1057 Efficient Lentivirus Mediated Gene Transfer of an Anti Sickling β Globin Gene Variant to CD34+ Bone Marrow Cells from Sickle Cell Disease Patients Molecular Therapy �������� ��� ���� �����������[.]
STEM CELL GENE THERAPY FOR GENETIC DISEASES transduction of HSC and stability of β87-globin expression in their erythroid progeny produced over a period of several months, lin(~60% CD34+, n=3) CB cells, or FACS-sorted CD34+ (>97% CD34+, n=2) or CD34+CD38- (>97% CD34+CD38-, n=1) CB cells were first prestimulated in serum-free medium containing TPO, Flt3ligand and SCF, then exposed to β87-globin virus for hr on retronectin-coated petri dishes and injected immediately into irradiated NOD/SCID mice (n=35) FACS analysis of bone marrow cells aspirated and 12 wk later showed 33 of the mice (94%) were engrafted with both human lymphoid (1-85%) and myeloid (0.48%) cells This multilineage engraftment with human cells remained stable for another 12 wk in all 10 mice followed for longer periods Thus we did not see any evidence of toxicity of the β87-globin virus DNA isolated from bone marrow cells aspirated from 25 of the 33 engrafted mice (76%) contained transduced cells, as indicated by a strong β87-globin-specific signal detectable by PCR A more quantitative measure of the efficiency of lenti-β87-globin gene transfer to human CB cells that repopulate NOD/SCID mice was obtained by a similar PCR analysis of individual colonies derived from regenerated human CFU-GM and BFU-E Assessment of 40 colonies obtained from human CD45+ cells isolated by FACS - 24 wk posttransplant from 15 engrafted mice (4 independent experiments) indicated an efficiency of β87-globin gene transfer to NOD/SCID repopulating cells of 42 ± 5% Importantly, high levels of β87-globin protein were again demonstrated in erythroblasts produced in vitro from human progenitors from different reconstituted mice isolated - 24 wk post-transplant and cultured without prior selection of transduced human progenitors (30 and 34% at wk, 33% at 16 wk, 10% at 23wk and 59% at 24 wk) These findings provide the first proof-of-principle evidence for the therapeutic potential in primary human cells of a safety-modified lentiviral vector encoding an antisickling β-globin gene Some of the authors are officers or consultants of Genetix Pharmaceutical Ltd 1056 Boosting Respiratory Burst-Positive Granulocytes in Chronic Granulomatous Disease Mice with a Second Generation Selective Amplifier Gene Takeshi Hara,1,2 Akihiro Kume,1 Yutaka Hanazono,1 Hiroaki Mizukami,1 Takashi Okada,1 Hisashi Tsurumi,2 Hisataka Moriwaki,2 Mamoru Hasegawa,3 Keiya Ozawa.1,4 1Division of Genetic Therapeutics, Jichi Medical School, Minamikawachi, Tochigi, Japan; 2First Department of Internal Medicine, Gifu University School of Medicine, Gifu, Gifu, Japan; DNAVEC Research Inc., Tsukuba, Ibaraki, Japan; 4Division of Hematology, Department of Medicine, Jichi Medical School, Minamikawachi, Tochigi, Japan Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytes in producing superoxide and derivative oxidants to kill microbes Responsible mutations are found in one of the four genes encoding essential subunits of the phagocyte NADPH oxidase, and CGD mouse models have been developed by gene targeting Although studies with retroviral vectors have demonstrated functional recovery in murine CGD, phase I clinical trials with analogous vectors showed that the low levels of gene transfer allowed the appearance of limited numbers of corrected neutrophils in the patients Thus, it may be required to boost corrected cells to treat human CGD, up to a sufficient level to clear pathogens For this purpose, we have developed a system for selective expansion of transduced cells In this system, a fusion protein will provide the target cell with a drug-induced growth signal, and the genes encoding such chimera were referred to as selective amplifier gene (SAG) The first generation SAG encoded a fusion protein between the GCSF receptor and the steroid binding domain, and a drug-induced Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy expansion of hematopoietic stem/progenitor cells was achieved In the present study, we tested another switching system for SAG; the extracellular domain of the erythropoietin receptor (EpoR) was fused to the cytoplasmic domain of the thrombopoietin receptor (Mpl) Erythropoietin (Epo), a therapeutic agent highly tolerable with repeated administration, will crosslink the EpoR-Mpl fusion protein to generate a potent growth signal We constructed a bicistronic retrovirus MGK/gp91-IRES-EpoR-Mpl encoding the human gp91 for therapeutic use and the EpoR-Mpl SAG for cell expansion First, IL-3-dependent Ba/F3 cells were transduced and examined for Epo-inducible growth Epo supported the proliferation of transduced Ba/F3 cells as robust as IL-3 indicating a potent growth signal via EpoR-Mpl Next, human gp91 knockout myeloid cells were transduced Recovery of respiratory burst activity in these cells was confirmed by a positive nitroblue tetrazolium reduction test Finally, we addressed whether respiratory burst-positive granulocytes were expandable in an X-CGD mouse model Lethally irradiated X-CGD recipients