342 correction of murine hemophilia a following nonmyeloablative transplantation of hematopoietic stem cells engineered to express a bioimproved human FVIII using a safety augmented, b lymphoid lineage restricted lentiviral vector

342 Correction of Murine Hemophilia A Following Nonmyeloablative Transplantation of Hematopoietic Stem Cells Engineered To Express a Bioimproved Human FVIII Using a Safety Augmented, B Lymphoid Lineag[.]

HEMATOLOGIC/IMMUNOLOGIC: BASIC 340 Development of an S/MAR Based Episomal Vector of a Specic Zinc-Finger Activator That Mediates Gamma-Globin Gene Activation, for the Gene Therapy of Hemoglobinopathies Eleana Stavrou,1 Eleni Lagadinou,2 Nikolas Zoumbos,2 Alexandros Spyridonidis,2 Carlos Barbas, III,3 Kenneth Peterson,4 Aglaia Athanassiadou.1 General Biology, School of Medicine, University of Patras, Patras, Rion, Greece; 2Internal Medicine, Hematology Unit, School of Medicine, University of Patras, Patras, Rion, Greece; Molecular Biology, Skaggs Institute for Chemical Biology, Scripps Research Institute, La Jolla, CA; 4Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City The increase of HbF through activation of gamma-globin gene is a valid strategy for the treatment of hemoglobinopathies Zif-VP64 is a selective, synthetic gamma-globin activator, containing a zinc-nger DNA protein that binds the gamma-globin promoter -117HPFH area and a transcription inducer that induces gamma globin gene in K562 cells, after viral transfer We report the study of an episomal vector of this activator, which is based on a Scaffold/Matrix attachment region (S/MAR) that supports retention of episomes in the nucleus of the host cell We constructed an episomal vector, Zif-VP64-Ep1, containing the activator Zif-VP64, the reporter gene cassette CMV-eGFP and the S/MAR element Gene transfer into cells was done by electroporation or nucleofection Expression of eGFP was documented by Florescent Microscopy and Flow Cytometry, while the fate of vector molecules in the cells was studied by Southern Blot and plasmid rescue experiments Real time PCR, Western blotting and Intracellular Flow Cytometry were used to investigate gamma-globin mRNA, gammaglobin protein and HbF protein levels respectively Binding specicity of the activator was determined by ChIP Gene transfer was done in K562 cells producing long term stable cell lines; murine beta-YAC cells, where the YAC contains the complete human, beta-globin gene locus; and human progenitor hemopoietic CD34+ cells from healthy, mobilized individuals, with transfection efciencies of 65%, 25% and 23% respectively In K562 cells, gamma-globin mRNA levels showed an increase of 250%, gamma-globin protein of 350% and HbF protein of 165% as compared to the corresponding levels in the untransfected K562 cells, at least 200 generations post-transfection In murine beta-YAC cells, vector Zif-VP64-Ep1 was able to mediate the activation of expression of the silent, human gamma-globin gene at a level matching the (active) human beta-globin gene of the YAC as well as the murine beta-globin gene, showing that it can efciently activate the gamma-globin gene from within a heterochromatic region Signicantly, vector Zif-VP64-Ep1 was able to transfect the human, hemopoietic progenitor CD34+ cells and to mediate a 3.0±1 fold increase of gamma globin mRNA, compared to untrasnfected CD34+ cells, as estimated in cultures of 7-8 days after transfection In conclusion, activation of human gamma-globin by episomal gene transfer of a synthetic activator, in three different hemopoietic cells, is documented, including the CD43+ cells, that are the target cells for gene therapy of the Hemoglobinopathies This is the rst time that an S/MAR based episomal vector is used for gene transactivation in a cell line and progenitor cells, aiming at specic gene therapy 341 Engineered Human Regulatory T Cells Expressing Lentiviral PDL1 under Cell Fate Control Prevent Lethal Xenogeneic GVHD Shoba Amarnath,1 James Wang,2 Courtney Mangus,1 James Riley,3 Bruce Levine,3 Carl June,3 Jeffrey Medin,2,4 Daniel Fowler.1 ETIB, CCR, NCI, Bethesda, MD; 2IMS, Toronto, ON, Canada; UPENN, Philadelphia, PA; 4UHN, Toronto, ON, Canada Programmed death ligand-1 (PD-L1) represents a regulatory T cell (Treg) mechanism that controls immunity mediated by effector S132 memory T cells expressing PD1 We hypothesized that geneticallyengineered human T cells forced to express PD-L1 would manifest a Treg phenotype, and thus be capable of inhibiting human-into-mouse xenogeneic graft-versus-host disease (x-GVHD) Advantages of this proposed therapy include a capacity to: (1) enforce long-term, stable expression of PD-L1; (2) utilize cellular delivery vehicles composed of functional T cells that persist in vivo; and (3) incorporate an enhanced ‘cell fate control’ cDNA to permit in vivo control of therapy We thus developed a recombinant lentiviral vector (LV) that encodes cDNA for a fusion protein consisting of human CD19 and mutated TMPK that activates the prodrug AZT, followed by full-length human PD-L1 We previously found that ex vivo T cell expansion in rapamycin induces an anti-apoptotic phenotype that permits in vivo T cell persistence in murine models and xenogeneic transplant models We thus manufactured human CD4+ T cells using co-stimulation (anti-CD3/28), Th1 polarization (IFN-α), and rapamycin After days of culture and subsequent ow sorting, >90% of transduced T cells expressed PD-L1 Next, we utilized a x-GVHD model to assess in vivo persistence of the modied T cells and transgene expression Two separate experiments demonstrated that gene-modied T cell recipients had increased numbers of human T cells in the spleen that co-expressed CD19 and PD-L1 relative to non-transduced T cell recipients (p=0.02) Harvested T cells were secondarily co-stimulated ex vivo and propagated for days: co-expression of CD19 and PD-L1 persisted in ∼ 50% of T cells (p=0.0001) To test the ability of PD-L1transduced T cells to prevent x-GVHD, a 3rd experiment involved cohorts that received: human Th1 cells alone (C#1) or in combination with PD-L1 LV-transduced T cells (C#2); puried human Treg cells (C#3); or control LV-transduced T cells (C#4) On day 5, mice were challenged with LPS to induce cytokine-mediated x-GVHD Cohorts did not differ with respect to human T cell engraftment Relative to C#1, both C#2 and C#3 had reduced numbers of human T cells that expressed IFN-γ (each comparison, p