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256 The Comparison of Various Promoters for Wiskott Aldrich Syndrome Gene Therapy Using Insulated Lentiviral Vectors Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Societ[.]
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I 254 Correction of RAG2 Severe Combined Immunodeficiency by Lentiviral Gene Therapy Requires Strong Expression of RAG2 Niek P van Til,1 Helen de Boer,1 Roya Sarwari,1 Trudi P Visser,1 Barbara Cassani,2 Anna Villa,3 Gerard Wagemaker.1 Department of Hematology, Erasmus University Medical Center, Rotterdam, Netherlands; 2Fondazione Humanitas per la Ricerca, Rozzano, Milan, Italy; 3CNR-ITB; Telethon Institute for Gene Therapy-HSR, Milan, Italy Recombination activating gene (RAG2) deficiency results in severe combined immunodeficiency (SCID) due to a lack of T and B lymphocytes, caused by a block in differentiation Patients succumb due to recurrent infections within the first year of life, unless treated with an allogeneic bone marrow (BM) transplant Unfortunately, the paucity of compatible donors affects the efficacy of treatment and the survival of treated patients Gene therapy using gammaretroviral vectors for X-linked SCID gene therapy and adenosine deaminase (ADA)-SCID has been shown to be highly effective, but RAG2 SCID may require more tightly controlled expression as its expression is heavily regulated during normal T and B cell development Previously, it has been shown that gammaretroviral vectors can effectively restore immune function in Rag2-/- mice To improve efficacy and reduce the risk of genotoxicity we developed and tested self-inactivating lentiviral vectors constituting a codon-optimised RAG2 (RAG2co) driven by the spleen focus forming virus promoter (SF) This vector was then compared to its native RAG2 counterpart as well as to weaker cell-restricted promoters based on minimum elements of the RAG2 and γc promoter 5x105 Rag2-/- transduced lineage negative BM cells were transplanted in sublethally irradiated Rag2-/- mice, monitored for six months Codon-optimization of RAG2 increased detectable RAG2 protein 3-fold, determined in HeLa cells The SF-RAG2co vector resulted in a significant reduction in the developmental block during T and B cell differentiation in thymus and BM, respectively, and in robust reconstitution of CD4 and CD8 T cell subsets, as well as mature IgM+IgD+ B cells in peripheral blood and spleen Reconstitution of T and B cells following transplantation of cells transduced with the vector carrying the native RAG2 was significantly slower, and most peripheral T cells expressed a single dominant T cell receptor subtype In contrast, the RAG2 and γc promoter reconstituted T cells effectively, but hardly any B cells Vector copy number per cell determined in bone marrow was considerable higher in the SFRAG2co (2.3±2.0, n=17) and SF-RAG2 treated mice (3.0±0.9, n=7) than in those treated with the RAG2 and γc promoters (0.5±0.5, n=9 and 0.5±0.4, n=11 respectively), and proportional to the levels of reconstitution In the SF-RAG2co treated mice, T cell responses to mitogens, plasma immunoglobulin levels and T cell dependent (tetanus toxoid) and independent (pneumococcal polysaccharide vaccine) antibody responses were fully restored, but incomplete in the other treatment groups We conclude that complete immune restoration can be achieved in Rag2-/- mice by use of the strong SF promoter driving constitutive RAG2co expression and that further development towards safe promoter cassettes is required for clinical application 255 Correction of X-SCID by Sleeping BeautyMediated Gene Transfer into Hematopoietic Cells Kendra A Hyland,1 Erik R Olson,1 Nicola Philpott,2 Xianzheng Zhou,3 R Scott McIvor.1 Discovery Genomics, Inc., Minneapolis, MN; 2Medicine, Rheumatic and Autoimmune Diseases, University of Minnesota, Minneapolis, MN; 3Pediatrics, University of Minnesota, Medical School, Minneapolis, MN Primary immune deficiencies comprise a group of inherited genetic disorders caused by interruption of normal lymphoid development Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy X-linked severe combined immunodeficiency (X-SCID) is one of the most common primary immunodeficiencies, and is caused by a mutation in the gene encoding the common gamma chain (γc) of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 In the absence of γc protein, cytokine signaling via the JAK-STAT pathway is interrupted, thereby blocking normal lymphoid development Clinical trials employing retroviral vectors for ex vivo treatment of X-SCID have resulted in complete restoration of patient immunity However, serious adverse events have emerged, highlighting the need for development of alternate and less genotoxic approaches for therapeutic gene transfer The Sleeping Beauty (SB) transposon system mediates stable integration of sequences into mammalian chromosomes, supporting long term expression of both reporter and therapeutic genes Advantages of the SB system include ease of manufacture, reduced immunogenicity in comparison with viral vectors, and a more random and potentially less genotoxic integrant profile in comparison with retroviral and lentiviral vectors We first demonstrated successful transposon-mediated expression of γc protein and restoration of JAK-STAT signaling in a T-cell line, lacking the γc gene Introduction of plasmid DNA by electroporation