- Email: [email protected]
Requirements for retroviral targeting of a suicide gene to alloreactive memory stem T cells for adoptive immunotherapy of leukemia
ABSTRACTS / Blood Cells, Molecules, and Diseases 40 (2008) 248–294
two additional transductions in American Fluoroseal bags were 44.6% and 57.6%, the former for therapeutic MGMT vector PhRD114-MND.GRS.P140Kc22 and the latter for therapeutic MGMT vector PhGALV-MND.GRS.P140Kc38. The latter method included pre-loading of Rn coated bag with virus twice prior to addition of cells and virus to bag. Subsequent sterility testing was negative, and the stability testing indicated good viability N 80% for 4 h at 23 °C, and = 75% at an additional 72 h at 4 °C. Thus, American Fluoroseal bags of GMP grade are suitable for clinical gene therapy trials. doi:10.1016/j.bcmd.2007.10.019
10 Novel lentivirus vectors for safe and efficient gene therapy of hemoglobin disorders David Bodine, Faith Harrow, Karina Laflame Hematopoiesis Section, NHGRI, National Institutes of Health, Bethesda, MD, USA Our long-term goal is to design gene transfer vectors for the treatment of beta-Thalassemia major and or Sickle Cell Disease. Both diseases could be effectively treated by expressing a betalike globin gene that is expressed at approximately 20% of the level of the endogenous alpha-globin genes the in 25–50% of red blood cells. Although great progress has been made toward this goal, before therapeutic application of gene therapy for the treatment of these diseases can be realized, three challenges must be solved: (1) Designing vectors that can produce therapeutic levels of beta-like globin that do not depend on enhancer elements in the viral vector or the transcription unit, which can lead to the activation of near by leukemia genes. (2) Designing vectors that are resistant to position dependent gene silencing. (3) Designing vectors that can transduce 20% or more of human HSC. To address the first challenge we have developed a novel strategy in which enhancer-independent promoters form nonglobin genes are used to express a human gamma-globin gene. To determine the location of enhancer and other regulatory elements in the Slc4a1 (which encodes the band 3 protein) ankyrin (ANK) and locus and alpha-spectrin (alpha-Sp) loci we have compiled a comprehensive map of all of the DNAse I Hypersensitive sites (DNase I HS) and assayed each DNase I HS for enhancer, enhancer blocking and barrier element activity. The DNase I HS in the minimal Slc4a1 promoter does NOT contain enhancer activity in erythroid cells. In transgenic mice the level of Slc4a1/gamma-globin mRNA and protein per transgene copy is approximately 20% the level of mouse alphaglobin (Frazar et al. MCB 23:4753–63, 2003). Our survey of the ANK locus revealed two DNase I HS associated with the erythroid specific promoter that do NOT have enhancer or enhancer blocking activity, but are powerful barrier elements in erythroid cell lines and transgenic mice. We have also identified a barrier element in the alpha-Sp locus using the same methods. To increase the output of ANK/gamma-globin vectors beyond the 8% of endogenous mouse alpha-globin level we reported
255
previously (Sabatino et al. PNAS 97:13294–9, 2000), we have screened a panel of 16,000 degenerate ANK promoters to identify those that are the most active. We have identified two sequence modifications in the ankyrin promoter activity that increase expression in transient transfection and in vitro transcription assays and show an increased ability to bind the transcription initiation complex, TFIID. The second challenge for globin gene therapy is to prevent gene silencing in erythroid cells. In transgenic mice, the Slc4a1/gamma-globin transgene required the chicken beta-globin 5′ hypersensitive site 4 (ch5′ HS4) insulator for uniform expression. In our vectors, the internal Slc4a1/gamma-globin gene is flanked by two different barrier elements (ch5’HS4/ANK, ANK/alpha-SP or ch5′HS4/ alpha-Sp). These viruses were pseudotyped with the ecotropic envelope for transduction of mouse primitive hematopoietic cells. All three vectors were generated at high titer and