530 Lentiviral Gene Therapy of Murine Hematopoietic Stem Cells Using Codon Optimized IL2RG cDNA A Comparison of Multiple Promoter Elements and Transplant Conditions Molecular Therapy Volume 18, Supple[.]
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II 528 Transgenic Expression of Cytidine Deaminase (CDD) for Myeloprotective Purposes Nico Lachmann,1 Nils Pfaff,1 Sebastian Brennig,1 Doreen Lüttge,1 Tobias Cantz,2 Christopher Baum,3 Axel Schambach,3 Thomas Moritz.1 REBIRTH Cluster of Excellence RG Reprogramming, Medical School Hannover, Hannover, Germany; 2REBIRTH Cluster of Excellence JRG Stem Cell Biology, Medical School Hannover, Hannover, Germany; 3Department of Experimental Hematology, Medical School Hannover, Hannover, Germany Introduction: Hematopoietic stem cells genetically modied to overexpress drug resistance genes such as O6-methylguanineDNA methyltransferase (MGMT) or multidrug-resistance gene (MDR1) have been advocated to overcome chemotherapy induced myelosuppression Cytidine deaminase (CDD) represents another drug resistance gene which deaminates cytosine nucleosides and their analogs such as cytarabine (Ara-C) and gemcitabine This prevents the intracellular accumulation of the triphosphate derivates, which act as the active metabolites of these cytotoxic drugs In this context we have recently shown in a murine in vivo model that CDD overexpression profoundly protects hematopoiesis from Ara-C and Gemcitabine toxicity (Rattmann, Blood, 2006) As these studies also revealed lymphoid toxicity and the lack of long term in vivo selection following CDD gene transfer and drug therapy, we now have generated novel and improved SIN lentiviral vector constructs for CDD expression in the hematopoietic system Methods: Vectors express the human CDD either from a spleen focus forming virus (SFFV) or a truncated elongation factor 1α (EFS) promoter/enhancer element In addition, inducible CDD expression based on a TET-on system was established CDD-mediated drug resistance was evaluated in murine myeloid (32D) and lymphoid (BWα-β-) cell lines as well as in primary murine (lineage negative) and human (cord blood CD34+) hematopoietic cells In these experiments cells transduced with CDD expressing lentiviral constructs (SFFV-CDD, EFS-CDD, TET-CDD) were sorted (GFP+) and treated with different concentrations of Ara-C or gemcitabine for 48h Subsequently, cell viability was determined utilizing propidium iodide and primary hematopoietic cells were analyzed in clonogenic assays Results: Irrespective of the lentiviral construct used, CDD transduced murine myeloid and lymphoid cell lines showed profound protection against Ara-C levels of up to 5000 nM, whereas untransduced control cells died at concentration of 200 nM With the TET-CDD construct protection in 32D cells was achieved within 24h of Doxycyclin treatment and upon Doxycyclin cessation CDD expression returned to baseline within three to ve days Similar protection was observed in primary clonogenic hematopoietic cells Here, cells transduced to yield EFS- or SFFV-driven CDD expression were protected from Ara-C levels of up to 300 and 600 nM, respectively, with untransduced controls dying at 50 nM Ara-C Conclusion: These data demonstrate profound protection from Ara-C toxicity in both, hematopoietic cell lines as well as primary hematopoietic cells by lentiviral vectors employing SFFV- or EFSdriven as well as TET-inducible CDD expression Next, the in vivo selection potential of these promising new vector constructs will be investigated in murine model systems employing optimized Ara-C application schedules S204 529 Development of a Pre-Clinical Model for the Gene Therapy Treatment of Wiskott-Aldrich Syndrome (WAS) Alexander Astrakhan,1 Byoung Ryu,2 Brigid Stirling,2 Blythe Sather,2 Jit Khim,2 Mikhail Garibov,2 Stephanie Humblet-Baron,2 Hans Ochs,2 David J Rawlings.1,2 Department of Immunology, University of Washington, Seattle, WA; 2Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA Wiskott-Aldrich Syndrome is an immunodeciency characterized by recurrent infections, thrombocytopenia, eczema, and increased susceptibility to autoimmunity and malignancy The disease is caused by defective function of the WAS protein (WASp), a crucial intermediary in receptor-mediated actin cytoskeletal rearrangement While prognosis is relatively good for patients receiving matched bone marrow transplants, overall outlook is poor for patients lacking suitable donors Lentiviral-mediated gene therapy is a promising therapeutic approach for the treatment of primary immunodeciencies We used WASp-decient mice to analyze the efcacy of lentiviral gene therapy in rescuing WAS-associated hematopoietic defects In initial experiments we utilized the strong, ubiquitous, retrovirallyderived MND promoter to drive WASp expression in vivo Lentiviral transduction of hematopoietic stem cells (HSCs) resulted in stable WASp expression in all hematopoietic lineages, including the myeloid, lymphoid and platelet compartments We observed selective outgrowth of WASp+ cells within T cell subsets, marginal zone (MZ) B cells and platelets Gene therapy treated mice demonstrated at least partial correction of WAS-associated