- Email: [email protected]
247. Long-Term Efficacy and Safety of Hematopoietic Stem Cell Gene Therapy in the Murine Model of Wiskott-Aldrich Syndrome
spleen of injected animals. 30-35% offemoral BM cells expressed AUI at 2w and 56w after receiving intramarrow SV(RNAiR51 RevM10). Percentages of marrow cells expressing AUI in other bones that were not injected with the vector (humerous, tibia and iliac crest) varied: in the humorus II % were positive for AUI; in the tibia, 31%; and in the iliac crest, 45%. The greatest percentages of AUI-positive cells were in granulocyte series and in cells that expressed stem cell antigen (SCA). Thus, 73.6% ofSCA+ cells in the femur expressedAU I 56 weeks after intramarrowinjection. At 56 weeks after intrafemoral injection with SV(RNAiR5/RevMIO), the percentagesofSCA-positive cells in the various bonesthat were also AUI-positive were: in the humerus, about 32%; in the tibia, 61.5%; and in the iliac crest 78.4%. Other organs were studied for AU1 expression 56 weeks after BM injection. Of note, about 50% of spleen cells, includingT lymphocytesof all lineages, B cells and cells bearing CDI4 macrophage/monocyte marker. Thus, direct intramarrow injection ofrSV40 vectors in vivo is highly effective in transducing HSC and preserving transgene expression in their differentiated progeny, both in the blood and in the tissues. The success of this approach lies in the high transduction efficiency of rSV40s for resting HSC and continued transgene expression in daughter cells for extended periods of time.
246. Very High Levels of Expression of the 0 6 Methylguanine-DNA-Methyltransferase P140K Mutant Results in an In Vivo Engraftment Defect
modified HSC. In competitive repopulation assays in which there was no 6BG/temozoiomide treatment, cells harboring SF-MGMT demonstrated an approximate 50% reduction in the production of gene marked peripheral bloodcells over the course of6 months(sec table). In contrast, cells expressing EFS or PGK-MGMTgenerated stable grafts over the same time period. We propose that the high level of MGMT(PI40K) expression driven by the SF promoter has a subtle yet reproducible deleteriouseffect upon hematopoiesisand that the use of weaker promoter elements should be considered for clinical applications. Compctetive engraftment of MGMT transducedcells . ~. .~ _~ Days. post-trans plan t ( 5..d.: ~ 7 0 .d __II.I." d .142d_ .p ..Id --; EFS,.MGMT 100-----l-100----490 --: 1 2 ~ I ~ Q----l PGK-MGMT 100 98---..479 93--\90 SF-MGMT 100- - '89 59 ' 4 8~ L:::J Chimerism expressed as percentageof cells in peripheral blood at 35d post-transplant
247. Long-Term Efficacy and Safety of Hematopoietic Stem Cell Gene Therapy in the Murine Model of Wiskott-Aldrich Syndrome
Francesco Marangoni.P Marita Bosticardo.! Samantha Scararnuzza,' Cristina Panaroni,' Sara Trifari,' Michela Locci,' Elena Draghici,I Anne Galy,' Luigi Naldini,1.2AlessandroAiuti,I Lore Dupre,1.4 Anna Villa.l-' Maria-Grazia Roncarolo.'-'
Michael D. Milsom,' Chad E. Harris,' Axel Schambach,' Jeff Bailey,' Emily Broun; Christopher Baum.P Geoffrey P. Margison,' Ina Rattmann,' Moran Jerabek-Willemsen;' Thomas Moritz.l-' Jurgen Thomale,' Elke Grassman; David A. Williams.'
'San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy; lVita-Salute San Raffaele University. San Raffaele Scientific Institute, Milan, Italy; JUMR CNRS 8115, Genethon, Evry, France; 4INSERM U563, Purpan University Hospital, Toulouse, France; JHuman Genome Department, Istituto di Tecnologie Biomediche (CNR-ITB), Segrate, Milan, Italy.
