261 Further Amelioration of the Late Effects of Total Body Irradiation (TBI) by Manganesium Superoxide Dismutase Plasmid/Liposome (MnSOD PL) Gene Therapy Supplemented by a Continuous Antioxidant Chemo[.]
HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I 259 Mouse Transplant Models for Evaluating the Safety of a SCID-X1 Lentiviral Vector Sheng Zhou,1 Zhijun Ma,1 Taihe Lu,1 Laura J Janke,1 John T Gray,1 Brian P Sorrentino.1 St Jude Children’s Research Hospital, Memphis The multiple cases of hematological malignancies in several gene therapy trials underscore the need for better preclinical safety models for assessing vector safety Mouse transplant models allow in vivo studies of vector safety and also testing in a SCID-X1 genetic background, which is known to increase transformation risk We also used a second model based on serial transplantation of wildtype cells using the SFFV gamma-retrovirus as a positive control Both of these studies lasted up to 14 months and a full necropsy is performed by a certified veterinary pathologist The test item was our self-inactivating CL20i4-EF1α-hγcOPT vector (EF1α) expressing a codon optimized gamma chain cDNA under control of a short EF1α promoter In the first experiment (Exp 1, table 1), none of the primary recipient mice developed any hematological malignancies during the 5-7 months of observation period Three secondary recipients from the same primary recipient donor in the EF1α group with SCID-X1 donor cells developed a clonal IgM+, Pax5+ B cell lymphoma Vector integration analysis identified a single copy vector insertion in the first intron of the Pkn2 gene, in the antisense orientation The Pkn2 gene encodes for a serine threonine kinase involved in cell cycle entry and mitosis, suggesting that its deregulation could be oncogenic Pkn2 mRNA levels measured by qRT-PCR from sorted leukemic cells was 58% of that of the normal IgM+ B cells and 30-43% of that of two B cell leukemic cells lines 70Z and CH12.1.6, showing that the B cell lymphoma was not caused by insertional activation of the Pkn2 gene No hematological malignancies occurred in any other groups so that this single case of malignancy was not statistically significant In the second transplant experiment (Exp 2, table1), no hematological malignancies occurred in any primary recipients However, peripheral blood lineage analysis by flow cytometry showed significant myeloid lineage skewing in the SFFV-DsRed group at 14 weeks to months Secondary transplants have recently been performed and three mice from a single primary recipient donor in the SFFV-DsRed group had high leukocyte count at 18 weeks, with blasts on blood smear All the mice will continue to be monitored for months and results will be reported at the meeting Altogether, our study showed no vector induced leukemias in as many as 79 primary recipients that received transplant of vector transduced bone marrow cells, supporting the notion that mouse models are insensitive for vector safety evaluation In the SCID-X1 model, we did not derive useful information and we conclude the single case of B cell lymphoma was not related to vector insertion The ongoing study with wildtype cells suggests that the EF1α lentiviral vector is less prone to inducing clonal skewing than the SFFV gamma-retroviral vector Table1 Design of two transplant experiments vector copy number in # Primary recipients PBMNC of Primary recipients Exp Rag2-/-IL2rg-/Mock 11 MSCV 15 1.4 ± 0.58 15 2.17 ± 0.74 EF1α # Secondary recipients Rag2-/-IL2rg-/23 36 39 Exp WT WT Mock 15 42 15 0.81 ± 0.31 42 MFG-γc SFFV-DsRed 19 1.81 ± 1.23 51 15 1.59 ± 0.39 45 EF1α MSCV: MSCV-γc-IRES-GFP MFG-γc: French SCID-X1 trial vector Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 260 Balancing Efficacy and Adverse Outcomes in Gene Therapy for Wiskott-Aldrich Syndrome Alexander Astrakhan,1,3 Blythe Sather,3 Sara Mamman,3 Michelle Brault,3 Socheath Khim,3 Byoung Ryu,3 Carol Miao,3 David J Rawlings.1,2,3 Immunology, University of Washington, Seattle, WA; 2Pediatrics, University of Washington, Seattle, WA; 3Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA Wiskott-Aldrich Syndrome (WAS) is a rare primary immunodeficiency caused by mutation of Wiskott-Aldrich Syndrome protein (WASp) Defective WASp functionality results in abnormal immunological synapse formation, impaired cellular migration and compromised cellular signaling As a result, WAS patients present with frequent infections, thrombocytopenia, autoimmunity and increased risk of malignancies The disease is usually fatal in the absence of bone marrow transplant; and gene therapy represents a promising alternative for patients lacking well-matched donors We investigated selective advantage and promoter activity as variables potentially regulating successful lentiviral (LV) - based gene therapy treatment in a murine model of WAS We compared in vivo efficacy of transplanted hematopoietic stem cells transduced using LV vectors containing either the gammaretroviral-derived enhancer/ promoter, MND, or the mammalian WAS endogenous promoter, WS1.6, to drive human WASp in WASp-/- recipient mice MNDhuWASp LV resulted in endogenous levels of WASp expression in all hematopoietic lineages and selection for WASp+ T, NKT and B cells MND-huWASp recipients exhibited normalized T cell