Dana Levasseur | Novartis - Academia.edu (original) (raw)

Papers by Dana Levasseur

Stem Cells, Sep 1, 2000

Lentiviral vectors efficiently transduce human CD34 + cells that mediate long-term engraftment of... more Lentiviral vectors efficiently transduce human CD34 + cells that mediate long-term engraftment of nonobese diabetic/severe combined immunodeficient mice. However, hematopoiesis in these animals is abnormal. Typically, 95% of the human cells in peripheral blood are B lymphocytes. To determine whether lentiviral vectors efficiently transduce stem cells that maintain normal hematopoiesis in vivo, we isolated Sca-1 + c-Kit + Linbone marrow cells from mice without 5-fluorouracil treatment, and transduced these cells in the absence of cytokine stimulation with a novel lentiviral vector containing a GFP (green flourescent protein) reporter gene. These cells were transplanted into lethally irradiated C57Bl/6 mice. In fully reconstituted animals, GFP expression was observed in 8.0% of peripheral blood mononuclear cells for 20 weeks posttransplantation. Lineage analysis demonstrated that a similar percentage (approximately 8.0%) of GFP-positive cells was detected in peripheral blood B cells, T cells, granulocytes and monocytes, bone marrow erythroid precursor cells, splenic B cells, and thymic T cells. In secondary transplant recipients, up to 20% of some lineages expressed GFP. Our results suggest that quiescent, hematopoietic stem cells are efficiently transduced by lentiviral vectors without impairing self-renewal and normal lineage specification in vivo. Efficient gene delivery into murine stem cells with lentiviral vectors will allow direct tests of genetic therapies in mouse models of hematopoietic diseases such as sickle cell anemia and thalassemia, in which corrected cells may have a selective survival advantage.

Investigative Ophthalmology & Visual Science, Dec 30, 2015

PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in ... more PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial (CECs), RPE, and photoreceptor cells. Stem cell-based treatment strategies designed to replace photoreceptor and RPE cells currently are a major scientific focus. However, the success of these approaches likely also will require replacement of the underlying, supportive choroidal vasculature. The purpose of this study was to generate stem cell-derived CECs to develop efficient differentiation and transplantation protocols. METHODS. Dermal fibroblasts from the Tie2-GFP mouse were isolated and reprogrammed into two independent induced pluripotent stem cell (iPSC) lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Tie2-GFP iPSCs were differentiated into CECs using a coculture method with either the RF6A CEC line or primary mouse CECs. Induced pluripotent stem cell-derived CECs were characterized via RT-PCR and immunocytochemistry for EC-and CEC-specific markers. RESULTS. Induced pluripotent stem cells generated from mice expressing green fluorescent protein (GFP) under control of the endothelial Tie2 promoter display classic pluripotency markers and stem cell morphology. Induced pluripotent stem cell-derived CECs express carbonic anhydrase IV, eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. CONCLUSIONS. Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.

Methods in molecular biology, 2013

Embryonic stem cells (ESCs) are defined by their simultaneous capacity for limitless self-renewal... more Embryonic stem cells (ESCs) are defined by their simultaneous capacity for limitless self-renewal and the ability to specify cells borne of all germ layers. The regulation of ESC pluripotency is governed by a set of core transcription factors that regulate transcription by interfacing with nuclear proteins that include the RNA polymerase II core transcriptional machinery, histone modification enzymes, and chromatin remodeling protein complexes. The growing adoption of systems biological approaches used in stem cell biology over last few years has contributed significantly to our understanding of pluripotency. Multilayered approaches coupling transcriptome profiling and proteomics (Nanog-, Oct4-, and Sox2-centered protein interaction networks or "interactomes") with transcription factor chromatin occupancy and epigenetic footprint measurements have enabled a more comprehensive understanding of ESC pluripotency and self-renewal. Together with the genetic and biochemical characterization of promising pluripotency modifying proteins, these systems biological approaches will continue to clarify the molecular underpinnings of the ESC state. This will most certainly contribute to the improvement of current methodologies for the derivation of pluripotent cells from adult tissues.