The drug rcsistaneegene ~mcthyguaninc-DNA-methyltransfer ase (MGMT) is of particular importancein the fieldof gene therapy due to its proposed use in hematopoietic stem cell (I-ISC) chemoprotective/chemoselectivestrategies. OverexpressionofMGMT in HSC confers protection against ~ alkylating agents to HSC and their matureprogeny. Since one MGMTmoleculecan only detoxify adduct, it has been previouslyassumedthat a single ~alkylguanine elevated expressionofMGMT (or mutantversionsofMGMT which are resistant to pseudosubstrateinhibitorssuch as ~benzylguanine (6BG), e.g. P140K) is beneficialfor HSC protection. Weattempted to assess whetherthe magnitudeofMGMT(P 140K)overexpression had any bearingupon HSC fate.Self inactivatingy-retroviral vectors wereconstructedwhichcontainedthe MGMT(P140K)cDNAdriven by either the spleen focus forming virus promoter (SF-MGMT) or the weaker human cellular promoters: elongation factor-In short form (EFS-MGMT) or phosphoglycerate kinase (PGK-MGMT). In vitro assays using 32D cells reproducibly demonstrated a proliferation defect in SF-MGMT transduced cells compared to EFS or PGK-MGMTtransduced cells. In primal)' murine bone marrow cells (BMC) the level of MGMT activity was 2169, 287 and 330 fmol/mg DNA for cells transduced with SF, EFS and PGK-MGMT respectively, compared to no detectable activity in control transduced BMC. We found that the approximate 7-fold lower level of MGMT activity mediated by the EFS and PGK driven vectors was still sufficient to protect progenitor cells against treatment with adducts formed 6BG/temozolomide, detoxify ~-methylguanine in vivo, and importantly also facilitated in vivo selection of gene
Wiskott-Aldrichsyndrome(WAS)is a severe X-linked immunodeficiencycharacterizedby recurrentinfections,thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. Hematopoietic stem cell (HSC) transplantation is the current therapy, however, it is not available tor all patients tor the lack of HLA-matched donors. Transplantation of genetically corrected autologous HSC could represent an alternative treatment. In a murine model of WAS (lJ'IIS"), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupre, Marangoni et al., Hum Gene Ther. 2006, 17]. The aim of the present study was to investigate the long-term efficacy and safety of our gene therapy approach in WAS' mice. Transduction of JE4S· HSC was performed with a Ientiviralvector encoding human WASPunder the control ofa 1.6Kb fragmentof the human WAS autologous promoter.Transduced HSC were transplanted into sub-lethally irradiated 11'1105" mice. Mice were sacrificed 7 and 12 months after gene therapy. Donor engraftment in the bone marrow and splenic T cells was >80%. WASP expression was detected in 30% of bone marrow CD45+ cells, and in similar percentages of peripheral myeloid cells. In the spleen, 75% ofT cells and 50% of B cells expressed WASP, suggesting a selective advantage for gene corrected cells in these lineages. Importantly, functional correction of splenic T lymphocytes after gene therapy was documented by the complete restoration ofTCR-driven proliferation and cytokine production.Safety of gene therapywas also demonstrated.No overexpression of WASPwas observed in bone marrow and splenic T cells, regardless of the lentiviral vector copy number. Absence of leukemias or lymphomas was demonstrated by blood count and FACS analysis performed on blood and spleen, Histopathologic analysis of thymus, spleen, lymph nodes and bone marrow did not show any abnormalities. Long-term survival of the gene therapy treated mice was comparable to that of control mice transplanted with untransduced WAS'-HSC. In conclusion, we provideevidence of engraftment of WASP-expressing cells and restoration ofTCRdriven T cell proliferation without toxicity, up to 12 months after
I Department ofExperimental Hematology, Cincinnati Children s Hospital Medical Center, Cincinnati; lDepartment ofExperimental Hematology. Hannover Medical School. Hannover, Germany; JCarcinogenesis Group, Paterson Institute for Cancer Research. Manchester; United Kingdom; 'lnternal Medicine (Cancer Research), University ofDuisburg-Essen Medical School, Essen, Germany; Jlnstitute ofCell Biology. University ofDuisburg-Essen Medical School, Essen, Germany.
