263 Long Term Multi Lineage Engraftment of NOD scid IL2Rgnull Mice after Transplantation with Genetically Modified b thalassemic CD34+ Cells Molecular Therapy Volume 19, Supplement 1, May 2011 Copyrig[.]
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 HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I achieve long-term multi-lineage engraftment using transduced patient specific thalassemic G-CSF mobilized CD34+ cells expressing human beta-globin These studies provide the basis for the upcoming phase I clinical trial of β-thalassemia major using LV transduced CD34+ cells encoding beta-globin 264 Uncovering Haemopoietic System Dynamics and Tracking Fate and Long-Term Survival of Single Progenitor Clones In Vivo in Humans by Retroviral Tagging Luca Biasco,1 Cristina Baricordi,2 Cynthia Bartholomae,3 Immacolata Brigida,1 Samantha Scaramuzza,1 Raffaele Fronza,4 Stefania Merella,1 Alessandro Ambrosi,5 Danilo Pellina,5 Clelia Di Serio,2,5 Eugenio Montini,1 Christof von Kalle,4 Manfred Schmidt,4 Alessandro Aiuti.1,6 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 2Vita-Salute University San Raffaele, Milan, Italy; National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany; 4Alma Mater Studiorum University, Bologna, Italy; 5CUSSB Vita-Salute University, Milan, Italy; 6University of Rome “Tor Vergata”, Rome, Italy Upon retroviral mediated gene correction of haemopoetic stem cells (HSC) each transduced progenitor is univocally marked by an integration site Long-term studies in gene therapy (GT) patients allow for the first time to track haemopoietic clonal dynamics and survival of single progenitor clones in humans by retroviral tagging We previously showed that ADA-SCID GT with CD34+ cells combined to reduced intensity conditioning resulted in full multilineage engraftment, in the absence of aberrant expansions We now performed a comprehensive multilineage longitudinal insertion profile of bone marrow (BM) (CD34+, CD15+, CD19+, Glycophorin+) and peripheral blood (PB) (CD15+, CD19+, CD4+, CD8+ cells, naïve and memory T cells) cells in patients 3-6 years after GT, retrieving to date 1055 and 1999 unique insertions from BM and PB cell lineages respectively We could shape the “insertional landscape” of each lineage through a tri-factorial analysis based on insertional variables allowing to uncover the effects of selective advantages of gene-corrected cells in the periphery and the frequency of identical integrants in different haematopoietic compartments BM cells and PB granulocytes displayed the highest proportion of shared integrants (up to 58.1%), reflecting the real-time repopulating activity of genecorrected progenitors Strikingly, we detected “core integrants”, shared between CD34+ cells and both lymphoid and myeloid lineages at multiple time points, stably tagging active long-term multipotent progenitors overtime Tracking two of these integrants (inside a fragile site of MLLT3 gene and downstream the LRRC30 gene) by specific PCRs we confirmed the multilineage contribution to haematopoiesis of these progenitor clones, showing fluctuating lineage outputs over a period of years in the patient We also retrieved 170 integrations from T cell subtypes (Naive, TEMRA, Central and Effector memory) from one patient who received infusions of transduced PBL showing evidences that naive T cell clones may survive in humans for up to 10 years while maintaining their differentiation capacity into different T cell subpopulations Additional analyses on transcriptional and chromatin status of integration sites collected from all lineages are ongoing In conclusion, through retroviral tagging, we can uniquely track single transduced haemopoietic cells directly in vivo in humans The application of mathematical models to our insertion datasets is allowing to uncover new information on the fate and activity of haematopoietic progenitors and their differentiated progeny years after transplantation in GT patients Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 265 Deep Sequencing Analyses of Small RNAs in HIV-1 Infected Cells Transduced with a Triple Inhibitory RNA Lentiviral Vector Haitang Li,1 John Rossi.1 Molecular and Cell Biology, City of Hope, Duarte, CA In an effort to develop a hematopoietic gene therapy approach