Frontiers in Bioscience, 2006

Proceedings of the National Academy of Sciences of the United States of America, Apr 29, 2008

Blood, Nov 13, 2019

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and functi... more Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have been developed that significantly increase the level of HbF following nucleotide conversion at key regulatory motifs within the HBG1 and HBG2 promoters. CD34+ hematopoietic stem and progenitor cells (HSPC) were purified at clinical scale and edited using a process designed to preserve self-renewal capacity. Editing at two independent sites with different ABEs reached 94 percent and resulted in up to 63 percent gamma globin by UPLC. The levels of HbF observed should afford protection to the majority of SCD and Beta thalassemia patients based on clinical observations of HPFH and non-interventional therapy that links higher HbF dosage with milder disease (Ngo et al, 2011 Brit J Hem; Musallam et al, 2012 Blood). Directly correcting the Glu6Val mutation of SCD has been a recent goal of genetic therapies designed for the SCD population. Current base editing technology cannot yet convert mutations like those that result from the A-T transversion in sickle beta globin; however, ABE variants have been designed to recognize and edit the opposite stranded adenine residue of valine. This results in the conversion of valine to alanine and the production of a naturally occurring variant known as Hb G-Makassar. Beta globin with alanine at this position does not contribute to polymer formation, and patients with Hb G-Makassar present with normal hematological parameters and red blood cell morphology. SCD patient fibroblasts edited with these ABE variants achieve up to 70 percent conversion of the target adenine. CD34 cells from healthy donors were then edited with a lead ABE variant, targeting a synonymous mutation in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. The average editing frequency was 40 percent. Donor myeloid chimerism documented at these levels in the allogeneic transplant setting exceeds the 20 percent that is required for reversing the sickle phenotype (Fitzhugh et al, 2017 Blood). These next generation editing approaches provide a promising new modality for treating patients with Beta thalassemia and SCD. Disclosures No relevant conflicts of interest to declare.

Leukemia, Dec 24, 2013

Letter to the Editor Increased aldehyde dehydrogenase (ALDH) activity has been found in murine an... more Letter to the Editor Increased aldehyde dehydrogenase (ALDH) activity has been found in murine and human hematopoietic stem cells (HSC) and human multiple myeloma stem cells (MMSCs). 1-3 ALDHs are a group of NAD(P)-dependent enzymes involved in drug resistance and retinoic acid metabolism, which are crucial for the protection of stem cells against toxic endogenous and exogenous aldehydes. Recently, the isolation and functional analysis of ALDH1positive (ALDH1 +) cells has been greatly facilitated by the development of the Aldefluor assay. 4 Matsui et al. reported that ALDH1 activity is increased in MMSCs. 3, 5 In the present study, we have addressed the question of whether ALDH1 + MM cells can initiate MM tumor formation and have evaluated the functional role of ALDH1 in MM cell growth, clonogenic ability, and cell signaling. To determine whether MM cell lines contain an ALDH1 stem cell like population, we evaluated ALDH1 activity using the Aldefluor assay in eleven human and one mouse MM cell lines. Flow cytometry demonstrated that all MM cell lines contained a very small subset of cells that were positive for ALDH1 (ALDH1 +) with a range from 0.1 to 4.85% of total cells (Figure 1A and 1B). Human MM cell lines (KMS12BM, XG6, U266, ARP1) and the mouse MM cell line (5TGM1) had the highest fraction of ALDH1 + cells with 4.85%, 3.6%, 2.1%, 1.7%, and 1.9% respectively (Figure 1B), while the remainder of the human MM cell lines (OPM1, JJN3, H929, KMS28, 8226, XG1, OCI-MY5 and OPM2) showed a very low percentage of ALDH1 + cells (< 1.5%; Figure 1B). We also analyzed ALDH1 + cells in primary MM samples by combing enzyme activity measurements with flow cytometric

Cancer Research, Sep 15, 2004

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively no... more Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.

Blood Cells Molecules and Diseases, Mar 1, 2007

Blood, 2019

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and functi... more Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have...

Cell reports, Mar 28, 2017

Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 prefer... more Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R2...

Investigative Opthalmology & Visual Science, 2015

PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in ... more PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial (CECs), RPE, and photoreceptor cells. Stem cell-based treatment strategies designed to replace photoreceptor and RPE cells currently are a major scientific focus. However, the success of these approaches likely also will require replacement of the underlying, supportive choroidal vasculature. The purpose of this study was to generate stem cell-derived CECs to develop efficient differentiation and transplantation protocols. METHODS. Dermal fibroblasts from the Tie2-GFP mouse were isolated and reprogrammed into two independent induced pluripotent stem cell (iPSC) lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Tie2-GFP iPSCs were differentiated into CECs using a coculture method with either the RF6A CEC line or primary mouse CECs. Induced pluripotent stem cell-derived CECs were characterized via RT-PCR and immunocytochemistry for EC-and CEC-specific markers. RESULTS. Induced pluripotent stem cells generated from mice expressing green fluorescent protein (GFP) under control of the endothelial Tie2 promoter display classic pluripotency markers and stem cell morphology. Induced pluripotent stem cell-derived CECs express carbonic anhydrase IV, eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. CONCLUSIONS. Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.