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Molecular Therapy Volume 15.Supplement I•.\by 20m Copyright © The American Society o t Gene Therapy
HSC gene therapy. Experiments aimed at investigating whether II"lS gene therapy can correct the defects ofplatelets, B cells and dendritic cells, and restore normal in vivo immune response to antigens are ongoing. Results from these studies will contribute to the design of a clinical trial for Wiskott-Aldrich syndrome.
248. Evidence of Globin Lentiviral VectorInduced Genotoxicity in a Murine Model of Sickle Cell Disease Pestina I. Pestina,' Hargrove W. Hargrove ,' Huifen Zhao; Kumar Srivastava,' Gray T. John, I Derek A. Persons .' 1Hematology. St. Jude Children s Research Hospital, Memphis , TN; 2Department ofBiostatistics, St. Jude Children's Research Hospital, Memphis. TN. Previously, we evaluated the therapeutic efficacy of a 3'd/4~' generation HIV-bascd, SIN lentiviral vector encoding the human y-globin gene driven by a ~-globin promoter and 3 kb of elements from the ~-globin LCR in murine ~-thalassemia (Hanawa et aI., Blood 104:2281-2290; 2004). This vector corrected the thalassemic phenotype at relatively low average vector copy number (1.3). Here, we describe testing ofthis vector containing the 250 bp x 2 HS4 core insulator fragment from the chicken ~.globin Locus Control Region in a " double copy" LTR format in the BERK sickle cell disease (SCD) mouse model. This vector and a control vector containing an MSCV LTR-driven YFP marker gene were used to transduce lineage negative SCD bone marrow (BM) . Thirteen of 16 CFU-S clones derived from cells transduced with the globin vector were positive for the vector genome by Southern blot analysis, consistent with high efficiency gene transfer into primitive cells. Lethally irradiated wild-type recipients (n = 10) of the same transduced SCD cell population expressed human y-globin with HbF'"cells ranging from 1-23% at 12 weeks post-transplant. Although the overall Hb level in these mice was not yet significantly improved, relative to the YFP transplanted control animals , the number of irreversibly sickle cells (ISCs) per high powered field (PHPF) was significantly reduced in the globin vector animals relative to YFP control animals (mean of2.2 ± 0.4 ISCslPHPF vs. 6.6 ±0.9; p < 0.009) . Further molecular analysis of the above globin vector-transduced CFU-S with indcpendent Southern blot analyses using different restriction enzymes showed an identical banding pattern in all 13 independently derived clones, consistent with 2 integrated copies of the vector. Assuming equal growth kinetics ofall transduced CPU-S, the probability ofthe same clone being identified 13 times is 1.8 x 10-34• This suggested that a significant growth advantage was endowed on this particular transduced, CFU-S-forming cell _LM-PCR analysis of several of the isolates ofthis clone, coupled with Blast sequence analysis using the NCB! mouse genome database, independently confirmed that the same genomic DNA-vector junction fragments were present in all, with one ofthe vector insertions being an intergenie integrantlocated in a gene dense region on chromosome 9. Some ofthe genes nearby the vector insertion included Myo Ie, a Rho GTPase which functions in intracellular signaling (70 kb away) , Ccnb2 (cyclin B2), a cyclindependent kinase important in controlling cell cycle progression (180 kb away) , and Bnip2, a Bcl-2 interacting protein (230 kb away) . The 250 bp x 2 HS4 core insulator fragm ent was completed deleted in the CFU-S clones as well as in independently transduced NIH3T3 cells . These results suggest that the 2 x 250 bp configuration of the insulator element is unstable . The globin LCR elements appear to have potential genotoxicity in primitive hematopoietic cells leading to the emergence of clonal dominance during in vitro and/or in vivo cell division while forming spleen colonies.
