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Safe lentiviral vectors with cellular and myeloid promoters driving glucocylceramidase expression corrects type 1 gaucher disease
S14
Oral Short Talk Presentations/ Experimental Hematology 42 (2014) S13–S21
O1005 - SAFE LENTIVIRAL VECTORS WITH CELLULAR AND MYELOID PROMOTERS DRIVING GLUCOCYLCERAMIDASE EXPRESSION CORRECTS TYPE 1 GAUCHER DISEASE ansson2, Hans Aerts3, Maria Dahl1, Alexander Doyle1, Karin Olsson1, Jan-Eric M Mats Ehinger4, Michael Rothe5, Ute Modlich5, Axel Schambach5, and Stefan Karlsson1 1 Lund University, Lund, Sweden; 2Sahlgrenska University Hospital, M€olndal, Sweden; 3University of Amsterdam, Amsterdam, Netherlands; 4Lund University Hospital, Lund, Sweden; 5Hannover Medical School, Hannover, Germany
O1007 - INFLAMMATION-DRIVEN FAST-TRACK DIFFERENTIATION OF HSCS INTO THE MEGAKARYOCYTIC LINEAGE Simon Haas1, Jenny Hansson2, Daniel Klimmeck1,2,3, Dirk L€offler4, Lars Velten2, Prendergast1,3, Timm Schroeder4, Hannah Uckelmann1, Stephan Wurzer1,3, Aine Andreas Trumpp1,3, Jeroen Krijgsveld2, and Marieke Essers1,3 1 German Cancer Research Center, Heidelberg, Germany; 2European Molecular Biology Laboratory, Heidelberg, Germany; 3The Heidelberg Institute for Stem Cell urich, Basel, Technology and Experimental Medicine, Heidelberg, Germany; 4ETH Z€ Switzerland
Gaucher disease (GD), the most common of the lysosomal storage disorders, is an autosomal recessive disorder affecting the enzyme glucosylceramidase (GCase). In patients, the mutated GBA gene encoding GCase gives rise to a deficient enzyme unable to degrade the cell membrane lipid glucosylceramide (GluCer), leading to a progressive build-up of GluCer in macrophages. Macrophages laden with unprocessed lipids cause hepatosplenomegaly, cytopenias and bone disease in patients. Our conditional mouse model of type 1 GD displaying key clinical symptoms can be cured with gene therapy using oncoretroviral vectors with a strong viral promoter (Enquist et al, PNAS 2006). To investigate whether safer vectors can correct the enzyme deficiency we utilized SIN lentiviral vectors harboring the human GBA transgene under the control of human phosphoglycerate kinase (PGK) and myeloid (CD68) promoters, respectively, for correction of disease in the GD1 model. Here we show correction of manifest symptoms using these vectors, resulting in enzyme restoration of 25-44% of wild type levels in bone marrow. Five months post transplant treated mice exhibit a robust decrease of GluCer in tissues, reversal of splenomegaly and Gaucher cell infiltration and a restoration in hematological parameters. By genetics and clonal analysis we find that as few as 6% functional macrophages are sufficient to reverse GD pathology. Furthermore, we conclude that these vectors exhibit a typical lentiviral integration pattern without any overrepresentation of hits in cancer gene databases. The findings demonstrate the feasibility of developing safe clinical vectors with cellular/tissue specific promoters to cure type 1 Gaucher patients.
During an acute stress situation, such as infection, distinct mature cell types are heavily consumed in order to combat invading pathogens. These include blood platelets- the immediate progeny of megakaryocytes (Mks). The coordination of the fast restoration of normal platelet levels, and the role of hematopoietic stem cells (HSCs) in this regulatory process, remain poorly characterized. While a high expression of certain Mk transcripts in HSCs has been suggested to be indicative of a platelet-primed HSC subpopulation, the functional role of such Mk lineage primingis not well understood. Here, we investigated the Mk differentiation from HSCs during homeostasis and inflammation. Single-cell transcriptomics revealed a stochastic bimodal expression of Mk transcripts in HSCs. Upon commitment towards the Mk lineage, a subset of HSCs switches to a highly coordinated unimodal Mk transcript expression program. Mk protein translation from Mk transcripts was largely silenced during homeostasis in HSCs, as inferred by proteomewide quantitative mass spectrometry and ribosome profiling. In contrast, transcripts were efficiently translated upon inflammatory signaling, resulting in a striking increase of Mk proteins. This inflammation-induced translation of Mk transcripts in HSCs was mainly instructed by a cell-autonomous, non-canonical type I interferon-mTOR crosstalk signaling. Enhanced Mk protein production was accompanied by a commitment of HSCs towards the Mk lineage as well as by indications of megakaryocytic maturation, such as appearance of alpha-granular structures, increased cell size and cell cycle induction. Our data reveal a highly efficient mechanism of inflammation-driven Mk maturation from HSCs, permitting rapid platelet recovery, demonstrating a functional role of Mk lineage priming in HSCs.