STEM CELLS, 2000

Lentiviral vectors efficiently transduce human CD34 + cells that mediate long-term engraftment of... more Lentiviral vectors efficiently transduce human CD34 + cells that mediate long-term engraftment of nonobese diabetic/severe combined immunodeficient mice. However, hematopoiesis in these animals is abnormal. Typically, 95% of the human cells in peripheral blood are B lymphocytes. To determine whether lentiviral vectors efficiently transduce stem cells that maintain normal hematopoiesis in vivo, we isolated Sca-1 + c-Kit + Linbone marrow cells from mice without 5-fluorouracil treatment, and transduced these cells in the absence of cytokine stimulation with a novel lentiviral vector containing a GFP (green flourescent protein) reporter gene. These cells were transplanted into lethally irradiated C57Bl/6 mice. In fully reconstituted animals, GFP expression was observed in 8.0% of peripheral blood mononuclear cells for 20 weeks posttransplantation. Lineage analysis demonstrated that a similar percentage (approximately 8.0%) of GFP-positive cells was detected in peripheral blood B cells, T cells, granulocytes and monocytes, bone marrow erythroid precursor cells, splenic B cells, and thymic T cells. In secondary transplant recipients, up to 20% of some lineages expressed GFP. Our results suggest that quiescent, hematopoietic stem cells are efficiently transduced by lentiviral vectors without impairing self-renewal and normal lineage specification in vivo. Efficient gene delivery into murine stem cells with lentiviral vectors will allow direct tests of genetic therapies in mouse models of hematopoietic diseases such as sickle cell anemia and thalassemia, in which corrected cells may have a selective survival advantage.

PLoS ONE, 2013

To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first ... more To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4 + cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4 + epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6β4-epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4 + epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs) serotypes, AAV2 and AAV8, capable of transducing α6β4 + cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4 + epithelial cells significantly rescued defects in Cltransport. Therefore, targeting the α6β4 + epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.

Journal of Biological Chemistry, 2004

A new recombinant, human anti-sickling ␤-globin polypeptide designated ␤ AS3 (␤Gly 16 3 Asp/␤Glu ... more A new recombinant, human anti-sickling ␤-globin polypeptide designated ␤ AS3 (␤Gly 16 3 Asp/␤Glu 22 3 Ala/␤Thr 87 3 Gln) was designed to increase affinity for ␣-globin. The amino acid substitutions at ␤22 and ␤87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The ␤16 substitution confers the recombinant ␤-globin subunit (␤ AS3) with a competitive advantage over ␤ S for interaction with the ␣-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (␣ 2 ␤ AS3 2) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that ␤ AS3 has a distinct advantage over ␤ S for dimerization with ␣-globin. When equal amounts of ␤ Sand ␤ AS3-globin monomers compete for limiting ␣-globin chains up to 82% of the tetramers formed is HbAS3. Knockout transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knockout transgenic sickle mice the ␤ AS3 polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of ␤ AS3-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit ␣-thalassemia. Therefore, constructs expressing the ␤ AS3-globin gene may be suitable for future clinical trials for sickle cell disease.

Stem Cells, Sep 1, 2000

Lentiviral vectors efficiently transduce human CD34 + cells that mediate long-term engraftment of... more Lentiviral vectors efficiently transduce human CD34 + cells that mediate long-term engraftment of nonobese diabetic/severe combined immunodeficient mice. However, hematopoiesis in these animals is abnormal. Typically, 95% of the human cells in peripheral blood are B lymphocytes. To determine whether lentiviral vectors efficiently transduce stem cells that maintain normal hematopoiesis in vivo, we isolated Sca-1 + c-Kit + Linbone marrow cells from mice without 5-fluorouracil treatment, and transduced these cells in the absence of cytokine stimulation with a novel lentiviral vector containing a GFP (green flourescent protein) reporter gene. These cells were transplanted into lethally irradiated C57Bl/6 mice. In fully reconstituted animals, GFP expression was observed in 8.0% of peripheral blood mononuclear cells for 20 weeks posttransplantation. Lineage analysis demonstrated that a similar percentage (approximately 8.0%) of GFP-positive cells was detected in peripheral blood B cells, T cells, granulocytes and monocytes, bone marrow erythroid precursor cells, splenic B cells, and thymic T cells. In secondary transplant recipients, up to 20% of some lineages expressed GFP. Our results suggest that quiescent, hematopoietic stem cells are efficiently transduced by lentiviral vectors without impairing self-renewal and normal lineage specification in vivo. Efficient gene delivery into murine stem cells with lentiviral vectors will allow direct tests of genetic therapies in mouse models of hematopoietic diseases such as sickle cell anemia and thalassemia, in which corrected cells may have a selective survival advantage.