Molecular Therapy Volume15.Suppkmcnll. M,')'2007
Cop)'rihht © The American Scciery of Gene "Il ler-Ill)'
249. Gene Therapy in Adenosine DeaminaseDeficient Children with Autologous Bone Marrow Cells Transduced with Two Retroviral Vectors after Withdrawal of Enzyme Replacement Treatment and Low-Dose Chemotherapy
Robert A. Sokol ic,I Greg Podsakoff, ' Linda Muu!, I Barbera Engel,' Elizabeth K. Garabedian; Denise Carbonaro.' Joanna Ireland.' Laura M. Tuschong,? Michael S. Hershfield,' Donald B. Kohn,' Fabio Candotti.' JDisorders 0/ Immunity Section , Genetics and Molecular Biology Branch. National Human Genome Research Institute, Bethesda. MD; lDivision ofResearch Immunology/Bon e Marrow Transplant, The Saban Research Institute ofChildrens Hospital Los Angeles. Department 0/Pediatrics, Los Angeles, CA; "Expertmen tal Transplantation and Immunology Branch, National Cancer Institute. Bethesda, MD; 'Department ofMedicine, Duke University Medical Center, Durham, NC. Adenosine deaminase (ADA) deficiency can result in severe combined immunodeficiency (SCID), a life-threatening pediatric emergency. Enzyme replacement treatment with polyethylene glycol-conjugated bovine ADA (PEG-ADA) is life-saving, but it usually does not restore normal immunity, its efficacy may decline with time and its costs are often prohibitive outside the USA . Allogeneic hematopoietic cell transplantation is curative, but a matched related donor is available for only a minority of patients , and alternative donor transplants carry a high rate oftoxicity. For these reasons, gene therapy has long been proposed as an alternative form of treatment for this disease and has recently proven beneficial in Europe and Japan. In November, 2005 we began a clinical gene therapy trial based on infusion of genetically corrected autologous bone marrow cells after withdrawal of PEG-ADA and after treatment with rnyclosupprcssive chemotherapy. The first treated patient (ADA302N) suffered from an unexpected prolonged marrow aplasia due to pre-existing trisomy 8 abnormalities of her bone marrow cells. No engrafiment ofgene corrected cells was documented, as reported (Blood 2007; 109:503). Our second treated patient (ADA-30IN) is a 2-year-old boy who was admitted to the NIH Clinical Center in January, 2007. Bone marrow harvest yielded 92.5x 10"6 CD34+ cells that, after pre-stimulation with SCF, MGDF and Flt3-L, were divided into two aliquots and separately transduced with the MND-ADA and GCsapM-ADA retroviral vectors . Two days before infusion of gene corrected cells, the patient received busulfan (37.5 mg/m2 IV x 2). Areas under the curve for the two doses were 1451 and 1583 mcM/minute, respectively. On day 0, the patient received 5.lx I0"6 CD34+ cell/kg. ADA activity in the GCsapM-ADA and MND-ADA transduced cells was >200 and 43 nmol/min/10"8 cells, respectively (ADA activity in healthy control CD34+ cells = -70 nmol/minll 0"8 cells) . Chemotherapy was well tolerated . The absolute neutrophil count fell below 50D/mcLon day 12, at which time the platelet count was still within normal limits. It is hoped that this protocol will allow us to follow the engraftrnent of cells corrected by each vector and determine if either one ultimately contributes more strongly to the recovery of lymphoid immunity.
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246. Very High Levels of Expression of the 0 6 Methylguanine-DNA-Methyltransferase P140K Mutant Results in an In Vivo Engraftment Defect
modified HSC. In competitive repopulation assays in which there was no 6BG/temozoiomide treatment, cells harboring SF-MGMT demonstrated an approximate 50% reduction in the production of gene marked peripheral bloodcells over the course of6 months(sec table). In contrast, cells expressing EFS or PGK-MGMTgenerated stable grafts over the same time period. We propose that the high level of MGMT(PI40K) expression driven by the SF promoter has a subtle yet reproducible deleteriouseffect upon hematopoiesisand that the use of weaker promoter elements should be considered for clinical applications. Compctetive engraftment of MGMT transducedcells . ~. .~ _~ Days. post-trans plan t ( 5..d.: ~ 7 0 .d __II.I." d .142d_ .p ..Id --; EFS,.MGMT 100-----l-100----490 --: 1 2 ~ I ~ Q----l PGK-MGMT 100 98---..479 93--\90 SF-MGMT 100- - '89 59 ' 4 8~ L:::J Chimerism expressed as percentageof cells in peripheral blood at 35d post-transplant
247. Long-Term Efficacy and Safety of Hematopoietic Stem Cell Gene Therapy in the Murine Model of Wiskott-Aldrich Syndrome
Francesco Marangoni.P Marita Bosticardo.! Samantha Scararnuzza,' Cristina Panaroni,' Sara Trifari,' Michela Locci,' Elena Draghici,I Anne Galy,' Luigi Naldini,1.2AlessandroAiuti,I Lore Dupre,1.4 Anna Villa.l-' Maria-Grazia Roncarolo.'-'
Michael D. Milsom,' Chad E. Harris,' Axel Schambach,' Jeff Bailey,' Emily Broun; Christopher Baum.P Geoffrey P. Margison,' Ina Rattmann,' Moran Jerabek-Willemsen;' Thomas Moritz.l-' Jurgen Thomale,' Elke Grassman; David A. Williams.'