proliferation and cytokine production and generated abundant marginal zone (MZ) B cells We also observed a single instance of viral-mediated myeloid clonal dominance with MND-huWASp, suggesting an enhanced risk of insertional mutagenesis using this promoter In contrast, WS1.6-huWASp LV exhibited substantially weaker promoter activity and resulted in only partial selection for WASp+ T cells and minimal selection for WASp+ B cells WS1.6-huWASp recipients exhibited minimal rescue of MZ B cell formation and an increase in autoimmune-prone B cells Our findings identify relative benefits and potential risks for each of these enhancer/promoter elements currently in use in clinical trials; and suggest approaches that may lead to a safer and more effective gene delivery platform for treating Wiskott-Aldrich Syndrome 261 Further Amelioration of the Late Effects of Total Body Irradiation (TBI) by Manganesium Superoxide Dismutase Plasmid/Liposome (MnSOD-PL) Gene Therapy Supplemented by a Continuous Antioxidant-Chemopreventive Diet Michael W Epperly,1 Hong Wang,1 Jeffrey A Jones,2,3 Tracy Dixon,1 Carlos A Montesinos,4 Joel S Greenberger.1 Department of Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2National Aeronautics and Space Administration, Johnson Space Center, Houston, TX; Department of Urology, Baylor College of Medicine, Houston, TX; 4AmeriSciences LP, Houston, TX Both acute and chronic effects of ionizing irradiation are mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS) which deplete antioxidant stores leading to cellular apoptosis, stem cell depletion, and accelerated aging TBI acute effects occur within the first month after irradiation Survivors may suffer late effects which occur months to years after irradiation Following irradiation, there is continued expression of ROS or RNS for weeks to months leading to accumulation of damage which contributes to late effects We tested the hypothesis that late effects of TBI may be reduced if increased antioxidants are continually present during prolonged S101 HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I expression of ROS and RNS following irradiation We demonstrated that C57BL/6NHsd mice receiving intravenous MnSOD-PL prior to 9.5 Gy total body irradiation (TBI) show increased survival from the acute hematopoietic syndrome as well as improved long term survival (Epperly, et al., Radiation Research, 170(4):437-444, 2008) We now evaluated whether an antioxidant-chemopreventive diet alone or with MnSOD-PL (compared to a regular diet) improved long-term survival Beginning seven days before TBI, two groups of eighty C57BL/6NHsd female mice were placed on either a control house diet or a diet enriched with antioxidants Twenty-four hours before the LD 50/30 dose of 9.5 Gy TBI subgroups of forty mice were injected intravenously with MnSOD-PL (100 µg plasmid DNA in 100 µl) Following irradiation each group remained on the house diet or on the antioxidant diet Mice treated with MnSOD-PL showed decreased deaths in the first 30 days following irradiation, independent of diet compared to irradiated mice on the house diet alone (p = 0.031 for the House Diet plus MnSOD-PL or 0.015 for antioxidant diet plus MnSOD-PL) Survival over the first 30 days was unaffected by either diet alone in the absence of MnSOD-PL (p = 0.823) Mice on the antioxidant-chemoprevention diet alone or combined with MnSOD-PL that survived 30 days had a significant increase in survival compared to TBI mice on the regular diet (p = 0.040 or 0.010, respectively) and mice treated with MnSOD-PL only and surviving 30 days after TBI had increased survival compared to those on the regular diet alone (p = 0.020) The best overall (acute and long term) survival was in mice receiving MnSOD-PL and antioxidant diet The data support continually increasing bioavailable antioxidants to reduce acute and late effects of irradiation 262 High Levels of Polyclonal, Multilineage Hematopoietic Cell Gene Marking Following Lentiviral Transduction and Transplantation of Steady-State Bone Marrow CD34+ Cells in a NonHuman Primate Model Phillip W Hargrove,1 Yoon-Sang Kim,1 Amy Taylor,1 Dana Simpson,1 Andrew M Davidoff,1 Perdeep K Mehta,1 Derek A Persons.1 St Jude Children’s Research Hospital, Memphis, TN Non-human primate autologous hematopoietic stem cell (HSC) transplantation models have been invaluable in developing the ex vivo transduction protocols used in human gene transfer clinical trials Previous studies of HSC gene transfer using gamma-retroviral and lentiviral vectors in these models have almost exclusively utilized cytokine-mobilized peripheral blood (PB) or bone marrow (BM) CD34+ cells as target cells The administration of the combination of granulocyte colony stimulating factor (G-CSF) plus stem cell factor (SCF) became standard as a priming method prior to HSC collection after it was shown that SCF coupled with G-CSF resulted in significantly improved long-term gene transfer levels, compared to poor gene transfer with G-CSF alone However, SCF is not available in the US for human gene transfer trials due to its history of causing severe anaphylactic reactions For this reason and to define a clinically relevant gene transfer protocol for sickle cell disease, in which G-CSF administration is also contraindicated, we evaluated