O1006 - IFN-GAMMA CAUSES APLASTIC ANEMIA BY ALTERING HSC COMPOSITION AND INTERRUPTING LINEAGE DIFFERENTIATION Fanching Lin1, Megan Karwan2, Bahara Saleh1, Debora Hodge1, Kimberly Boelte1, Tim Chan1, Jonathan Keller1, and Howard Young1 1 National Cancer Institute, Frederick, Maryland, USA; 2Leidos, Frederick, Maryland, USA
O1008 - TYPE II INTERFERON PROMOTES DIFFERENTIATION OF MYELOID-BIASED HEMATOPOIETIC STEM CELLS Katie Matatall1, Ching-Chieh Shen2, Grant Challen2, and Katherine King1 1 Baylor College of Medicine, Houston, Texas, USA; 2Washington University, St. Louis, Missouri, USA
Aplastic anemia (AA) is characterized by hypocellular marrow and pancytopenia with largely unknown etiology. Since IFN-g and T-bet can be detected in peripheral blood of AA patients, it was believed that autoreactive T lymphocytes play a major role in destroying the hematopoietic stem cells (HSCs) in the bone marrow (BM). Thus, AA has been treated as an autoimmune disease. We have observed AA-like symptoms in our IFN-g AU-rich element (ARE) - deleted (del) mice. These mice constitutively express low level of IFN-g under physiological condition as the deletion increase the half-life of IFN-g mRNA. Here, we used ARE-del mice as a model to understand the possible etiology of AA. Upon examining the BM, we did not observe infiltration of T cells in ARE-del mice. BM T cells from these mice appeared to be non-pathogenic, since they failed to respond to anti-CD3/anti-CD28 stimulation. When studying HSC and progenitor composition, we observed dramatic decreases in common myeloid progenitors (CMP), megakaryocyte/erythrocyte progenitors (MEP) and granulocyte/monocyte progenitors (GMP) despite there was a significant increase in serum levels of cytokines involved in hematopoietic differentiation and maturation. Furthermore, there was a disruption in erythropoiesis and B cell differentiation. The same HSC and progenitor composition as well as lineage disruption were also observed in WT recipients of ARE-del BM, further confirming the role of IFN-g in the etiology of AA. In conclusion, our data suggests that AA occurs when IFN-g inhibits the generation of CMP, GMP and MEP from multipotent progenitors and prevents lineage differentiation, as opposed to infiltration of autoreactive T cells into the BM. The finding in this study provides a possible pathogenesis of AA in certain patient populations and a probable cause for disease relapse. These results would be useful in improving treatment as well as maintaining a disease free status.
Background: Inflammatory conditions affect hematopoietic stem cell (HSC) quiescence. Indeed, both interferon alpha (IFNa) and interferon gamma (IFNg) have been shown to promote cell division of HSCs. IFNg-induced cell division does not expand HSC number and it impairs HSC self-renewal, suggesting a role in differentiation. However, the mechanisms by which inflammatory cytokines affect HSC division and differentiation are not well understood. Hypothesis: We postulated that IFNg stimulates myeloid differentiation of HSCs as part of the innate immune response. Methods: We utilized HSCs lacking receptors for interferon alpha or interferon gamma to characterize the requirement for these receptors in HSC repopulating activity. Further, we conducted in vivo infections (both lymphochoriomeningitis virus and Mycobacterium avium) to characterize patterns of hematopoiesis during infection. We separately evaluated myeloid-biased and lymphoid-biased HSCs using molecular and adoptive transfer studies to understand their distinct biology in the context of these infections. Results: Here we report that IFNg signaling is required, both at baseline and during an animal model of LCMV infection, to maintain normal myeloid development. We found that myeloid-biased HSCs express higher levels of IFNg receptor, are specifically activated to divide after recombinant IFNg exposure in vivo, and show increased expression of the transcription factor C/EBPb after infection. Administration of IFNa did not show lineage-specific effects. Furthermore, myeloid-biased HSCs are transiently depleted from the marrow during the IFNg-mediated immune response to Mycobacterium avium infection, as measured both functionally and phenotypically. These findings indicate that IFNg selectively promotes differentiation of myeloid-biased HSCs during an innate immune response to infection. Conclusion: This represents the first report of a context and mechanism for discriminate utilization of alternate HSC subtypes. Terminal differentiation, at the expense of self-renewal, may compromise HSC populations with age or during states of chronic inflammation.