'San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy; lVita-Salute San Raffaele University. San Raffaele Scientific Institute, Milan, Italy; JUMR CNRS 8115, Genethon, Evry, France; 4INSERM U563, Purpan University Hospital, Toulouse, France; JHuman Genome Department, Istituto di Tecnologie Biomediche (CNR-ITB), Segrate, Milan, Italy.
The drug rcsistaneegene ~mcthyguaninc-DNA-methyltransfer ase (MGMT) is of particular importancein the fieldof gene therapy due to its proposed use in hematopoietic stem cell (I-ISC) chemoprotective/chemoselectivestrategies. OverexpressionofMGMT in HSC confers protection against ~ alkylating agents to HSC and their matureprogeny. Since one MGMTmoleculecan only detoxify adduct, it has been previouslyassumedthat a single ~alkylguanine elevated expressionofMGMT (or mutantversionsofMGMT which are resistant to pseudosubstrateinhibitorssuch as ~benzylguanine (6BG), e.g. P140K) is beneficialfor HSC protection. Weattempted to assess whetherthe magnitudeofMGMT(P 140K)overexpression had any bearingupon HSC fate.Self inactivatingy-retroviral vectors wereconstructedwhichcontainedthe MGMT(P140K)cDNAdriven by either the spleen focus forming virus promoter (SF-MGMT) or the weaker human cellular promoters: elongation factor-In short form (EFS-MGMT) or phosphoglycerate kinase (PGK-MGMT). In vitro assays using 32D cells reproducibly demonstrated a proliferation defect in SF-MGMT transduced cells compared to EFS or PGK-MGMTtransduced cells. In primal)' murine bone marrow cells (BMC) the level of MGMT activity was 2169, 287 and 330 fmol/mg DNA for cells transduced with SF, EFS and PGK-MGMT respectively, compared to no detectable activity in control transduced BMC. We found that the approximate 7-fold lower level of MGMT activity mediated by the EFS and PGK driven vectors was still sufficient to protect progenitor cells against treatment with adducts formed 6BG/temozolomide, detoxify ~-methylguanine in vivo, and importantly also facilitated in vivo selection of gene
Wiskott-Aldrichsyndrome(WAS)is a severe X-linked immunodeficiencycharacterizedby recurrentinfections,thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. Hematopoietic stem cell (HSC) transplantation is the current therapy, however, it is not available tor all patients tor the lack of HLA-matched donors. Transplantation of genetically corrected autologous HSC could represent an alternative treatment. In a murine model of WAS (lJ'IIS"), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupre, Marangoni et al., Hum Gene Ther. 2006, 17]. The aim of the present study was to investigate the long-term efficacy and safety of our gene therapy approach in WAS' mice. Transduction of JE4S· HSC was performed with a Ientiviralvector encoding human WASPunder the control ofa 1.6Kb fragmentof the human WAS autologous promoter.Transduced HSC were transplanted into sub-lethally irradiated 11'1105" mice. Mice were sacrificed 7 and 12 months after gene therapy. Donor engraftment in the bone marrow and splenic T cells was >80%. WASP expression was detected in 30% of bone marrow CD45+ cells, and in similar percentages of peripheral myeloid cells. In the spleen, 75% ofT cells and 50% of B cells expressed WASP, suggesting a selective advantage for gene corrected cells in these lineages. Importantly, functional correction of splenic T lymphocytes after gene therapy was documented by the complete restoration ofTCR-driven proliferation and cytokine production.Safety of gene therapywas also demonstrated.No overexpression of WASPwas observed in bone marrow and splenic T cells, regardless of the lentiviral vector copy number. Absence of leukemias or lymphomas was demonstrated by blood count and FACS analysis performed on blood and spleen, Histopathologic analysis of thymus, spleen, lymph nodes and bone marrow did not show any abnormalities. Long-term survival of the gene therapy treated mice was comparable to that of control mice transplanted with untransduced WAS'-HSC. In conclusion, we provideevidence of engraftment of WASP-expressing cells and restoration ofTCRdriven T cell proliferation without toxicity, up to 12 months after
I Department ofExperimental Hematology, Cincinnati Children s Hospital Medical Center, Cincinnati; lDepartment ofExperimental Hematology. Hannover Medical School. Hannover, Germany; JCarcinogenesis Group, Paterson Institute for Cancer Research. Manchester; United Kingdom; 'lnternal Medicine (Cancer Research), University ofDuisburg-Essen Medical School, Essen, Germany; Jlnstitute ofCell Biology. University ofDuisburg-Essen Medical School, Essen, Germany.
