674 Long Term Safety of Clinical Grade LentiGlobin Vectors in a γ Thalassemic and Normal Mice Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Thera[.]
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II 672 Transduction of Human CD34+ Cells in the NOD SCIDγ (NSG) Mouse Model Using LentihWASP-Vectors Derived from Stable Producer Clones Matthew M Wielgosz,1 Yoon S Kim,1 Robert E Throm,1 Gael G Carney,1 John T Gray,1 Arthur W Nienhuis.1 Experimental Hematology, St Jude Children’s Research Hospital, Memphis, TN We have developed several producer clones for a Lenti-human Wiskott Aldrich Syndrome Protein (hWASP) vector We previously demonstrated (14th ASGCT Meeting, 2012) that concentrated vector preparations from these clones transduced human CD34+ cells at levels generally higher than preparations derived by concentrating culture media from transfected cells, as assessed by PCR of clones derived from transduced CFU-Cs Here we demonstrate that vector preparations from different producer clones transduced CD34+ cells at levels (23%, 40%, and 46%) comparable to transiently derived GFP and hWASP vector preparations (26% and 36%, respectively), with MOIs ranging from 50-100 Following transplantation of human cells into NSG mice (tail vain injection), harvest of human CD45+ cells from murine bone marrow (BM) months post transplantation (tibia and fibula), and inoculation of 100,000 BM-derived human CD45+ cells into methocult cultures, the percentages of hWASP PCR(+) or GFP(+) CFU-Cs were higher for producer cell-derived vector preparations (22%, 37%, and 57%) than those from transiently-derived preparations (3.3% and 8.4% for hWASP and GFP, respectively), despite similar levels of human CD45+ engraftment (ranging 11.3 to 20%) We used a vector preparation from one producer clone (hWASP1) in a second CD34+-NSG transplant experiment, to examine the effects of several small molecules and varied MOI on CD34+ transduction and engraftment, months post-transplant Nicotinamide (NAM) and EX527 (both SIRT1 inhibitors) increased human CD45+ engraftment levels (26% and 30%, respectively) over the untreated control group (16.3%), but yielded decreased transduction levels (25% and 7%, respectively, vs 37%) Interestingly, the addition of stromal derived factor alpha (SDF-1) to NAM-treated CD34+ cells appeared to reverse the NAM-transduction defect (31% vs 17%), and enhance human CD45+ engraftment levels (23% vs 14%) We also observed that CD34+ cells that underwent a second transduction had decreased human CD45+ engraftment levels (2.9%) over the control group (16%), but had the highest human CD45+ cell transduction level (55%) In addition, when WPRE mRNA expression levels were measured by qRT-PCR (a surrogate to estimate hWASP expression in CD45+ cells sorted from NSG BM), WPRE expression levels averaged 81% of an RNA control derived from a transduced Jurkat cell line (MOI 100), whereas the single transduction control group averaged 44%, normalized to VCN These results confirm that vector preparations derived from our lenti-hWASP producer clone transduce CD34+ cells at levels higher than those obtained from transient vector preparations as assayed in the NSG mouse transplant model Importantly, we demonstrate that the evaluation of the transduction and engraftment of stem repopulating cells (SRCs) in the CD34+NSG mouse transplant model can be used to optimize transduction conditions These findings support our plans to utilize this producer clone to make vector for our clinical trial 673 Pre-Clinical Modeling of Foamy Virus Vector Based Gene Therapy for SCID-X1 Gabrielle M Curinga,1 Iram Khan,1 Swati Singh,1 Joey Pangallo,1 Brian Beard,4,5 Troy Torgerson,1,2 Andrew Scharenberg,1,2 Grant Trobridge,6,7 Hans-Peter Kiem,4,5 David Rawlings.1,2 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle; 2Immunology and Pediatrics, University of Washington School of Medicine, Seattle; 3Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle; 4University of Washington School of Medicine, Seattle; Pharmaceutical Sciences, Washington State University, Pullman, Washington; 6School of Molecular Biosciences, Pullman X-linked severe-combined immune deficiency (SCID-X1) results from inactivating mutations in the gene encoding