showed faithful transmission to cultured cells with no evidence of rearrangement or deletion. Analysis of both CFU-S and long term repopulated mice demonstrated that the Slc4a1/gammaglobin vectors expressed gamma-globin mRNA and protein at levels of ∼ 17% the level of endogenous mouse alpha-globin. The ANK promoter carries with it a barrier element, and we have generated a double copy mutant ANK/gamma-globin vector. To improve gene transfer to human cells (challenge 3), we are pseudotyping our lentivirus vectors with the Feline Leukemia Virus type C (FeLV-C) envelope (Lucas et al. Blood 106:51–9, 2005), which we have previously shown recognizes a receptor expressed at high levels on human HSC. At this point, we have successfully generated FeLV-C pseudotyped vectors at high titer that can faithfully transfer the Slc4a1/gamma-globin lentivirus vectors to cultured cells. We plan to test our vectors in mouse models of beta-thal and SCD as well as patient cells using the fetal sheep transplantation model. doi:10.1016/j.bcmd.2007.10.020
11 Requirements for retroviral targeting of a suicide gene to alloreactive memory stem T cells for adoptive immunotherapy of leukemia A. Bondanza1, S. Kaneko1, L. Hambach2, S. Mastaglio1, B. Nijmeijer2, A. Van Halteren2, M. Ponzoni3, L. Aldrighetti4, S. Toma5, M. Radrizzani5, F. Ciceri1, C. Bordignon1, E. Goulmy2, C. Bonini1 1 Cancer Immunotherapy and Gene Therapy Program, S. Raffaele Scientific Institute, Milan, Italy 2 Immunohematology and Blood Bank, Leiden University Medical Center, The Netherlands 3 Pathology Unit, Raffaele Scientific Institute, Milan, Italy 4 Division of Surgery, S. Raffaele Scientific Institute, Milan, Italy 5 Molmed S.p.A., Milan, Italy In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of
256
ABSTRACTS / Blood Cells, Molecules, and Diseases 40 (2008) 248–294
haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA−CD62L−, CD28±CD27+, IL-2± IFN-γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA−CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generate central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHagspecific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect. doi:10.1016/j.bcmd.2007.10.021
12 Cancer stem cells: New therapeutic targets? Dominique Bonnet Cancer Research UK, London Research Institute, London, UK Acute myeloid leukemia (AML) is a clonal disorder defined by the accumulation of abnormally differentiated myeloid blasts. Because leukemic blasts have very limited proliferative capacity, it is believed that leukemic clone is maintain by a rare
population of leukemic stem cells (LSC) that have extensive proliferation and self-renewal capacities. Elucidating the nature of the target cell that undergoes leukemic transformation and characterizing the LSC is essential for both the understanding of the leukemogenic process and for the design of effective therapies. The development of an in vivo model that replicates many aspects of human AML had provide a mean to identify leukemic stem cells (termed the SCID-Leukemia Initiating Cells, SL-IC). SL-IC is defined by the ability of that cell to initiate AML in NOD/SCID mice. This in vivo assay provides the foundation of an assay to define the biological and molecular properties of such leukemic stem cells (LSC). Despite the clear importance of the LSC in the genesis and perpetuation of leukemic disease, little is currently known about the biological and molecular properties that make LSCs distinct from normal hematopoietic stem cells. The presentation will summarize the work done using the xenograft system to characterise the nature of the leukemic clone and will specifically highlights the advances made in phenotypically, molecularly and functionally defining LSC. It will also discuss why leukemic stem cells represent new therapeutic targets and what means we might be able to use to targets them. doi:10.1016/j.bcmd.2007.10.022
