TCRz and CD4-28z CAR were shuttled into PG13 packaging cells (GaLV-pseudotyped) We also packaged MLV-vector expressing the maC46 fusion inhibitor in Phoenix (amphotropic-pseudotyped) clones We stimulated rhesus PBMCs with αCD3αCD28 and cotransduced T cells with CD4-CAR and maC46 vectors To evaluate the CTL activity of the dTc, we measured 1) HIV Env-specific cytotoxicity using a novel real-time cytotoxicity assay as changes in electrical impedance and 2) cytokine production using IFN-g ELIspot Therefore, HEK 293T cells were transiently transfected with an HIV-1 Env expression plasmid (or control) and plated with CD4-CAR and control-transduced populations at effector:target ratio of 1:1 Results: We observed gene transfer frequency up to 60-70% of rhesus CD3+CD8+ T cells with the individual vectors and cotransduction of 35% of the cells with both vectors In two experiments, CD4-CAR populations specifically killed 293T-Env+ cells Additionally, transduced populations showed increased frequency of INF-γ spot-forming cells Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy Conclusions: In these studies, we showed that genetically modified T cells were redirected to target HIV Env+ cells Additional intracellular signaling domains may increase CTL activity and/ or in vivo persistence Control of viremia without HAART would revolutionize treatment for HIV patients Somatic Stem Cells 708 A Novel Method for Longitudinal and NonInvasive Imaging of Transplanted Cells in a Mouse Model of Hereditary Tyrosinemia Type Raymond D Hickey,1 Shennen A Mao,1 Jaime Glorioso,1 Bruce Amiot,1 Lukkana Suksanpaisan,2 Kah Whye Peng,1 Scott L Nyberg,1 Stephen J Russell.1 Mayo Clinic, Rochester, MN; 2Imanis Life Sciences, Rochester, MN Hepatocyte transplantation is a potential treatment for myriad metabolic liver disorders that are currently only curable by liver transplantation One major limiting factor is an inability to monitor cells longitudinally and non-invasively after transplantation to determine their biodistribution We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to monitor transplanted hepatocytes and set out to test this reporter system in a rodent model of metabolic liver disease Wild-type C57Bl/6J mouse hepatocytes were transduced ex vivo using a lentiviral vector containing the mouse Slc5a5 (NIS) gene under the control of a liver specific promoter Transduction efficiencies of 75-80% were achieved and NIS-labeled cells could robustly concentrate radiolabeled iodine in vitro Next, NIS-labeled hepatocytes were transplanted into congenic fumarylacetoacetate hydrolase knockout (Fah-/-) mice, a small animal model of hereditary tyrosinemia type NIS-labeled hepatocytes were readily imaged in vivo non-invasively by single-photon emission computed tomography imaging with an increase in radiolabeled tracer detection correlating with an increase in liver repopulation over time after intrasplenic injection of cells Additionally, NIS-imaging was able to unambiguously identify the extrahepatic biodistribution of transplanted hepatocytes in Fah-/- mice after intraperitoneal injection This work demonstrates the efficacy of NIS-labeling in the field of cell transplantation and we anticipate that NIS-labeling of cells will allow further testing of hepatocyte and stem cell therapies for various liver disorders, not only in small animals, but in larger preclinical models also 709 Phase 1-2 Clinical Trial in Patients With Decompensated Liver Cirrhosis Treated With Bone-Marrow Derived Endotelial Progenitor Cells: Preliminary Safety and Efficacy Analysis Delia D’Avola,1 Veronica Fernandez-Ruiz,2 Francisco Carmona de la Torre,5 Miriam Mendez,2 Felipe Prosper,3 Enrique Andreu,3 Jose Ignacio Herrero,1 Mercedes Iñarrairaegui,1 Carmen Fuertes,5 Jose Ignacio Bilbao,1 Bruno Sangro,1 Jesus Prieto,1 Jorge Quiroga.1 Liver Unit and Ciberehd, Clinica Universidad de Navarra, Pamplona, Spain; 2Department of Hepatology and Gene Therapy, Centro Investigación Médica Aplicada (CIMA), Pamplona, Spain; Cell Therapy, Clinica Universidad de Navarra, Pamplona, Spain; Radiology, Clinica Universidad de Navarra, Pamplona, Spain; Liver Unit, Clinica Universidad de Navarra, Pamplona, Spain Background and aims Bone marrow-derived endothelial progenitor cells (EPC) promote liver regeneration and improve survival in animal models of acute and chronic liver disease S273 SOMATIC STEM CELLS This phase 1-2 clinical trial aimed to evaluate the safety, feasibility and efficacy of the treatment with autologous EPC in decompensated cirrhosis Matherials and methods Child-Pugh>7 cirrhotic patients underwent aspiration of 50 ml of bone marrow (BM) blood for ex vivo differentiation and selection of EPC The final product was resuspended in 50 ml and injected via hepatic artery Patients were followed-up for 12 months Results Between May-11 and September-13, 14 patients (Child-Pugh 9, range 8-11; MELD 17, range 8-27) were evaluated One patient died before BM aspiration due to cause not related to liver disease and another was a screening failure Among the 12 patients who underwent BM aspiration, the feasibility of production of EPC was 84% and 91% with and punctions, respectively 11 patients were treated Three patients died at day 25, 85 and 166 No treatment-related severe adverse events after a median follow-up of 166 days (range 25-364) were observed MELD and Child-Pugh scores improved in 67% and 44% of patients at 30 days (n= 9) and in 66% and 44% at 90 days (n=9) Compared to baseline, hepatic vein pressure gradient improved at 90 days in 55% of patients Conclusion Treatment with autologous BM-EPC is feasible and safe in decompensated cirrhosis and may have beneficial effects on liver function 710 Genetic Modification of Human Airway Basal Stem/Progenitor Cells to Skew Differentiation Towards the Secretory Cell Lineage Matthew S Walters,1 Kazunori Gomi,1 Ann E Tilley,1 Ben-Gary Harvey,1 Robert J Kaner,1 Ronald G Crystal.1 Weill Cornell Medical College, New York The human airway epithelium is a complex multi-cellular tissue composed of basal, Clara, secretory and ciliated cells that line the airway lumen Basal cells (BC) are the stem/progenitor cells of the airway epithelium that differentiate into the other specialized epithelial cell types during physiological turnover and repair In response to environmental insult, including cigarette smoke, the normal differentiation response of BC becomes dysregulated, resulting in a disordered epithelium that characterizes chronic obstructive pulmonary disease (COPD) Based on the concept that gene transfer might be used to reestablish the normal differentiation of the BC stem/progenitor cells, the objective of this study was to assess whether it would be possible to shift the differentiation of primary human airway BC to mucus-producing cells by genetic manipulation To evaluate this concept, lentivirus gene transfer vectors were used to infect human airway BC as they were differentiating on air-liquid interface (ALI) culture Lentivirus reporter (GFP) constructs mediated high transduction efficiency (>90% cells GFP positive) and long-term reporter expression (28 days on ALI culture) Quantification of BC differentiation at ALI Day 28 revealed no significant deleterious effects of lentivirus infection on differentiation of BC into a mucociliated epithelium Based on the knowledge that the Notch pathway is involved in a wide variety of cellular processes, including turnover and repair of tissues and known to be dysregulated in the airway epithelium of smokers and smokers with COPD relative to healthy nonsmokers, as a demonstration of principal, we genetically modified BC activation of the Notch signaling pathway For sustained Notch activation during BC differentiation, the constitutively active intracellular form of the Notch 1, 2, or receptor (NICD1, 2, or 4, respectively) was expressed from lentivirus vectors and differentiation assessed at ALI Day 28 Compared to control lentivirus-infected cells, expression of NICD2 or had no effect on differentiation of BC into secretory and ciliated cells In marked contrast, expression of NICD1 or resulted in a significant increase in the levels of secretory S274 cell genes and number of secretory cells, with a parallel decrease in expression of ciliated cell genes and number of ciliated cells These data suggest Notch and mediated signaling have no effect on human BC differentiation whereas activation of the Notch and pathways have a major effect on shifting differentiation towards the secretory