defects, with normal numbers of marginal zone B cells and functional T cell proliferation and IL-2 production following T cell receptor (TCR) engagement Due to the strong oncogenic potential of the MND promoter in vivo, we next compared MND to mammalian promoters, including the minimal WAS promoter (WS1.6) and the short elongation factor alpha (sEF1a) promoter Surprisingly, the WS1.6 promoter was poorly active in all hematopoietic lineages, and was particularly ineffective in driving WASp expression in B cells The reduced expression levels was specic to the WAS promoter, as the sEF-1a promoter exhibited robust activity in all hematopoietic lineages Combined, these ndings suggest that lentiviral-mediated gene therapy can lead to successful correction of WAS-associated hematopoietic defects in the murine model of WAS Our observations argue against the use of the endogenous WAS promoter in human clinical trials; rather, we propose that the EF-1a promoter is likely to be more effective and potentially safer promoter for transition into clinical testing 530 Lentiviral Gene Therapy of Murine Hematopoietic Stem Cells Using Codon Optimized IL2RG cDNA: A Comparison of Multiple Promoter Elements and Transplant Conditions Marshall W Huston,1 Niek P van Til,1 Trudi P Visser,1 Roya Sawari,1 Shazia Arshad,1 Gerard Wagemaker.1 Hematology, Erasmus University Medical Center, Rotterdam, Netherlands Gene therapy for X-linked severe combined immunodeciency (X-SCID) by gammaretroviral vectors to deliver a functional copy of the IL2RG gene into the host genome has been very effective in the clinical setting, but carries the risk of insertional oncogenesis (Hacein-Bey-Abina, JCI 2008) HIV-1 derived lentiviral vectors have advantages over gammaretroviruses in transducing quiescent cells, such as long-term repopulating hematopoietic stem cells (HSC), are less likely to integrate near transcription start sites, and are thought to have lower genotoxicity (Montini, Nat Biotechnol 2006) To test efcacy and safety of lentiviral IL2RG gene therapy for X-SCID, a self-inactivating lentiviral vector was constructed containing a Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II codon optimized human IL2RG cDNA (coγc) Coγc expression was driven by the SFFV viral promoter, PGK cellular promoter or a native human IL2RG promoter region (γcPROM) Lineage negative HSC of Il2rg-/- mice were transduced with these vectors or a GFP control vector, resulting in to transgene copies per transduced cell The transduced cells were transplanted into Il2rg-/- mice and compared to Il2rg-/- recipients of healthy wild type cells Blood collected monthly and analyzed by differential cell counting and immunophenotyping demonstrated that mice transplanted with cells transduced with the coγc transgene reconstituted mature T and B cell populations, whereas recipients of cells transduced with GFP vectors did not TCRβ repertoire, IL-2/Con-A spleen cell proliferation assays, basal IgM/IgG1 serum levels and T-cell dependent immune responses in coγc treated mice yielded results similar to wild type controls Polyclonal integration patterns were conrmed by LAMPCR eight months post-transplant Further detailed studies with the γcPROM-coγc vector demonstrated that reducing the viral MOI to reach an average copy number/cell of and the number of cells transplanted to 5x107/kg body weight had a negligible effect on the efcacy of the gene therapy treatment Reducing pre-transplant irradiation conditioning from Gy to Gy likewise had little effect, but mice given no pre-transplant conditioning had a more protracted immune reconstitution in both coγc-treated and wild type groups We conclude that lentiviral-based coγc gene therapy is an effective alternative to gammaretroviral gene delivery even with low levels of conditioning and cell dosage and that the PGK promoter and native IL2RG promoter region are efcacious candidates for future clinical gene therapy development Current experiments focus on integration analyses of treated mice and long-term follow-up monitoring of potential adverse effects 531 Human Fetal Liver HSCs Demonstrate High Transduction Rates and Sustained Transgene Expression Following Implantation Niraja Dighe,1 Maroun Khoury,4 Mark Chong,1 Citra Mattar,1 Michael N Antoniou,5 Jianzhu Chen,4 Mahesh Choolani,1 Jerry Chan.1,2,3 Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; 2Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore, Singapore; 3Cancer and Stem Cell Program, Duke-NUS Graduate Medical School, Singapore, Singapore; Interdisciplinary Research Group in Infectious Diseases, Singapore-Massachusetts Institute of Technology Alliance in Research and Technology, Singapore, Singapore; 5Department of Medical and Molecular Genetics, King’s College London School of Medicine, Guys Hospital, London, United Kingdom Introduction: Hematopoietic Stem Cells (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects Recently, it has been shown that fetal stem cells demonstrate a higher propensity for transduction compared to adult cells We hypothesized that fetal tissue-derived HSC is more amenable to transduction and hence a suitable candidate for targeted gene transfer Here, we report