13 Long-term efficacy and safety of hematopoietic stem cell gene therapy mediated by lentiviral vectors in the murine model of Wiskott-Aldrich syndrome Marita Bosticardo1,∗, Francesco Marangoni1,2,∗, Michela Locci2, Elena Draghici2, Samantha Scaramuzza2, Cristina Panaroni2, Anne Galy3, Luigi Naldini1,2, Alessandro Aiuti2, Loic Dupré2,4, Maria Grazia Roncarolo1,2, Anna 1 Vita-Salute San Raffaele University, Milan, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy 3 Généthon UMR CNRS 8115, Evry, France 4 INSERM U563, Toulouse, France 5 Istituto di Tecnologie Biomediche, Segrate, Milan, Italy *Contributed equally to this work. Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by thrombocytopenia, eczema and increased risk of autoimmunity and malignancies. HLAmatched bone marrow transplantation is curative; however, hematopoietic stem cell (HSC) gene therapy could represent an alternative treatment for patients lacking a suitable donor. The aim of the present study was to investigate the long-term efficacy and safety of gene therapy in a large cohort of WAS−/− mice. WAS−/− bone marrow-derived HSC untransduced or transduced with a human WAS promoter/cDNA encoding lentiviral vector at low or high multiplicity of infection (MOI 10–20 or 200) were injected into sub-lethally irradiated WAS−/−
two additional transductions in American Fluoroseal bags were 44.6% and 57.6%, the former for therapeutic MGMT vector PhRD114-MND.GRS.P140Kc22 and the latter for therapeutic MGMT vector PhGALV-MND.GRS.P140Kc38. The latter method included pre-loading of Rn coated bag with virus twice prior to addition of cells and virus to bag. Subsequent sterility testing was negative, and the stability testing indicated good viability N 80% for 4 h at 23 °C, and = 75% at an additional 72 h at 4 °C. Thus, American Fluoroseal bags of GMP grade are suitable for clinical gene therapy trials. doi:10.1016/j.bcmd.2007.10.019
10 Novel lentivirus vectors for safe and efficient gene therapy of hemoglobin disorders David Bodine, Faith Harrow, Karina Laflame Hematopoiesis Section, NHGRI, National Institutes of Health, Bethesda, MD, USA Our long-term goal is to design gene transfer vectors for the treatment of beta-Thalassemia major and or Sickle Cell Disease. Both diseases could be effectively treated by expressing a betalike globin gene that is expressed at approximately 20% of the level of the endogenous alpha-globin genes the in 25–50% of red blood cells. Although great progress has been made toward this goal, before therapeutic application of gene therapy for the treatment of these diseases can be realized, three challenges must be solved: (1) Designing vectors that can produce therapeutic levels of beta-like globin that do not depend on enhancer elements in the viral vector or the transcription unit, which can lead to the activation of near by leukemia genes. (2) Designing vectors that are resistant to position dependent gene silencing. (3) Designing vectors that can transduce 20% or more of human HSC. To address the first challenge we have developed a novel strategy in which enhancer-independent promoters form nonglobin genes are used to express a human gamma-globin gene. To determine the location of enhancer and other regulatory elements in the Slc4a1 (which encodes the band 3 protein) ankyrin (ANK) and locus and alpha-spectrin (alpha-Sp) loci we have compiled a comprehensive map of all of the DNAse I Hypersensitive sites (DNase I HS) and assayed each DNase I HS for enhancer, enhancer blocking and barrier element activity. The DNase I HS in the minimal Slc4a1 promoter does NOT contain enhancer activity in erythroid cells. In transgenic mice the level of Slc4a1/gamma-globin mRNA and protein per transgene copy is approximately 20% the level of mouse alphaglobin (Frazar et al. MCB 23:4753–63, 2003). Our survey of the ANK locus revealed two DNase I HS associated with the erythroid specific promoter that do NOT have enhancer or enhancer blocking activity, but are powerful barrier elements in erythroid cell lines and transgenic mice. We have also identified a barrier element in the alpha-Sp locus using the same methods. To increase the output of ANK/gamma-globin vectors beyond the 8% of endogenous mouse alpha-globin level we reported
255