Investigative Ophthalmology & Visual Science, Dec 30, 2015

PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in ... more PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial (CECs), RPE, and photoreceptor cells. Stem cell-based treatment strategies designed to replace photoreceptor and RPE cells currently are a major scientific focus. However, the success of these approaches likely also will require replacement of the underlying, supportive choroidal vasculature. The purpose of this study was to generate stem cell-derived CECs to develop efficient differentiation and transplantation protocols. METHODS. Dermal fibroblasts from the Tie2-GFP mouse were isolated and reprogrammed into two independent induced pluripotent stem cell (iPSC) lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Tie2-GFP iPSCs were differentiated into CECs using a coculture method with either the RF6A CEC line or primary mouse CECs. Induced pluripotent stem cell-derived CECs were characterized via RT-PCR and immunocytochemistry for EC-and CEC-specific markers. RESULTS. Induced pluripotent stem cells generated from mice expressing green fluorescent protein (GFP) under control of the endothelial Tie2 promoter display classic pluripotency markers and stem cell morphology. Induced pluripotent stem cell-derived CECs express carbonic anhydrase IV, eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. CONCLUSIONS. Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.

Methods in molecular biology, 2013

Embryonic stem cells (ESCs) are defined by their simultaneous capacity for limitless self-renewal... more Embryonic stem cells (ESCs) are defined by their simultaneous capacity for limitless self-renewal and the ability to specify cells borne of all germ layers. The regulation of ESC pluripotency is governed by a set of core transcription factors that regulate transcription by interfacing with nuclear proteins that include the RNA polymerase II core transcriptional machinery, histone modification enzymes, and chromatin remodeling protein complexes. The growing adoption of systems biological approaches used in stem cell biology over last few years has contributed significantly to our understanding of pluripotency. Multilayered approaches coupling transcriptome profiling and proteomics (Nanog-, Oct4-, and Sox2-centered protein interaction networks or &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;interactomes&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;) with transcription factor chromatin occupancy and epigenetic footprint measurements have enabled a more comprehensive understanding of ESC pluripotency and self-renewal. Together with the genetic and biochemical characterization of promising pluripotency modifying proteins, these systems biological approaches will continue to clarify the molecular underpinnings of the ESC state. This will most certainly contribute to the improvement of current methodologies for the derivation of pluripotent cells from adult tissues.

Frontiers in Bioscience, 2006

Proceedings of the National Academy of Sciences of the United States of America, Apr 29, 2008

Blood, Nov 13, 2019

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and functi... more Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have been developed that significantly increase the level of HbF following nucleotide conversion at key regulatory motifs within the HBG1 and HBG2 promoters. CD34+ hematopoietic stem and progenitor cells (HSPC) were purified at clinical scale and edited using a process designed to preserve self-renewal capacity. Editing at two independent sites with different ABEs reached 94 percent and resulted in up to 63 percent gamma globin by UPLC. The levels of HbF observed should afford protection to the majority of SCD and Beta thalassemia patients based on clinical observations of HPFH and non-interventional therapy that links higher HbF dosage with milder disease (Ngo et al, 2011 Brit J Hem; Musallam et al, 2012 Blood). Directly correcting the Glu6Val mutation of SCD has been a recent goal of genetic therapies designed for the SCD population. Current base editing technology cannot yet convert mutations like those that result from the A-T transversion in sickle beta globin; however, ABE variants have been designed to recognize and edit the opposite stranded adenine residue of valine. This results in the conversion of valine to alanine and the production of a naturally occurring variant known as Hb G-Makassar. Beta globin with alanine at this position does not contribute to polymer formation, and patients with Hb G-Makassar present with normal hematological parameters and red blood cell morphology. SCD patient fibroblasts edited with these ABE variants achieve up to 70 percent conversion of the target adenine. CD34 cells from healthy donors were then edited with a lead ABE variant, targeting a synonymous mutation in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. The average editing frequency was 40 percent. Donor myeloid chimerism documented at these levels in the allogeneic transplant setting exceeds the 20 percent that is required for reversing the sickle phenotype (Fitzhugh et al, 2017 Blood). These next generation editing approaches provide a promising new modality for treating patients with Beta thalassemia and SCD. Disclosures No relevant conflicts of interest to declare.