the efcient transduction of primitive human CD34+ fetal liver cells with a lentiviral vector encoding an A2UCOE-eGFP cassette, compared to cord bloodderived HSC Methods: CD34+ cells were isolated from second trimester human liver (HSC) and umbilical cord blood (UCBHSC) by MACS, and subsequently infected with UCOE-GFP at a multiplicity of infection (MOI) from to 20 FACS, CFU assays and vector copy number analyses were done to evaluate the effects of transduction on HSC character and efciency of transduction respectively Subsequently, cells infected at MOI 20 were transplanted Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy into sublethally-irradiated neonatal NOD/SCID/Il2rg-/- mice via intracardiac injection, to study multilineage engraftment and secondary transplantation capacity of transduced HSC In parallel, some cells were expanded in culture to study the continued expression of GFP over 21 days Results: HSC were transduced with a maximal transduction rate of 85% at MOI 10 Increasing MOI beyond 10 failed to raise the transduction rates appreciably (87% at MOI 20) In contrast, transduction of UCB-HSC was poor, with only 2% transduced at MOI of 20 CFU assays demonstrated that transduced HSC were able to differentiate toward multiple lineages Finally, transduced HSC were found to be capable of long term multi-lineage engraftment of sublethally-irradiated mice, and retained expression of eGFP for more than weeks post-transplantation This correlates with in vitro data showing sustained expression of eGFP (64% at day 3, 61% at day 21) during continuous culture Conclusions: Our data suggest that CD34+ cells from human fetal liver represent a suitable source of HSC for targeted gene transfer, capable of efcient lentiviral transduction rates compared to UCB-HSC, These encouraging results lay the foundation for the use of lentiviral vectors containing β-globin locus control region (βLCR)-driven therapeutic gene cassettes to effect lineage-restricted expression for the treatment of various hematopoietic disorders, including the hemoglobinopathies 532 FANG Autologous Tumor Cell Vaccine Development and Manufacturing Phillip B Maples,1 Padmasini Kumar,1 Yang Yu,1 Beena O Pappen,1 Chris M Jay,1 Zhaohui Wang,1 Donald D Rao,1 Joseph Kuhn,2 John Nemunaitis,1,3,4,5 Neil Senzer.1,3,4,5 Gradalis, Inc., Dallas, TX; 2General and Oncology Surgery Associates, Dallas, TX; 3Mary Crowley Cancer Research Centers, Dallas, TX; 4Baylor Sammons Cancer Center, Dallas, TX; 5Texas Oncology, P.A., Dallas, TX Gene modied cell-based cancer vaccines have demonstrated durable responses in selected patients We have developed the FANG expression vector which we believe, when transfected into tumor cells, will evoke an enhanced immune recognition /stimulation versus our previous TAG vaccine vector The FANG nonviral vector system expresses both GM-CSF and a proprietary bifunctional shRNA to furin Preclinical data demonstrated that blocking furin protein expression in turn blocked the activation of both TGFβ1 and TGFβ2 In contrast, our TAG vector expressed both GM-CSF and a TGFβ2 antisense Data from our TAG Phase I autologous vaccine clinical trial and others indicate that TFG β1 overexpression is present in a wide range of cancers In fact our data suggest that TGFβ1 tends to be about tenfold higher than TGFβ2 expression in the more than thirty tumors we examined in that study So while the TAG vector blocked TGFβ2 expression, there was no effect on TGFβ1 expression The FANG expression vector is identical to the TAG expression vector except that the TGFβ2 antisense coding sequence has been replaced with the furin shRNA sequence FANG plasmid DNA was GMP-S manufactured We generated nonclinical and clinical vaccines under cGMP as part of our IND submission data (4 melanoma, colorectal, gall bladder, NSCLC and breast cancer) All vaccine manufacturing processes met specications (no contamination or failure to meet nal dose or quality requirements) Average cell viability is 91.5+5.3%, median 93.5% and range 78-96% (values taken on Day of manufacturing) Average GM-CSF expression is 657+550pg/1x106 cells/ml, median 602pg and range 80-1870pg The mean pretransfection TGFβ1 is 1241+1115pg/1x106 cells/ml, median 1039pg The mean posttransfection TGFβ1 is 211+421pg/1x106 cells/ ml, median 20.1pg The average percent knockdown of TGFβ1 was 89+20%, median 97% and range 36-100% The mean pretransfection TGFβ2 is 293+189pg/1x106 cells/ml, median 257pg The mean posttransfection TGFβ2 is 9.1+12pg/1x106 cells/ml, median 4pg The average percent knockdown of TGFβ2 was 94+12 %, median 99% and S205 ... gene therapy is an effective alternative to gammaretroviral gene delivery even with low levels of conditioning and cell dosage and that the PGK promoter and native IL2RG promoter region are efcacious... Inc., Dallas, TX; 2General and Oncology Surgery Associates, Dallas, TX; 3Mary Crowley Cancer Research Centers, Dallas, TX; 4Baylor Sammons Cancer Center, Dallas, TX; 5Texas Oncology, P .A. , Dallas,... nonclinical and clinical vaccines under cGMP as part of our IND submission data (4 melanoma, colorectal, gall bladder, NSCLC and breast cancer) All vaccine manufacturing processes met specications