were transplanted with transduced XCGD marrow After hematological recovery, superoxide production was monitored by flow-cytometry measuring reduced dihydrorhodamine-123 In the animals treated with Epo, the percentage of the oxidase-positive granulocytes was 27.5 ± 6.8 % (n = 4), which was significantly higher than that in the unstimulated mice (8.4 ± 3.1 %) (n = 3) (p < 0.03) This expansion effect was maintained for more than 26 weeks by repeated stimulation So far no hematological abnormality has been observed in the treated animals expect for mild to moderate polycythemia These results suggest that the second generation SAG would augment the efficacy of gene therapy for CGD The EpoR-based fusion system provides a versatile molecular switch, for the ligand is a well characterized drug without a serious adverse effect 1057 Efficient Lentivirus Mediated Gene Transfer of an Anti-Sickling β-Globin Gene Variant to CD34+ Bone Marrow Cells from Sickle Cell Disease Patients Bruno Dalle,1 Emmanuel Payen,1 Robert Pawliuk,2 Karen Westerman,2 Yves Beuzard,1 Philippe Leboulch.1,3 INSERM EMI 0111, Hopital Saint-Louis, Paris, France; Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, United States; 3Department of Medicine, Harvard Medical School and Brigham and Womens Hospital, Boston, MA, United States We have previously demonstrated correction of Sickle Cell Disease (SCD) in transgenic mouse models by Lentivirus mediated transfer of an anti-sickling human β-globin gene variant [βA-T87Q] under the control of large regulatory elements of the human β-globin Locus Control Region (LCR) to hematopoietic stem cells (HSCs) Towards clinical gene therapy for SCD we have investigated the efficacy of our anti-sickling lentiviral vector to transduce CD34+ bone marrow cells isolated from SCD patients and to subsequently express in erythroid progeny VSV-G pseudotyped Lentivirus supernatants were generated by transient transfection of 293T cells with a novel, recently developed multi-plasmid packaging system and concentrated by rounds of ultracentrifugation Virus titer, as determined by quantitative Southern blot analysis, was 8.3 x 108 CFU/ml Bone marrow was isolated from an SCD patient undergoing hip surgery and CD34+ cells purified by immunomagnetic separation Following 16 hours of culture in X-Vivo 20 serum free medium supplemented with Interleukin-3, Flt-3 Ligand, Stem Cell Factor and Thrombopoietin cells were harvested and exposed to concentrated Lentivirus preparations supplemented with the above growth factors and protamine sulfate on Retronectin coated dishes for hours Transduced cells were immediately transplanted into irradiated NOD/SCID recipient mice to assess transduction of NOD/ S407 STEM CELL GENE THERAPY FOR GENETIC DISEASES SCID repopulating cells, or plated in liquid culture or methylcellulose medium to assess gene transfer to LTC-IC and progenitors, respectively Following weeks culture in methylcellulose, individual colonies were plucked and genomic DNA isolated for PCR PCR amplification of vector specific sequences showed integrated βA-T87Q provirus in 62% of colonies analyzed (n = 45) Expression of human βA-T87Q protein in erythroid progenitors was ascertained by HPLC analysis of hemoglobin (Hb) extracted from individual BFU-E (n = 46) On average, HbβA-T87Q was expressed at 24.2 ± 9.2% (SEM) the level of HbS, demonstrating high level expression of the Lentivirus encoded βA-T87Q gene in erythroid progenitors of an SCD patient Assessment of gene transfer efficiency to NOD/SCID repopulating cells and LTC-IC, as well as βA-T87Q expression in the erythroid progeny of these cells is in progress 1058 Cytotoxic T Cell Defects in X-Linked Lymphoproliferative Disease and Reconstitution of Abnormalities by SAP Gene Transfer Reza Sharifi,1 Jo Buddle,1 Kimberly C Gilmour,1 Christine Kinnon,1 Adrian J Thrasher,1 Bobby Gaspar.1 Molecular Immunology Unit, Institute of Child Health, London, United Kingdom X-linked lymphoproliferative (XLP) disease is a rare immunodeficiency characterised by a dysregulated immune response to Epstein-Barr infection leading to fulminant infectious mononucleosis, lymphoma development or agammaglobulinaemia The gene defective in XLP has recently been identified and designated as SAP for SLAM (signalling lymphocyte associated molecule) Associated Protein The cellular pathogenesis of XLP is unclear and in particular abnormalities in the cytotoxic T cell (CTLs) response has not been clearly demonstrated In this study we analysed cytokine responses and cytolytic activity in CTLs from XLP patients (n=2) and compared them to CTLs from normal donors (n=2) We then used a spleen focus forming (SFFV) retroviral construct encoding the SAP gene (in combination with eGFP for selection of transduced cells) to transduce XLP CTLs to determine whether SAP gene transfer could reconstitute observed functional defects We found that CTLs from XLP patients showed the same phenotypic profile as CTLs from normal donors and thus demonstrated that XLP patients can indeed generate EBV-specific CTLs Stimulation of cells by a variety of stimuli including PMA, CD3, CD28, CD3/CD28, SLAM, CD3/SLAM and analysis of INFγ expression using ELISPOT assays showed no difference between XLP patients and normal donors However, when stimulated by CD3/2B4, 2B4 alone and in particular autologous EBV transformed lymphoblastoid cell lines (LCLs), there was a marked decrease in INF-γ production We then tested the ability of EBV-CTLs to kill autologous LCLs using standard LDH release assays Again, XLP CTLs showed markedly decreased cytolytic activity XLP-CTLs transduced by the SAP retroviral vector after stimulation with autologous LCLs and IL-2 were selected and tested for functionality We observed that introduction of SAP restored the ability of XLPCTLs to produce INF-γ in response to CD3/2B4, 2B4 and autologous LCL stimulation and also restored cytolytic activity against autologous LCLs These experiments demonstrate for the first time in a physiologically relevant human model, functional defects of T effector cells in XLP and also show that these defects are SAP dependent and can be restored by SAP gene transfer S408 1059 Gene Therapy of Chronic Granulomatous Disease: Results from Pre-Clinical and Phase I Clinical Studies Marion G Ott,1 Ulrike Koehl,2 Mohammed A Sadat,3 Heike Merget-Millitzer,4 Stefan Stein,5 Sandra Saulnier,6 Johann Peter Hossle,6 Mary C Dinauer,3 Reinhard Seger,6 Dieter Hoelzer,1 Manuel Grez.5 Hematology/Oncology, University Hospital, JW-Goethe University, Frankfurt, Germany; 2Pediatric Hematology/ Oncology, University Hospital, JW-Goethe University, Frankfurt, Germany; 3Pediatric Hematology/Oncology, Wels Center for Pediatric Research, Indiana University School, Indianapolis, IN, United States; 4MainGen Biotechnology, Frankfurt, Germany; Molecular Virology, Georg-Speyer-Haus, Frankfurt, Germany; Immunology/Hematology, Childrens Hospital, University of Zurich, Zurich, Switzerland Chronic Granulomatous Disease (CGD) as a molecular defined genetic defect of phagocytes is an excellent candidate for a gene replacement therapy, in which autologous hematopoietic or myeloid stem cells are genetically modified and reintroduced into affected patients As part of our efforts to develop strategies for somatic gene therapy of the X-linked form of CGD (X-CGD), we developed a bicistronic retroviral vector (SPsLdS) containing elements of the murine stem cell virus (MSCV) and the spleen focus-forming virus (SFFV) to drive expression of both the human gp91phox gene and a truncated form of the low affinity nerve growth factor receptor (dLNGFR) as a selectable marker Pre-clinical data was obtained in a murine model of X-CGD Bone marrow cells from X-CGD mice were transduced with SPsLdS and transplanted either with or without prior sorting for dLNGFR expressing cells into lethally irradiated syngeneic animals Expression of gp91phox and dLNGFR was maintained for at least 12 months in primary transplants, and was detected in secondary and tertiary transplanted animals, consistent with transduction of long-term repopulating cells dLNGFR was detected on granulocytes, B-lymphocytes, and erythrocytes, but was less in T lymphocytes Silencing of gene expression was observed in some secondary and tertiary transplanted animals No obvious adverse consequences of transgenic protein expression were observed Based on this and other in vitro experiments a Phase I clinical trial was initiated G-CSF mobilized peripheral blood CD34+ cells were obtained from a 30 years old X-CGD patient 3x108 selected CD34+ cells were transduced in retronectin-coated bags in the presence of X-VIVO10 containing SCF, Flt3-L, TPO and IL6 The transduction efficiency was 49,3% as estimated from dLNGFR expression Cells were frozen after transduction until safety testing was completed (3 months) Cells were transfused into the patient in June, 2002 at a cell dose of 6.2 x 106 cells per kg (4.2 x 108 total cell numbers) Prior to transplantation, the patient was subjected to a nonmyeloablative conditioning with endoxan given at a dose of 1g/ m²/day at three consecutive days starting at day -4 The patient had no major side effects from endoxan and recovered within days to absolute neutrophil count (ANC) >500 cells per μl The percentage of gp91phox positive cells in peripheral blood ranged between 1% at day and 0.002% at day 61 as estimated by TaqMan PCR A weak dLNGFR signal was detected in 0.2% of the leukocytes at day 211 Similarly, NADPH oxidase activity was detected at low levels in a small fraction of the granulocytes The patient is well with no sign of bacterial or fungi infections Toxicity due to transgene expression has not been observed Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy ...STEM CELL GENE THERAPY FOR GENETIC DISEASES SCID repopulating cells, or plated in liquid culture or methylcellulose medium to assess gene transfer to LTC-IC and progenitors, respectively... of HbS, demonstrating high level expression of the Lentivirus encoded βA-T87Q gene in erythroid progenitors of an SCD patient Assessment of gene transfer efficiency to NOD/SCID repopulating cells. .. cells in XLP and also show that these defects are SAP dependent and can be restored by SAP gene transfer S408 1059 Gene Therapy of Chronic Granulomatous Disease: Results from Pre-Clinical and Phase