into mouse hematopoietic stem cells (HSC) is not currently feasible, so we will deliver the SB system to mouse HSC using a non-integrating lentiviral vector system After transplantation of transduced HSC in γc-deficient mice, we will assay for correction of lymphoid development by measuring the presence of B, T, and NK cells as well as restoration of normal immunity Transposition will be verified by recovery and molecular characterization of transposon integrants and flanking chromosomal sequence Results from these studies will provide a platform for establishment of SB-mediated transposition of HSC in the treatment of human X-linked SCID 256 The Comparison of Various Promoters for Wiskott Aldrich Syndrome Gene Therapy Using Insulated Lentiviral Vectors Rachel M Koldej,1 Gael Carney,1 Matthew Wielgosz,1 Satyanarayana Pondugula,1 Arthur W Nienhuis.1 Hematology, St Jude Children’s Research Hospital, Memphis, TN Wiskott Aldrich Syndrome (WAS) is a severe disease which affects virtually all lineages of the hematopoietic system Thrombocytopenia, eczyma and recurrent or persistent infections are common disease manifestations Patients with a matched sibling donor can be cured by bone marrow transplantation but transplantation from alternative donors leads to a poorer outcome Development of effective lentiviral vector mediated gene therapy for WAS would provide alternative curative therapy Our efforts to develop a safe and effective selfinactivating lentiviral vector for gene therapy of WAS have focused on the evaluation of vectors with an insulator element in the integrated long terminal repeats (LTRs) and various internal promoters driving the WAS protein (WASp) cDNA The ideal promoter for WAS gene therapy would achieve physiological expression levels at the majority of integration sites in all relevant blood lineages, particularly T-cells, B-cells, monocytes and megakaryocytes Several promoters including a γ-retroviral LTR (MND), a short fragment (240 bp) from the elongation factor 1-α (EF1α) promoter and various length fragments of the proximal WAS gene promoter are being evaluated These promoters were cloned into non-insulated, 400bp or 650bp chicken HS4 insulated lentiviral vectors and examined in mouse bone marrow transplant studies using GFP as a surrogate marker for hWASp expression Lineage negative B6.SJL-Ptprca Pepcb/ BoyJ mouse bone marrow was prestimulated for 28 hours prior to transduction on retronectin coated plates for 21 hours with virus at an MOI of 20 after which the transduced cells were administered via tail vein to irradiated recipient C57BL/6J mice The mice were monitored with periodic eyebleeds and sacrificed 22 weeks post transplant In peripheral blood all mice exhibited equal levels of S99 HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I engraftment (>80%) with percent GFP positive ranging from 40100% in CD45.1 positive white blood cells The presence of the 400bp insulator increased relative GFP expression in lymphoid lineages when combined with the short (500bp) WAS promoter fragment but not the full length (1.6kb) WAS promoter or the EF1α promoter Platelet counts stabilized at 600 to 1,200 x 103/ul and GFP expression, when normalized to bone marrow copy number, showed that the presence of the 400bp insulator increased expression in the platelets independent of the promoter utilized In bone marrow, the 400bp insulator decreased GFP expression from the EF1α promoter in every lineage examined In contrast, expression from either the short or full length WAS promoter was not affected Secondary transplants are ongoing In vitro studies in WAS patient peripheral blood mononuclear cells using insulated hWASp expression vectors have demonstrated that while all the promoters examined will express to wild-type levels in transduced cells, the MND promoter gives a higher proportion of cells expressing at this level 257 In Vivo, Lineage-Directed Transgene Expression after Hematopoietic Stem Cell Gene Transfer in a Large Animal Model Aravind Ramakrishnan,1,2 Beverly Torok-Stord,1 Mortimer Poncz,3 Nathaniel P Williams,1 Christina Ironside,1 Hans-Peter Kiem,1,2 Brian C Beard.1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Department of Medicine, University of Washington, Seattle, WA; 3Pediatric Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA Efficacy of gene-modified hematopoietic stem cell (HCS) transplantation for the treatment of patients with single allele genetic disease has unequivocally been demonstrated for adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID), X-linked severe combined immunodeficiency (SCID-X1), chronic granulomatous disease (CGD), adrenoleukodystrophy, (ALD), and Wiskott-Aldrich Syndrome (WAS) For a subset of these patients proto-oncogene activation as a result of insertional mutagenesis have resulted in both clonal dominance and frank leukemia The strong promoter enhancer elements in the long terminal repeats (LTR) were likely responsible for the adverse events seen in these patients In order to maintain the efficacy of these pioneering clinical trials while improving the safety for future patients a promising strategy is to eliminate the LTR promoter and enhancers and instead use inducible or lineage-directed promoters In mouse and human cells, the platelet factor (PF4) promoter is largely megakaryocyte lineage-specific and has been used to study megakaryopoiesis