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Molecular Therapy Volume 15.Supplement I•.\by 20m Copyright © The American Society o t Gene Therapy
HSC gene therapy. Experiments aimed at investigating whether II"lS gene therapy can correct the defects ofplatelets, B cells and dendritic cells, and restore normal in vivo immune response to antigens are ongoing. Results from these studies will contribute to the design of a clinical trial for Wiskott-Aldrich syndrome.
248. Evidence of Globin Lentiviral VectorInduced Genotoxicity in a Murine Model of Sickle Cell Disease Pestina I. Pestina,' Hargrove W. Hargrove ,' Huifen Zhao; Kumar Srivastava,' Gray T. John, I Derek A. Persons .' 1Hematology. St. Jude Children s Research Hospital, Memphis , TN; 2Department ofBiostatistics, St. Jude Children's Research Hospital, Memphis. TN. Previously, we evaluated the therapeutic efficacy of a 3'd/4~' generation HIV-bascd, SIN lentiviral vector encoding the human y-globin gene driven by a ~-globin promoter and 3 kb of elements from the ~-globin LCR in murine ~-thalassemia (Hanawa et aI., Blood 104:2281-2290; 2004). This vector corrected the thalassemic phenotype at relatively low average vector copy number (1.3). Here, we describe testing ofthis vector containing the 250 bp x 2 HS4 core insulator fragment from the chicken ~.globin Locus Control Region in a " double copy" LTR format in the BERK sickle cell disease (SCD) mouse model. This vector and a control vector containing an MSCV LTR-driven YFP marker gene were used to transduce lineage negative SCD bone marrow (BM) . Thirteen of 16 CFU-S clones derived from cells transduced with the globin vector were positive for the vector genome by Southern blot analysis, consistent with high efficiency gene transfer into primitive cells. Lethally irradiated wild-type recipients (n = 10) of the same transduced SCD cell population expressed human y-globin with HbF'"cells ranging from 1-23% at 12 weeks post-transplant. Although the overall Hb level in these mice was not yet significantly improved, relative to the YFP transplanted control animals , the number of irreversibly sickle cells (ISCs) per high powered field (PHPF) was significantly reduced in the globin vector animals relative to YFP control animals (mean of2.2 ± 0.4 ISCslPHPF vs. 6.6 ±0.9; p < 0.009) . Further molecular analysis of the above globin vector-transduced CFU-S with indcpendent Southern blot analyses using different restriction enzymes showed an identical banding pattern in all 13 independently derived clones, consistent with 2 integrated copies of the vector. Assuming equal growth kinetics ofall transduced CPU-S, the probability ofthe same clone being identified 13 times is 1.8 x 10-34• This suggested that a significant growth advantage was endowed on this particular transduced, CFU-S-forming cell _LM-PCR analysis of several of the isolates ofthis clone, coupled with Blast sequence analysis using the NCB! mouse genome database, independently confirmed that the same genomic DNA-vector junction fragments were present in all, with one ofthe vector insertions being an intergenie integrantlocated in a gene dense region on chromosome 9. Some ofthe genes nearby the vector insertion included Myo Ie, a Rho GTPase which functions in intracellular signaling (70 kb away) , Ccnb2 (cyclin B2), a cyclindependent kinase important in controlling cell cycle progression (180 kb away) , and Bnip2, a Bcl-2 interacting protein (230 kb away) . The 250 bp x 2 HS4 core insulator fragm ent was completed deleted in the CFU-S clones as well as in independently transduced NIH3T3 cells . These results suggest that the 2 x 250 bp configuration of the insulator element is unstable . The globin LCR elements appear to have potential genotoxicity in primitive hematopoietic cells leading to the emergence of clonal dominance during in vitro and/or in vivo cell division while forming spleen colonies.