the common gamma chain (c), a receptor sub-unit required for lymphoid development and cell function Unless successfully treated by hematopoietic stem cell transplantation, SCID-X1 is fatal within the first years of life While HLA matched allogenic stem cell transplantation boasts survival rates exceeding 90%, such donors are rarely available and use of alternative stem cells sources results in poorer outcomes Thus, gene replacement therapy has emerged as a promising alternative for SCID-X1 patients lacking suitable donors Pioneering c gene replacement clinical trials demonstrated efficacy, but also the need for improved safety due to adverse events associated with the gamma-retroviral delivery platform Although new self-inactivating (SIN) gamma-retroviral and lentiviral vectors appear to offer improved safety profiles relative to the original gamma-retroviral vector, the lack of native pathogenicity of foamy viruses and an integration site profile that is much less focused on either active genes or promoter regions than lentiviruses or gamma-retroviruses suggest that FV vectors are the safest available platform for c gene replacement therapy We therefore initiated studies to determine whether clinical FV vectors driving transcription of human c will rescue lymphocyte development and function in SCID-X1 mice and in human cells; and whether this can be achieved without evidence of toxicity Transduction of a human T cell line lacking c with FV c vectors demonstrated high-level sustained c expression Further, FV transduction restored IL2, IL7, or IL21 stimulation-dependent phospho-STAT3 or pSTAT5 signals Next, we established protocols resulting in consistent marking of purified lineage depleted (lin-) murine hematopoietic stem cells (HSC) Co-culture of FV-transduced SCID-X1 HSC on a Notchligand expressing stromal cell line, OP9-Delta 1, led to efficient in vitro T cell differentiation Thus, transduction with the FV c vector facilitates rescue of c expression and signaling in human cell lines and in vitro T cell development of murine SCID-X1 HSC In vivo functional recovery and integration site profiling is currently being assessed using in SCID-X1 mice transplanted with FV c vector transduced HSC 674 Long Term Safety of Clinical Grade LentiGlobin Vectors in a β-Thalassemic and Normal Mice Olivier Negre,1 Cynthia Bartholoma,2 Robert Kutner,3 Beatrix Gillet-Legrand,1 Celine Courne,1 Anais Paulard,1 Byoung Ryu,3 Maria Denaro,3 Christof von Kalle,2 Emmanual Payan,4 Michell Finer,3 Gabor Veres.3 Bluebird Bio Inc, MA USA, Fontenay aux Roses, France; DKFZ, National Center for Tumor Diseases (NCT), Heilderberg, Germany; 3Bluebird Bio Inc, Cambridge; 4Institute of Emerging Diseases and Innovative Therapies (iMETI), CEA, Fontenay aux Roses, France The long term safety of an improved LentiGlobin vector was evaluated by transplanting transduced Lin-depleted bone marrow cells isolated from-thalassemia (Hbbth1/th1) mice into syngeneic Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S257 HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II myeloablated recipients The therapeutic efficacies and potential toxicity (histopathology, hematology) and insertion site analyses were analyzed in 54 primary transplants A significant improvement of hemoglobin concentration, hematocrit level, red blood cell counts and reticulocyte percentage were observed in mice transplanted with bone marrow cells transduced with the LentiGlobin HPV569 lentiviral vector or the LentiGlobin BB305 lentiviral vector compared to the group transplanted with untransduced cells in the primary recipients The improvement of -thalassemic phenotype was similar between mice transplanted with cells transduced with LentiGlobin HPV569 lentiviral vector and mice transplanted with cells transduced with LentiGlobin BB305 lentiviral vector No toxicological changes were observed in the treated groups A comparative large scale integration site analysis has been performed on mouse bone marrow samples transduced with the LentiGlobin HPV569 vector and with the LentiGlobin BB305 lentiviral vector and the analyses of the pre transplant samples showed a highly polyclonal vector integration profile in both LentiGlobin vectors (BB305, HPV569) IS analyses