previously (Sabatino et al. PNAS 97:13294–9, 2000), we have screened a panel of 16,000 degenerate ANK promoters to identify those that are the most active. We have identified two sequence modifications in the ankyrin promoter activity that increase expression in transient transfection and in vitro transcription assays and show an increased ability to bind the transcription initiation complex, TFIID. The second challenge for globin gene therapy is to prevent gene silencing in erythroid cells. In transgenic mice, the Slc4a1/gamma-globin transgene required the chicken beta-globin 5′ hypersensitive site 4 (ch5′ HS4) insulator for uniform expression. In our vectors, the internal Slc4a1/gamma-globin gene is flanked by two different barrier elements (ch5’HS4/ANK, ANK/alpha-SP or ch5′HS4/ alpha-Sp). These viruses were pseudotyped with the ecotropic envelope for transduction of mouse primitive hematopoietic cells. All three vectors were generated at high titer and showed faithful transmission to cultured cells with no evidence of rearrangement or deletion. Analysis of both CFU-S and long term repopulated mice demonstrated that the Slc4a1/gammaglobin vectors expressed gamma-globin mRNA and protein at levels of ∼ 17% the level of endogenous mouse alpha-globin. The ANK promoter carries with it a barrier element, and we have generated a double copy mutant ANK/gamma-globin vector. To improve gene transfer to human cells (challenge 3), we are pseudotyping our lentivirus vectors with the Feline Leukemia Virus type C (FeLV-C) envelope (Lucas et al. Blood 106:51–9, 2005), which we have previously shown recognizes a receptor expressed at high levels on human HSC. At this point, we have successfully generated FeLV-C pseudotyped vectors at high titer that can faithfully transfer the Slc4a1/gamma-globin lentivirus vectors to cultured cells. We plan to test our vectors in mouse models of beta-thal and SCD as well as patient cells using the fetal sheep transplantation model. doi:10.1016/j.bcmd.2007.10.020
11 Requirements for retroviral targeting of a suicide gene to alloreactive memory stem T cells for adoptive immunotherapy of leukemia A. Bondanza1, S. Kaneko1, L. Hambach2, S. Mastaglio1, B. Nijmeijer2, A. Van Halteren2, M. Ponzoni3, L. Aldrighetti4, S. Toma5, M. Radrizzani5, F. Ciceri1, C. Bordignon1, E. Goulmy2, C. Bonini1 1 Cancer Immunotherapy and Gene Therapy Program, S. Raffaele Scientific Institute, Milan, Italy 2 Immunohematology and Blood Bank, Leiden University Medical Center, The Netherlands 3 Pathology Unit, Raffaele Scientific Institute, Milan, Italy 4 Division of Surgery, S. Raffaele Scientific Institute, Milan, Italy 5 Molmed S.p.A., Milan, Italy In a phase I/II clinical trial investigating the prophylactic infusion of suicide gene-modified donor T cells in the context of
256
ABSTRACTS / Blood Cells, Molecules, and Diseases 40 (2008) 248–294
haploidentical hemopoietic cell transplantation (haplo-HCT) for the treatment of high-risk leukemia, we observed a rapid and effective immune reconstitution. After activation with anti-CD3 antibodies, genetic modification of donor T cells was accomplished with a retroviral vector encoding for the Herpes Simplex thymidine kinase (TK). In vitro before infusion and in vivo at immune reconstitution, TK+ cells displayed an effector memory (EM) phenotype (CD45RA−CD62L−, CD28±CD27+, IL-2± IFN-γ+). The graft-versus-leukemia (GvL) effect was substantial in patients transplanted in remission, but failed to cure patients in relapse. Gene targeting with retroviral vectors is limited to memory T cells. Central memory (CM) T cells (CD45RA−CD62L+, CD28+CD27+, IL-2+IFN-γ±) share many characteristics with stem cells, namely the ability to self-renew and to differentiate into a progeny of effector cells. EM TK+ cells have a reduced alloreactivity. Recently, it has been proposed that alloreactivity may be confined to memory T cells with stem cell-features. Since alloantigens are the target not only of graft-versus-host disease (GvHD), but also of the GvL effect, crucial to the success of the strategy is the suicide gene-modification of this subset of memory T cells. We found that addition of CD28 costimulation on cell-sized beads and the use of homeostatic cytokines, such as IL-7 and