Molecular Therapy Volume15.Suppkmcnll. M,')'2007
Cop)'rihht © The American Scciery of Gene "Il ler-Ill)'
249. Gene Therapy in Adenosine DeaminaseDeficient Children with Autologous Bone Marrow Cells Transduced with Two Retroviral Vectors after Withdrawal of Enzyme Replacement Treatment and Low-Dose Chemotherapy
Robert A. Sokol ic,I Greg Podsakoff, ' Linda Muu!, I Barbera Engel,' Elizabeth K. Garabedian; Denise Carbonaro.' Joanna Ireland.' Laura M. Tuschong,? Michael S. Hershfield,' Donald B. Kohn,' Fabio Candotti.' JDisorders 0/ Immunity Section , Genetics and Molecular Biology Branch. National Human Genome Research Institute, Bethesda. MD; lDivision ofResearch Immunology/Bon e Marrow Transplant, The Saban Research Institute ofChildrens Hospital Los Angeles. Department 0/Pediatrics, Los Angeles, CA; "Expertmen tal Transplantation and Immunology Branch, National Cancer Institute. Bethesda, MD; 'Department ofMedicine, Duke University Medical Center, Durham, NC. Adenosine deaminase (ADA) deficiency can result in severe combined immunodeficiency (SCID), a life-threatening pediatric emergency. Enzyme replacement treatment with polyethylene glycol-conjugated bovine ADA (PEG-ADA) is life-saving, but it usually does not restore normal immunity, its efficacy may decline with time and its costs are often prohibitive outside the USA . Allogeneic hematopoietic cell transplantation is curative, but a matched related donor is available for only a minority of patients , and alternative donor transplants carry a high rate oftoxicity. For these reasons, gene therapy has long been proposed as an alternative form of treatment for this disease and has recently proven beneficial in Europe and Japan. In November, 2005 we began a clinical gene therapy trial based on infusion of genetically corrected autologous bone marrow cells after withdrawal of PEG-ADA and after treatment with rnyclosupprcssive chemotherapy. The first treated patient (ADA302N) suffered from an unexpected prolonged marrow aplasia due to pre-existing trisomy 8 abnormalities of her bone marrow cells. No engrafiment ofgene corrected cells was documented, as reported (Blood 2007; 109:503). Our second treated patient (ADA-30IN) is a 2-year-old boy who was admitted to the NIH Clinical Center in January, 2007. Bone marrow harvest yielded 92.5x 10"6 CD34+ cells that, after pre-stimulation with SCF, MGDF and Flt3-L, were divided into two aliquots and separately transduced with the MND-ADA and GCsapM-ADA retroviral vectors . Two days before infusion of gene corrected cells, the patient received busulfan (37.5 mg/m2 IV x 2). Areas under the curve for the two doses were 1451 and 1583 mcM/minute, respectively. On day 0, the patient received 5.lx I0"6 CD34+ cell/kg. ADA activity in the GCsapM-ADA and MND-ADA transduced cells was >200 and 43 nmol/min/10"8 cells, respectively (ADA activity in healthy control CD34+ cells = -70 nmol/minll 0"8 cells) . Chemotherapy was well tolerated . The absolute neutrophil count fell below 50D/mcLon day 12, at which time the platelet count was still within normal limits. It is hoped that this protocol will allow us to follow the engraftrnent of cells corrected by each vector and determine if either one ultimately contributes more strongly to the recovery of lymphoid immunity.
S95