lentiviral stem cell gene transfer using steady-state BM CD34+ cells in the pigtail macaque transplantation model Bone marrow CD34+ cells were enriched using an anti-CD34 antibody, immunomagetic beads and prestimulated overnight in serum-free medium containing 100 ng/ml of FLT-3 ligand, thrombopoietin, and stem cell factor Cells were then exposed for 14 hours to a VSV-G pseudotyped, HIV-based lentiviral vector encoding GFP at an MOI of 40 After 24 hours of culture in serum-free medium, cells were transplanted into an animal (15 x 106/ kg) which had been conditioned with 950 cGy The marked cells, as assessed by flow cytometry for GFP expression, was 74% and all hematopoietic colonies obtained in methylcellulose cultures were GFP S102 positive Neutrophil recovery (ANC > 500) was observed on day 20 At 96 weeks post-transplant, PB leukocyte marking levels are: 16%, B cells 16% and T cells 17% Vector copy number in PB leukocytes at 80 weeks was 0.4 Insertion site analysis using LAM-PCR coupled with next generation sequencing technology identified 950-2200 unique vector insertion sites (clones) present at each of multiple, individual time points post-transplantation We observed some clones with a large contribution to hematopoiesis early on, but much less frequently at subsequent time points and a highly polyclonal vector insertion site set of more than 2000 unique insertions was identified at months post-transplant We are currently obtaining insertion site data from the 80 week time point Two additional animals were transplanted with transduced steady-state BM CD34+ cells (11 x 106 cells/kg and 6.7 x 106 cells/kg) One animal displayed granulocyte marking of 75% two weeks post-transplant but died unexpectedly due to a “bloat” syndrome unrelated to the transplant procedure The third animal at day 46 showed myeloid marking of 35% These data are critical as they suggest that therapeutic levels of lentiviral vector gene transfer can be obtained using CD34+ cells from steady-state BM as the HSC cell source 263 Long-Term Multi-Lineage Engraftment of NOD-scid IL2Rgnull Mice after Transplantation with Genetically Modified b-thalassemic CD34+ Cells Leda Ferro,1 Jinrong Qu,1 Clare Taylor,1 Xiuyan Wang,1 Aurelio Maggio,2 Farid Boulad,1 Sadelain Michel,3 Isabelle Riviere.1 Gene Transfer Facility, MSKCC, New York; 2Hematology II, V Cervello Hospital, Palermo, Italy; 3Center of Stem Cell Engineering, MSKCC, New York The β-thalassemias are genetic disorders caused by β-globin mutations that reduce or abolish β-globin gene expression The transplantation of autologous hematopoietic stem cells (HSCs) modified with recombinant lentiviral vectors (LVs) encoding the human β-globin gene represents a potentially curative therapy for patients lacking an HLA-matching donor We previously showed stable erythroid specific and therapeutic levels of human β-globin expression in mice transplanted with TNS9 transduced HSCs genetically modified with the TNS9 LV Here we sought to optimize the transductions of G-CSF mobilized-CD34+ cells derived from thalassemic patients using the LV TNS9 and analyzed the engraftment potential of the genetically modified patient HSCs We tested the transduction efficiency of G-CSF mobilized-CD34+ cells derived from patients with β-thalassemia using various prestimulation cytokine cocktails, MOIs, time in culture and single versus double transductions We were able to obtain an average of 0.6-1.2 vector copies/cell and 1-2 copies/cell in a methocult based clonogenic assay β-globin expression was demonstrated by HPLC We further investigated the engraftment efficiency of the genetically modified HSCs using the NOD-scid IL2Rgnull mouse model Transduced thalassemic CD34+ cells, harvested after or days in culture, were injected into sublethally conditioned mice at various doses We detected 5-50% of CD45+ cells in the bone marrow of the transplanted mice up to six months after bone marrow transplantation Detectable amount of CD45+ cells were also found in peripheral blood, spleen and liver of this animals Multi-lineage engraftment was obtained as demonstrated by the presence of both myeloid (up to 20%) and lymphoid (up to 90%) cell types The majority of the CD45+ cells detected in the bone marrow were of the lymphoid lineage with mature B cells (≤ 70% CD19/CD10+ cells), immature B cells (≤ 15% CD10+ cells) and/or T lymphocytes (≤ 3% CD3+) Additionally, we found cells of the myeloid lineage such as early erythroblasts (≤ 6% CD71+), megakaryocytes (≤ 3% CD61+) and monocytes (≤ 7% CD14+) β-globin vector sequences were also detected in the engrafted human cells Our findings demonstrated that we could Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... unrelated to the transplant procedure The third animal at day 46 showed myeloid marking of 35% These data are critical as they suggest that therapeutic levels of lentiviral vector gene transfer... bioavailable antioxidants to reduce acute and late effects of irradiation 262 High Levels of Polyclonal, Multilineage Hematopoietic Cell Gene Marking Following Lentiviral Transduction and Transplantation... compared to those on the regular diet alone (p = 0.020) The best overall (acute and long term) survival was in mice receiving MnSOD-PL and antioxidant diet The data support continually increasing