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HbE Disease
STEM CELL GENE THERAPY FOR GENETIC DISEASES In Summary, although HoxB4 may greatly enhance haemopoietic reconstitution and facilitate improved gene therapy protocols involving transduction of HSCs, careful thought needs to be given as to how to deliver this transcription factor whilst protecting against the adverse possibility of cellular transformation.
1065. Humanised Mouse Models for βThalassemia/HbE Disease Duangporn Jamsai,1,2 Jim Vadolas,1 Faten Zaibak,1 Michael Orford,1 Mikhail Nefedov,1 Suthat Fucharoen,2 Robert Williamson,1 Panos A. Ioannou.1 1 CAGT Research Group, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Victoria, Australia; 2 Thalassemia Research Center, Institute of Science and Technology and Institute of Molecular Biology and Genetics, Mahidol University, Nakornpathom, Bangkok, Thailand. HbE is the most common hemoglobin variant, caused by a substitution of Glutamic acid by Lysine at codon 26 of the β-globin. This mutation also activates a cryptic splice site in exon 1, resulting in weak aberrant splicing. Individuals homozygous for HbE exhibit mild thalassemia, while combination of HbE with a β-thalassemia mutation results in β-thalassemia/HbE disease with a highly variable severity of presentation. We have used the GET Recombinationsystem in combination with novel counterselection markers to introduce the HbE and the codons 41/42 4 bp deletion mutations into a 205 kb BAC containing the entire human β-globin locus. The normal and modified β-globin BAC clones were used to generate transgenic mice. Transgenic mice were bred with heterozygous knockout mice carrying a deletion of the mouse βglobin genes. Mice hemizygous for the HbE transgene on a heterozygous knockout background have 25-35% heterotetrameric (αm2/βE2) hemoglobin and exhibit mild thalassemia phenotype. The heterotetrameric hemoglobin thus shows significant complementation of the intermediate thalassemia phenotype that is normally found in the heterozygous knockout mice, while mice carrying the 4 bp deletion are similar to heterozygous knockout mice, with a βthalassemia intermedia phenotype. Further breeding of these mice has resulted in mice carrying the HbE transgene on a homozygous knockout background with 100% αm2/βE2 hemoglobin. These mice survive to adulthood with mild thalassemia phenotype similar to human HbE homozygotes. These are the first mouse models to recapitulate the main features of β-thalassemia/HbE disease and should be invaluable for the development of various therapeutic approaches.
1066. High Level Lentiviral Transduction of LowDose SDF-1-Exposed Human Hematopoietic Progenitors under Conditions of Reduced Proliferation Steven P. Zielske,1,2 Karen T. Lingas,2 Eric J. Arts,1,3 Stanton L. Gerson.1,2 1 Molecular Virology Program; 2Division of Hematology/ Oncology, Comprehensive Cancer Center; 3Division of Infectious Diseases, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH. Multicomponent cytokine cocktails used to stimulate cell division for oncoretroviral transduction have been translated to lentiviral transduction protocols, however, the biology of lentiviral vectors is such that different conditions may offer unique advantages. SDF-1 is a chemokine which has been found to have effects on human hematopoietic stem cells (HSC), notably the ability of ex vivo exposure to enhance engraftment of NOD/SCID mice. Since SDF-1 has not been evaluated for its effect on lentiviral transduction of human HSC, we analyzed transduction and cell cycle characteristics Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
of human hematopoietic progenitors following preincubation with low or high concentration SDF-1. Human CD34+ cells were enriched from umbilical cord blood and the expression of surface CXCR4 was analyzed by flow cytometry Overnight incubation without cytokines, with SCF alone, or with 0.05 ng/ml SDF-1 resulted in increased surface expression of CXCR4. Expression either remained the same or was slightly increased when cultures were incubated with 100 ng/ml SDF-1 or a cocktail of IL-3, IL-6, SCF, and Flt-3L (36SF). The effect of culture with SDF-1 at 0.05 ng/ml or 100 ng/ml concentration on cell survival and recruitment into the cell cycle was then evaluated. SDF-1 at low or high concentration acted as a survival factor by suppressing apoptosis, similar to the cytokine cocktail. The effect on cell cycle, however, was markedly different to 36SF. While 36SF incubation induced 37% of the cells to proliferate by 2 days, SDF-1 exposure resulted in 8-14% proliferation. These data indicate that SDF-1 acts as a survival factor similar to other cytokines, but fails to induce proliferation to the same extent. Given the detrimental effects of cell cycle progression on the stem cell phenotype, SDF-1 may be superior to 36SF in this regard. Transductions using an HIV-based lentiviral vector were carried out following overnight incubation with no cytokines, with 36SF, or with SDF-1 at 0.05 or 100 ng/ml. SDF-1 had no effect on transduction (measured by expression) of mass culture, with 20% of 36SF-exposed cultures transduced while 9.8% of 0.05 ng/ml and 6.1% of 100 ng/ml SDF-1 exposed cultures were transduced, compared to 9.2% without cytokines. However, this was not the case when analysis of progenitor transduction by PCR of CFU was determined. This showed 71% of CFU carried the transgene when transduced in the presence of 0.05 ng/ml SDF-1, compared to 77% with 36SF. This was significantly higher (P=0.04) than transduction without cytokines, which was 39%. Transduction was not significantly enhanced by incubation with 100 ng/ml SDF-1. These data show that low concentration SDF-1 enhances lentiviral transduction of CFC progenitors, but not more differentiated hematopoietic cells in CD34+ enriched mass culture. The enhancement was equivalent to a standard cytokine cocktail. SDF-1 may offer the advantage of increasing lentiviral transduction without inducing rates of high proliferation, a property which may be beneficial for maintaining the progenitor phenotype.