We hypothesized that the PF4 promoter could potentially be used as a tool to target transgene expression specifically to the megakaryocytic lineage In theory, this could be used to correct underlying genetic defects that affect platelets or utilize platelets as delivery vehicles to deposit specific gene products at sites of thrombosis/injury To effectively test hematopoietic lineage expression of the PF4 promoter in a large animal, we used the clinically relevant dog model Specifically, autologous CD34-selected bone marrow cells were transduced with a VSVG-pseudotyped HIV-derived self-inactivating (SIN) lentivirus vector with PF4 expressing green fluorescent protein (GFP) using a 24-hour protocol at a multiplicity of infection of ∼10 Following ablative conditioning ∼1.0E+06 cells/kg were infused and the dog displayed normal gene-modified hematopoietic engraftment kinetics At the most recent follow-up, 77 days after transplantation, the dog has stable in vivo gene marking of ∼5.4% in granulocytes, ∼2.4% in red blood cells, and ∼19.0% in platelets Importantly, compared to historical controls at similar time points for dogs that received cells gene-modified with HIV-derived SIN lentivirus vectors expressing GFP from a constitutive promoter (phosphoglycerate kinase-PGK) the fold difference in mean fluorescence intensity of GFP+ relative S100 to GFP- gene marked granulocytes was 34.9 (PGK) compared to 12.2 (PF4) and gene marked platelets was 12.9 (PGK) compared to 72.1 (PF4) These data shows that the PF4 promoter is preferentially active in platelet lineages and far less active in myeloid lineages and have important implications for improving the safety and efficacy of gene therapy strategies by limiting transgene expression primarily to affected subsets or specifically for this application, sites of thrombosis/ injury 258 Non-Myeloablative Conditioning with Busulfex mg/kg or 200 cGy TBI Is Equivalent in Gene Therapy of Canine Leukocyte Adhesion Deficiency Using Foamy Viral Vectors Thomas R Bauer, Jr.,1 Erik M Olson,2 Laura M Tuschong,1 Tanya H Burkholder,3 David W Russell,2 Dennis D Hickstein.1 Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD; Department of Hematology, University of Washington, Seattle, WA; 3Division of Veterinary Service, Office of Research Services, National Institutes of Health, Bethesda, MD The optimal non-myeloablative regimen for hematopoietic stem cell gene therapy for treatment of phagocytic diseases remains unclear In particular, the use of low doses of ionizing radiation, although widely use in reduced intensity allogeneic hematopoietic conditioning regimens, has not been used in human gene therapy clinical trials To address this issue, we compared the results with non-myeloablative conditioning with Busulfex mg/kg to conditioning with 200 cGy total body irradiation (TBI) in the engraftment and chimerism of foamy viral (FV) vector transduced CD34+ cells in animals with canine leukocyte adhesion deficiency (CLAD) Similar to children with LAD-1, dogs with CLAD suffer from recurrent bacterial infections and early death within the first six months of life due to infections resulting from the absence of the CD18 leukocyte integrin on the neutrophil surface and the resultant lack of adherence and migration of neutrophils lacking CD18 Initially, four CLAD pups were treated using a non-myeloablative conditioning regimen of 200cGy total body irradiation (TBI) administered the day before receiving genetically-corrected cells The four dogs survived for four years, at which time the study was completed Of note, CD18+ neutrophil levels ranged from 2.9 to 3.9% in the peripheral blood, and CD18+ T-cell levels ranged from 10.7 to 22.9% at one-year post-transplant In the companion study four CLAD pups received a reduced intensity conditioning regimen with Busulfex mg/kg given IV two days prior to infusion of genetically-corrected cells All four dogs survived more than year, free of severe CLAD disease CD18+ neutrophil levels ranged from 1.8 to 4.5% and CD18+ T-cell levels ranged from 14.1 to 21.6% at one-year post-transplant Thus, comparison of the chimerism levels in the dogs in each group revealed little difference between the conditioning regimens at year post-transplant The slight variation in chimerism could be attributed to the corrected cell doses infused No animal with either regimen became neutropenic nor required platelet infusions due to conditioning These results demonstrate the efficacy of both conditioning regimens for achieving sufficient levels of CD18+ cells to reverse the CLAD disease phenotype, and further show that Busulfex mg/kg appears to represent an effective substitute for 200 cGy TBI as a conditioning regimen for the treatment of myeloid disease such as LAD by hematopoietic stem cell gene therapy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... regimen for the treatment of myeloid disease such as LAD by hematopoietic stem cell gene therapy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy. .. Bethesda, MD The optimal non-myeloablative regimen for hematopoietic stem cell gene therapy for treatment of phagocytic diseases remains unclear In particular, the use of low doses of ionizing... and Wiskott- Aldrich Syndrome (WAS) For a subset of these patients proto-oncogene activation as a result of insertional mutagenesis have resulted in both clonal dominance and frank leukemia The