IL-15, generate central memory (CM) TK+ cells. CM TK+ cells are highly alloreactive, both in vitro and in vivo in a humanized animal model of GvHD based on the grafting of human skin onto NOD/scid mice. Interestingly, CM TK+ cells express the IL7Rα, a marker associated with the stem cell-features of memory T cells. Moreover, IL7Rα expression is maintained after stimulation with alloantigens. Stimulation of CM, but not of EM TK+ cells with autologous dendritic cells pulsed with restricted peptides from the minor histocompatibility alloantigen (mHag) HA-1 or H-Y efficiently induces mHag-specific effector T cells that lyse natural ligand expressing HLA-A2+ targets. TK+ mHagspecific effector T cells also lysed mHag+HLA-A2+ leukemic cells and, when infused in conditioned NOD/scid mice harboring human leukemia, significantly delayed disease progression. Altogether, these data suggest that optimal T-cell receptor triggering and homeostatic cytokines are required for retroviral targeting of a suicide gene to alloreactive memory stem T cells and warrant their use for a safe and powerful GvL effect. doi:10.1016/j.bcmd.2007.10.021
12 Cancer stem cells: New therapeutic targets? Dominique Bonnet Cancer Research UK, London Research Institute, London, UK Acute myeloid leukemia (AML) is a clonal disorder defined by the accumulation of abnormally differentiated myeloid blasts. Because leukemic blasts have very limited proliferative capacity, it is believed that leukemic clone is maintain by a rare
population of leukemic stem cells (LSC) that have extensive proliferation and self-renewal capacities. Elucidating the nature of the target cell that undergoes leukemic transformation and characterizing the LSC is essential for both the understanding of the leukemogenic process and for the design of effective therapies. The development of an in vivo model that replicates many aspects of human AML had provide a mean to identify leukemic stem cells (termed the SCID-Leukemia Initiating Cells, SL-IC). SL-IC is defined by the ability of that cell to initiate AML in NOD/SCID mice. This in vivo assay provides the foundation of an assay to define the biological and molecular properties of such leukemic stem cells (LSC). Despite the clear importance of the LSC in the genesis and perpetuation of leukemic disease, little is currently known about the biological and molecular properties that make LSCs distinct from normal hematopoietic stem cells. The presentation will summarize the work done using the xenograft system to characterise the nature of the leukemic clone and will specifically highlights the advances made in phenotypically, molecularly and functionally defining LSC. It will also discuss why leukemic stem cells represent new therapeutic targets and what means we might be able to use to targets them. doi:10.1016/j.bcmd.2007.10.022
13 Long-term efficacy and safety of hematopoietic stem cell gene therapy mediated by lentiviral vectors in the murine model of Wiskott-Aldrich syndrome Marita Bosticardo1,∗, Francesco Marangoni1,2,∗, Michela Locci2, Elena Draghici2, Samantha Scaramuzza2, Cristina Panaroni2, Anne Galy3, Luigi Naldini1,2, Alessandro Aiuti2, Loic Dupré2,4, Maria Grazia Roncarolo1,2, Anna 1 Vita-Salute San Raffaele University, Milan, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy 3 Généthon UMR CNRS 8115, Evry, France 4 INSERM U563, Toulouse, France 5 Istituto di Tecnologie Biomediche, Segrate, Milan, Italy *Contributed equally to this work. Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by thrombocytopenia, eczema and increased risk of autoimmunity and malignancies. HLAmatched bone marrow transplantation is curative; however, hematopoietic stem cell (HSC) gene therapy could represent an alternative treatment for patients lacking a suitable donor. The aim of the present study was to investigate the long-term efficacy and safety of gene therapy in a large cohort of WAS−/− mice. WAS−/− bone marrow-derived HSC untransduced or transduced with a human WAS promoter/cDNA encoding lentiviral vector at low or high multiplicity of infection (MOI 10–20 or 200) were injected into sub-lethally irradiated WAS−/−