Oral Short Talk Presentations/ Experimental Hematology 42 (2014) S13–S21
O1005 - SAFE LENTIVIRAL VECTORS WITH CELLULAR AND MYELOID PROMOTERS DRIVING GLUCOCYLCERAMIDASE EXPRESSION CORRECTS TYPE 1 GAUCHER DISEASE ansson2, Hans Aerts3, Maria Dahl1, Alexander Doyle1, Karin Olsson1, Jan-Eric M Mats Ehinger4, Michael Rothe5, Ute Modlich5, Axel Schambach5, and Stefan Karlsson1 1 Lund University, Lund, Sweden; 2Sahlgrenska University Hospital, M€olndal, Sweden; 3University of Amsterdam, Amsterdam, Netherlands; 4Lund University Hospital, Lund, Sweden; 5Hannover Medical School, Hannover, Germany
O1007 - INFLAMMATION-DRIVEN FAST-TRACK DIFFERENTIATION OF HSCS INTO THE MEGAKARYOCYTIC LINEAGE Simon Haas1, Jenny Hansson2, Daniel Klimmeck1,2,3, Dirk L€offler4, Lars Velten2, Prendergast1,3, Timm Schroeder4, Hannah Uckelmann1, Stephan Wurzer1,3, Aine Andreas Trumpp1,3, Jeroen Krijgsveld2, and Marieke Essers1,3 1 German Cancer Research Center, Heidelberg, Germany; 2European Molecular Biology Laboratory, Heidelberg, Germany; 3The Heidelberg Institute for Stem Cell urich, Basel, Technology and Experimental Medicine, Heidelberg, Germany; 4ETH Z€ Switzerland
Gaucher disease (GD), the most common of the lysosomal storage disorders, is an autosomal recessive disorder affecting the enzyme glucosylceramidase (GCase). In patients, the mutated GBA gene encoding GCase gives rise to a deficient enzyme unable to degrade the cell membrane lipid glucosylceramide (GluCer), leading to a progressive build-up of GluCer in macrophages. Macrophages laden with unprocessed lipids cause hepatosplenomegaly, cytopenias and bone disease in patients. Our conditional mouse model of type 1 GD displaying key clinical symptoms can be cured with gene therapy using oncoretroviral vectors with a strong viral promoter (Enquist et al, PNAS 2006). To investigate whether safer vectors can correct the enzyme deficiency we utilized SIN lentiviral vectors harboring the human GBA transgene under the control of human phosphoglycerate kinase (PGK) and myeloid (CD68) promoters, respectively, for correction of disease in the GD1 model. Here we show correction of manifest symptoms using these vectors, resulting in enzyme restoration of 25-44% of wild type levels in bone marrow. Five months post transplant treated mice exhibit a robust decrease of GluCer in tissues, reversal of splenomegaly and Gaucher cell infiltration and a restoration in hematological parameters. By genetics and clonal analysis we find that as few as 6% functional macrophages are sufficient to reverse GD pathology. Furthermore, we conclude that these vectors exhibit a typical lentiviral integration pattern without any overrepresentation of hits in cancer gene databases. The findings demonstrate the feasibility of developing safe clinical vectors with cellular/tissue specific promoters to cure type 1 Gaucher patients.
During an acute stress situation, such as infection, distinct mature cell types are heavily consumed in order to combat invading pathogens. These include blood platelets- the immediate progeny of megakaryocytes (Mks). The coordination of the fast restoration of normal platelet levels, and the role of hematopoietic stem cells (HSCs) in this regulatory process, remain poorly characterized. While a high expression of certain Mk transcripts in HSCs has been suggested to be indicative of a platelet-primed HSC subpopulation, the functional role of such Mk lineage primingis not well understood. Here, we investigated the Mk differentiation from HSCs during homeostasis and inflammation. Single-cell transcriptomics revealed a stochastic bimodal expression of Mk transcripts in HSCs. Upon commitment towards the Mk lineage, a subset of HSCs switches to a highly coordinated unimodal Mk transcript expression program. Mk protein translation from Mk transcripts was largely silenced during homeostasis in HSCs, as inferred by proteomewide quantitative mass spectrometry and ribosome profiling. In contrast, transcripts were efficiently translated upon inflammatory signaling, resulting in a striking increase of Mk proteins. This inflammation-induced translation of Mk transcripts in HSCs was mainly instructed by a cell-autonomous, non-canonical type I interferon-mTOR crosstalk signaling. Enhanced Mk protein production was accompanied by a commitment of HSCs towards the Mk lineage as well as by indications of megakaryocytic maturation, such as appearance of alpha-granular structures, increased cell size and cell cycle induction. Our data reveal a highly efficient mechanism of inflammation-driven Mk maturation from HSCs, permitting rapid platelet recovery, demonstrating a functional role of Mk lineage priming in HSCs.