1067. Critical Parameters and Molecular Analysis of Lentiviral Vector-Mediated Gene Transfer into Human Self-Renewing Multilineage NOD/SCID Repopulating Hematopoietic Stem Cells Francesca R. Santoni de Sio,1,2 Manfred Schmidt,3 Laurie Ailles,1,4 Stefania Bruno,1 Wanda Piacibello,1 Christof von Kalle,3,5 Luigi Naldini.1,2 1 Institute for Cancer Research and Treatment, University of Torino Medical School, Candiolo, Turin, Italy; 2Telethon Institute for Gene Therapy, San Raffaele, Milan, Italy; 3Department I of Internal Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany; 4 Department of Pathology, Stanford University School of Medicine, Stanford, CA; 5Program for Molecular and Gene Therapy, Division of Experimental Hematology, Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH. The major challenges in hematopoietic stem cells (HSC) gene therapy are to achieve efficient transduction and, as shown by recent adverse events in a X-linked SCID gene therapy trial using retroviral vectors, to limit the risk of insertional mutagenesis when an integrating vector is used. Others and we previously showed that VSVpseudotyped lentiviral vectors (LV) transduce efficiently human cord blood-derived NOD/SCID mouse repopulating cells (SRC). We first studied the effect of cytokines during the short ex vivo S411
1065. Humanised Mouse Models for βThalassemia/HbE Disease Duangporn Jamsai,1,2 Jim Vadolas,1 Faten Zaibak,1 Michael Orford,1 Mikhail Nefedov,1 Suthat Fucharoen,2 Robert Williamson,1 Panos A. Ioannou.1 1 CAGT Research Group, Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, Victoria, Australia; 2 Thalassemia Research Center, Institute of Science and Technology and Institute of Molecular Biology and Genetics, Mahidol University, Nakornpathom, Bangkok, Thailand. HbE is the most common hemoglobin variant, caused by a substitution of Glutamic acid by Lysine at codon 26 of the β-globin. This mutation also activates a cryptic splice site in exon 1, resulting in weak aberrant splicing. Individuals homozygous for HbE exhibit mild thalassemia, while combination of HbE with a β-thalassemia mutation results in β-thalassemia/HbE disease with a highly variable severity of presentation. We have used the GET Recombinationsystem in combination with novel counterselection markers to introduce the HbE and the codons 41/42 4 bp deletion mutations into a 205 kb BAC containing the entire human β-globin locus. The normal and modified β-globin BAC clones were used to generate transgenic mice. Transgenic mice were bred with heterozygous knockout mice carrying a deletion of the mouse βglobin genes. Mice hemizygous for the HbE transgene on a heterozygous knockout background have 25-35% heterotetrameric (αm2/βE2) hemoglobin and exhibit mild thalassemia phenotype. The heterotetrameric hemoglobin thus shows significant complementation of the intermediate thalassemia phenotype that is normally found in the heterozygous knockout mice, while mice carrying the 4 bp deletion are similar to heterozygous knockout mice, with a βthalassemia intermedia phenotype. Further breeding of these mice has resulted in mice carrying the HbE transgene on a homozygous knockout background with 100% αm2/βE2 hemoglobin. These mice survive to adulthood with mild thalassemia phenotype similar to human HbE homozygotes. These are the first mouse models to recapitulate the main features of β-thalassemia/HbE disease and should be invaluable for the development of various therapeutic approaches.