in samples derived from the transplanted - thalassemic (Hbb th1/th1) mice revealed an oligo-/polyclonal hematopoietic repopulation for both LentiGlobin (BB305: 3508 unique IS; HPV569: 4257 unique IS) vectors Secondary transplants were also conducted (total of 108 mice) to evaluate the toxicity of LentiGlobin vectors BB305 and HPV569 in CD45.1 C57BL/6 mice following a single intravenous administration of “secondary” bone marrow obtained from -thalassemic Hbb th1/ th1mice originally transplanted four months earlier with syngeneic Lin depleted mouse bone marrow cells transduced with LentiGlobin BB305 and Lentiglobin HPV 569 lentiviral vectors The secondary transplant mice are sacrificed and necropsied six months after transplantation and an extensive histopathology evaluation are performed to assess hematopoietic homeostatis, clinical chemistry, hematology and bone marrow cytology In addition, comprehensive IS analyzes is being performed on DNA samples isolated from the bone marrow of the secondary recipients.A comprehensive data set will be presented on both the primary and secondary transplant animals Fr-MuLV-U3, the housekeeping hPGK or the FANCA homologous promotor (FANCendo) that we have engineered The latter mediates low basal expression levels with EGFP1 mean fluorescence reduced by one and two ranges compared to hPGK or viral promoters, respectively In primary MSCs cells grown under Mitomycin C while retrieving protection from oxidative stress, the earliest and most significant recovery is monitored in cells transduced with FANCendo Data will be shown in CD34+ cells efficiently transduced with the FANCendoInsLV and grown over weeks in LTCIC without toxicity and patients’ samples This new insulated lentivector driven by FANCA-homologous promoter shows improved safety and efficacy profile towards gene therapy in Fanconi’s anemia 675 A Safety and Efficacy Improved New Insulated LV Driven by FANCA-Promoter towards GT in Fanconi’s Anemia Emilie Bayart,1 Caroline Duros,1 Alexandre Artus,1 Stephanie Lemaire,1 Odile Y D Cohen-Haguenauer.1,2 CNRS UMR 8113, Ecole Normale Superieure de Cachan, Cachan, France; 2Oncogenetics-Medical Oncology, Hopital St-Louis Sorbonne Paris-Cité, Faculté de Médecine Paris-Diderot, Paris, France Fanconi anemia is a rare inherited genomic instability syndrome, with severe bone-Marrow (BM) deficiency developing around age The 15 known genes are involved in DNA repair, redox metabolism and differentiation FA patients’ cells exhibit hypersensitivity to DNA crosslinking agents such as Mitomycin C (MMC) In a previous study, we have: (i) demonstrated that BM dysfunction can be ameliorated in protecting cells from oxidative stress in growing cells with both hypoxia and N-Actyl-Cystein in the culture medium and (ii) achieved in vitro and in vivo long-term reconstitution with retrovirus transduced cells from unfractionated patients’ BM (Cohen-Haguenauer et al, 2006) With view to reducing the risk for GT induced malignancies in this cancer-prone disease, we have developed a new generation of genetically stable insulated vectors which have been used to shuttle the FANCA cDNA Transduction of HSC72 FA cells resulted in functional correction with overcoming the G2M block on high doses MMC and restoring FancD2 mono-ubiquitination In genetically corrected primary mesenchymal cells from FANCA-/- patients the transgene is stably expressed and the insulator sequences preserved after 18 months of sustained growth Three different internal promoters – with distinct potentials- have been tested: the strong viral derived S258 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... improved safety and efficacy profile towards gene therapy in Fanconi’s anemia 675 A Safety and Efficacy Improved New Insulated LV Driven by FANCA-Promoter towards GT in Fanconi’s Anemia Emilie Bayart,1... with LentiGlobin BB305 and Lentiglobin HPV 569 lentiviral vectors The secondary transplant mice are sacrificed and necropsied six months after transplantation and an extensive histopathology evaluation... IS) vectors Secondary transplants were also conducted (total of 108 mice) to evaluate the toxicity of LentiGlobin vectors BB305 and HPV569 in CD45.1 C57BL/6 mice following a single intravenous administration