O1006 - IFN-GAMMA CAUSES APLASTIC ANEMIA BY ALTERING HSC COMPOSITION AND INTERRUPTING LINEAGE DIFFERENTIATION Fanching Lin1, Megan Karwan2, Bahara Saleh1, Debora Hodge1, Kimberly Boelte1, Tim Chan1, Jonathan Keller1, and Howard Young1 1 National Cancer Institute, Frederick, Maryland, USA; 2Leidos, Frederick, Maryland, USA
O1008 - TYPE II INTERFERON PROMOTES DIFFERENTIATION OF MYELOID-BIASED HEMATOPOIETIC STEM CELLS Katie Matatall1, Ching-Chieh Shen2, Grant Challen2, and Katherine King1 1 Baylor College of Medicine, Houston, Texas, USA; 2Washington University, St. Louis, Missouri, USA
Aplastic anemia (AA) is characterized by hypocellular marrow and pancytopenia with largely unknown etiology. Since IFN-g and T-bet can be detected in peripheral blood of AA patients, it was believed that autoreactive T lymphocytes play a major role in destroying the hematopoietic stem cells (HSCs) in the bone marrow (BM). Thus, AA has been treated as an autoimmune disease. We have observed AA-like symptoms in our IFN-g AU-rich element (ARE) - deleted (del) mice. These mice constitutively express low level of IFN-g under physiological condition as the deletion increase the half-life of IFN-g mRNA. Here, we used ARE-del mice as a model to understand the possible etiology of AA. Upon examining the BM, we did not observe infiltration of T cells in ARE-del mice. BM T cells from these mice appeared to be non-pathogenic, since they failed to respond to anti-CD3/anti-CD28 stimulation. When studying HSC and progenitor composition, we observed dramatic decreases in common myeloid progenitors (CMP), megakaryocyte/erythrocyte progenitors (MEP) and granulocyte/monocyte progenitors (GMP) despite there was a significant increase in serum levels of cytokines involved in hematopoietic differentiation and maturation. Furthermore, there was a disruption in erythropoiesis and B cell differentiation. The same HSC and progenitor composition as well as lineage disruption were also observed in WT recipients of ARE-del BM, further confirming the role of IFN-g in the etiology of AA. In conclusion, our data suggests that AA occurs when IFN-g inhibits the generation of CMP, GMP and MEP from multipotent progenitors and prevents lineage differentiation, as opposed to infiltration of autoreactive T cells into the BM. The finding in this study provides a possible pathogenesis of AA in certain patient populations and a probable cause for disease relapse. These results would be useful in improving treatment as well as maintaining a disease free status.
Background: Inflammatory conditions affect hematopoietic stem cell (HSC) quiescence. Indeed, both interferon alpha (IFNa) and interferon gamma (IFNg) have been shown to promote cell division of HSCs. IFNg-induced cell division does not expand HSC number and it impairs HSC self-renewal, suggesting a role in differentiation. However, the mechanisms by which inflammatory cytokines affect HSC division and differentiation are not well understood. Hypothesis: We postulated that IFNg stimulates myeloid differentiation of HSCs as part of the innate immune response. Methods: We utilized HSCs lacking receptors for interferon alpha or interferon gamma to characterize the requirement for these receptors in HSC repopulating activity. Further, we conducted in vivo infections (both lymphochoriomeningitis virus and Mycobacterium avium) to characterize patterns of hematopoiesis during infection. We separately evaluated myeloid-biased and lymphoid-biased HSCs using molecular and adoptive transfer studies to understand their distinct biology in the context of these infections. Results: Here we report that IFNg signaling is required, both at baseline and during an animal model of LCMV infection, to maintain normal myeloid development. We found that myeloid-biased HSCs express higher levels of IFNg receptor, are specifically activated to divide after recombinant IFNg exposure in vivo, and show increased expression of the transcription factor C/EBPb after infection. Administration of IFNa did not show lineage-specific effects. Furthermore, myeloid-biased HSCs are transiently depleted from the marrow during the IFNg-mediated immune response to Mycobacterium avium infection, as measured both functionally and phenotypically. These findings indicate that IFNg selectively promotes differentiation of myeloid-biased HSCs during an innate immune response to infection. Conclusion: This represents the first report of a context and mechanism for discriminate utilization of alternate HSC subtypes. Terminal differentiation, at the expense of self-renewal, may compromise HSC populations with age or during states of chronic inflammation.