1066. High Level Lentiviral Transduction of LowDose SDF-1-Exposed Human Hematopoietic Progenitors under Conditions of Reduced Proliferation Steven P. Zielske,1,2 Karen T. Lingas,2 Eric J. Arts,1,3 Stanton L. Gerson.1,2 1 Molecular Virology Program; 2Division of Hematology/ Oncology, Comprehensive Cancer Center; 3Division of Infectious Diseases, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, OH. Multicomponent cytokine cocktails used to stimulate cell division for oncoretroviral transduction have been translated to lentiviral transduction protocols, however, the biology of lentiviral vectors is such that different conditions may offer unique advantages. SDF-1 is a chemokine which has been found to have effects on human hematopoietic stem cells (HSC), notably the ability of ex vivo exposure to enhance engraftment of NOD/SCID mice. Since SDF-1 has not been evaluated for its effect on lentiviral transduction of human HSC, we analyzed transduction and cell cycle characteristics Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
of human hematopoietic progenitors following preincubation with low or high concentration SDF-1. Human CD34+ cells were enriched from umbilical cord blood and the expression of surface CXCR4 was analyzed by flow cytometry Overnight incubation without cytokines, with SCF alone, or with 0.05 ng/ml SDF-1 resulted in increased surface expression of CXCR4. Expression either remained the same or was slightly increased when cultures were incubated with 100 ng/ml SDF-1 or a cocktail of IL-3, IL-6, SCF, and Flt-3L (36SF). The effect of culture with SDF-1 at 0.05 ng/ml or 100 ng/ml concentration on cell survival and recruitment into the cell cycle was then evaluated. SDF-1 at low or high concentration acted as a survival factor by suppressing apoptosis, similar to the cytokine cocktail. The effect on cell cycle, however, was markedly different to 36SF. While 36SF incubation induced 37% of the cells to proliferate by 2 days, SDF-1 exposure resulted in 8-14% proliferation. These data indicate that SDF-1 acts as a survival factor similar to other cytokines, but fails to induce proliferation to the same extent. Given the detrimental effects of cell cycle progression on the stem cell phenotype, SDF-1 may be superior to 36SF in this regard. Transductions using an HIV-based lentiviral vector were carried out following overnight incubation with no cytokines, with 36SF, or with SDF-1 at 0.05 or 100 ng/ml. SDF-1 had no effect on transduction (measured by expression) of mass culture, with 20% of 36SF-exposed cultures transduced while 9.8% of 0.05 ng/ml and 6.1% of 100 ng/ml SDF-1 exposed cultures were transduced, compared to 9.2% without cytokines. However, this was not the case when analysis of progenitor transduction by PCR of CFU was determined. This showed 71% of CFU carried the transgene when transduced in the presence of 0.05 ng/ml SDF-1, compared to 77% with 36SF. This was significantly higher (P=0.04) than transduction without cytokines, which was 39%. Transduction was not significantly enhanced by incubation with 100 ng/ml SDF-1. These data show that low concentration SDF-1 enhances lentiviral transduction of CFC progenitors, but not more differentiated hematopoietic cells in CD34+ enriched mass culture. The enhancement was equivalent to a standard cytokine cocktail. SDF-1 may offer the advantage of increasing lentiviral transduction without inducing rates of high proliferation, a property which may be beneficial for maintaining the progenitor phenotype.
1067. Critical Parameters and Molecular Analysis of Lentiviral Vector-Mediated Gene Transfer into Human Self-Renewing Multilineage NOD/SCID Repopulating Hematopoietic Stem Cells Francesca R. Santoni de Sio,1,2 Manfred Schmidt,3 Laurie Ailles,1,4 Stefania Bruno,1 Wanda Piacibello,1 Christof von Kalle,3,5 Luigi Naldini.1,2 1 Institute for Cancer Research and Treatment, University of Torino Medical School, Candiolo, Turin, Italy; 2Telethon Institute for Gene Therapy, San Raffaele, Milan, Italy; 3Department I of Internal Medicine and Institute for Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany; 4 Department of Pathology, Stanford University School of Medicine, Stanford, CA; 5Program for Molecular and Gene Therapy, Division of Experimental Hematology, Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH. The major challenges in hematopoietic stem cells (HSC) gene therapy are to achieve efficient transduction and, as shown by recent adverse events in a X-linked SCID gene therapy trial using retroviral vectors, to limit the risk of insertional mutagenesis when an integrating vector is used. Others and we previously showed that VSVpseudotyped lentiviral vectors (LV) transduce efficiently human cord blood-derived NOD/SCID mouse repopulating cells (SRC). We first studied the effect of cytokines during the short ex vivo S411