to treat HIV infection, we created a lentiviral vector containing three different inhibitory agents including a short hairpin RNA (shRNA) targeting the HIV tat/rev region, a nucleolar localizing Tat binding decoy and a ribozyme against the endogenous CCR5 chemokine receptor mRNA This lentiviral construct, has proven to be highly effective in inhibiting HIV replication in cell culture and is currently in use in gene therapy trial for AIDS/lymphoma patients The potential deregulation of miRNA expression by ectopic expression of a short hairpin RNA or HIV-1 infection has been a matter of concern for gene therapy applications of RNAi We therefore took advantage of next generation sequencing to monitor endogenous microRNA changes during the time course of HIV-1 infection in CEM T-cells transduced with the triple construct vector in the presence and absence of HIV-1 infection Consistent with the idea that HIV replication was effetely inhibited, microRNAs generated from HIV-1 TAR and Nef were greatly reduced in triple construct treated CEM cells relative to vector backbone alone transduced cells, HIV-1 infected cells Interestingly, the level of several endogenous microRNAs changed dramatically at different time points in the face of HIV-1 challenge, but the changes were less dramatic in cells expressing the anti-tat/rev shRNA We also analyzed the ratios of the anti-HIV tat/rev guide strand versus the passenger strand over time in the triple construct transduced cells infected with HIV versus the uninfected triple construct transduced cells Our results show that the guide strand/passenger strand ratio increases dramatically in HIV-1 infected cells whereas it remains stable in uninfected vector transduced cells These results support a model in which engagement of an siRNA guide strand via targeting of the HIV transcripts leads to its stabilization Finally, we analyzed the microRNA profile from a patient in the clinical trial at two time points-one pre-transduction and the other one year post transduction and post autologous stem cell transplant The results of these analyses show marked changes in the miRNA profile as well, most likely a consequence of the stem cell transplant We also see the HIV-1 TAR miRNA pre-transplant but it is absent post-transplant/transduction In summary, our results provide the first in depth look at the microRNA profiles of HIV-1 infected cells in the presence of a protective gene therapy vector 266 Decade-Long Persistence of Adoptively Transferred Autologous Redirected CD4ζ Chimeric Receptor T Cells in HIV+ Study Subjects John Scholler,1 Naomi Aaronson,2 Gabriela Plesa,1 Richard Carroll,1 Gwendolyn Binder,1 Kristen Hege,3 Steven Deeks,4 Ronald Mitsuyasu,5 Ashley Vogel,1 Zhaohui Zheng,1 Julio Cotte,1 Wendy Bernstein,6 Troy Brady,1 Bruce Levine,1 Carl June.1 University of Pennsylvania, Philadelphia, PA; 2Walter Reed Army Medical Center, Washington, DC; 3Cell Genesys, South San Francisco; 4University of California, San Francisco; 5University of California, Los Angeles; 6National Cancer Institute, Bethesda The ability of adoptively transferred T cells to persist long term and function in a homologous or enhanced fashion is a central goal of immunotherapy To test the hypothesis that genetically modified T cells can have long term persistence, we measured the frequency of CD4ζ modified T cells in patients with HIV in three trials following adoptive transfer of CD4 and CD8 T cells transduced with the CD4ζ chimeric receptor over time We developed a quantitative PCR (qPCR) assay to measure the persistence of adoptively transferred T lymphocytes transduced with a chimeric receptor consisting of S103 ... the absence of aberrant expansions We now performed a comprehensive multilineage longitudinal insertion profile of bone marrow (BM) (CD34+, CD15+, CD19+, Glycophorin+) and peripheral blood (PB)... THERAPY I achieve long- term multi- lineage engraftment using transduced patient specific thalassemic G-CSF mobilized CD34+ cells expressing human beta-globin These studies provide the basis for the... survival of single progenitor clones in humans by retroviral tagging We previously showed that ADA -SCID GT with CD34+ cells combined to reduced intensity conditioning resulted in full multilineage engraftment,