lineage, i.e., genetic modification of NICD or components of the Notch pathway of airway BC stem/progenitor cells can modulate the proportions of mucin-producing cells Overall, this study demonstrates the feasibility of genetic modification of airway BC to alter the proportion of mucin-producing cells, a strategy that may be used to suppress the increased numbers of mucin-producing cells that characterize the disordered airway epithelium in COPD 711 Genetic Editing To Preclude HLA-A Expression on Hematopoietic Stem Cells -Improving the Chance of Finding an HLA-Matched Donor Hiroki Torikai,1 Tiejuan Mi,1 Loren Gragert,2 Martin Maiers,2 Amer Najjar,1 Sonny Ang,1 Sourindra Maiti,1 Kirsten C Switzer,1 Helen Huls,1 Andreas Reik,3 Edward Rebar,3 Michael C Holmes,3 Philip D Gregory,3 Richard E Champlin,1 Elizabeth J Shpall,1 Laurence J N Cooper.1 MD Anderson Cancer Center, Houston; 2National Marrow Donor Program, Minneapolis; 3Sangamo BioSciences, Inc, Richmond Allogeneic hematopoietic stem cells (HSCs) are infused to restore or replace absent or dysfunctional HSCs in the recipient and serve as a source for generating specific hematopoietic cells The diversity of the human leukocyte antigen (HLA) system poses a hurdle to find an HLA-compatible donor which is exacerbated by the impact of racial genetic polymorphism Furthermore, despite pre-banking umbilical cord blood (UCB) units and access to registered adult donors through the National Marrow Donor Program (NMDP), finding a suitable HLA-matched product remains challenging for many recipients, especially those from racial and ethnic minorities who are underrepresented in the donor pool We hypothesized that the clinical application of donors already registered would be markedly increased if allogeneic HSCs were genetically edited to eliminate expression of the HLA-A locus Modeling from NMDP shows that the chance of an African American recipient finding a HLA-matched donor increases from 18% to 73% when the need for a match at HLA-A is eliminated leaving HLA-B, C and DR We have previously shown that engineered zinc finger nucleases (ZFNs) can eliminate HLA-A expression in genetically edited T cells and cell lines To extend genetic editing to HSCs, we sought to disrupt HLA-A expression by introducing ZFNs targeting this locus CD34+ lineageneg HSCs (99% purity) were isolated using paramagnetic beads from UCB Electrotransfer of in vitro-transcribed mRNA species encoding the HLAA-specific ZFNs generated 30% HLA-Aneg HSCs after one week ex vivo culture with defined cytokines (FLT3-L, SCF, TPO, and IL-6) and an aryl hydrocarbon receptor antagonist (stem reginin-1, SR-1) DNA sequence analysis revealed that HLA-Aneg HSCs display the expected nucleotide changes at the ZFN target site In vitro assays showed no significant difference in lineage specific colony formation between HLA-Apos HSC and HLA-Aneg HSC Moreover, an in vivo engraftment assay, using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, demonstrated that engineered HLA-Aneg HSCs maintain the capability of engraftment and differentiation into HLA-Aneg multilineage hematopoietic cells Methods to improve access to the existing donor pool are anticipated to have clinical impact Indeed, the NMDP calculates that approximately 10,000 patients in the US could benefit from unrelated allogeneic HSCT The elimination of HLA-A expression in HSCs using ZFNs provides an appealing approach to increase the chance for finding HLA-matched donors This is anticipated to broaden the clinical application of allogeneic HSCT, especially for patients belonging to ethnic minority groups Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... phase 1- 2 clinical trial aimed to evaluate the safety, feasibility and efficacy of the treatment with autologous EPC in decompensated cirrhosis Matherials and methods Child-Pugh>7 cirrhotic patients. .. -Improving the Chance of Finding an HLA-Matched Donor Hiroki Torikai ,1 Tiejuan Mi ,1 Loren Gragert ,2 Martin Maiers ,2 Amer Najjar ,1 Sonny Ang ,1 Sourindra Maiti ,1 Kirsten C Switzer ,1 Helen Huls ,1 Andreas... screening failure Among the 12 patients who underwent BM aspiration, the feasibility of production of EPC was 84% and 91% with and punctions, respectively 11 patients were treated Three patients