Leukemia, Dec 24, 2013

Letter to the Editor Increased aldehyde dehydrogenase (ALDH) activity has been found in murine an... more Letter to the Editor Increased aldehyde dehydrogenase (ALDH) activity has been found in murine and human hematopoietic stem cells (HSC) and human multiple myeloma stem cells (MMSCs). 1-3 ALDHs are a group of NAD(P)-dependent enzymes involved in drug resistance and retinoic acid metabolism, which are crucial for the protection of stem cells against toxic endogenous and exogenous aldehydes. Recently, the isolation and functional analysis of ALDH1positive (ALDH1 +) cells has been greatly facilitated by the development of the Aldefluor assay. 4 Matsui et al. reported that ALDH1 activity is increased in MMSCs. 3, 5 In the present study, we have addressed the question of whether ALDH1 + MM cells can initiate MM tumor formation and have evaluated the functional role of ALDH1 in MM cell growth, clonogenic ability, and cell signaling. To determine whether MM cell lines contain an ALDH1 stem cell like population, we evaluated ALDH1 activity using the Aldefluor assay in eleven human and one mouse MM cell lines. Flow cytometry demonstrated that all MM cell lines contained a very small subset of cells that were positive for ALDH1 (ALDH1 +) with a range from 0.1 to 4.85% of total cells (Figure 1A and 1B). Human MM cell lines (KMS12BM, XG6, U266, ARP1) and the mouse MM cell line (5TGM1) had the highest fraction of ALDH1 + cells with 4.85%, 3.6%, 2.1%, 1.7%, and 1.9% respectively (Figure 1B), while the remainder of the human MM cell lines (OPM1, JJN3, H929, KMS28, 8226, XG1, OCI-MY5 and OPM2) showed a very low percentage of ALDH1 + cells (< 1.5%; Figure 1B). We also analyzed ALDH1 + cells in primary MM samples by combing enzyme activity measurements with flow cytometric

Cancer Research, Sep 15, 2004

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively no... more Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.

Blood Cells Molecules and Diseases, Mar 1, 2007

Blood, 2019

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and functi... more Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have...

Cell reports, Mar 28, 2017

Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 prefer... more Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R2...

Investigative Opthalmology & Visual Science, 2015

PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in ... more PURPOSE. Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial (CECs), RPE, and photoreceptor cells. Stem cell-based treatment strategies designed to replace photoreceptor and RPE cells currently are a major scientific focus. However, the success of these approaches likely also will require replacement of the underlying, supportive choroidal vasculature. The purpose of this study was to generate stem cell-derived CECs to develop efficient differentiation and transplantation protocols. METHODS. Dermal fibroblasts from the Tie2-GFP mouse were isolated and reprogrammed into two independent induced pluripotent stem cell (iPSC) lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Tie2-GFP iPSCs were differentiated into CECs using a coculture method with either the RF6A CEC line or primary mouse CECs. Induced pluripotent stem cell-derived CECs were characterized via RT-PCR and immunocytochemistry for EC-and CEC-specific markers. RESULTS. Induced pluripotent stem cells generated from mice expressing green fluorescent protein (GFP) under control of the endothelial Tie2 promoter display classic pluripotency markers and stem cell morphology. Induced pluripotent stem cell-derived CECs express carbonic anhydrase IV, eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. CONCLUSIONS. Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.

STEM CELLS, 2000

Lentiviral vectors efficiently transduce human CD34 + cells that mediate long-term engraftment of... more Lentiviral vectors efficiently transduce human CD34 + cells that mediate long-term engraftment of nonobese diabetic/severe combined immunodeficient mice. However, hematopoiesis in these animals is abnormal. Typically, 95% of the human cells in peripheral blood are B lymphocytes. To determine whether lentiviral vectors efficiently transduce stem cells that maintain normal hematopoiesis in vivo, we isolated Sca-1 + c-Kit + Linbone marrow cells from mice without 5-fluorouracil treatment, and transduced these cells in the absence of cytokine stimulation with a novel lentiviral vector containing a GFP (green flourescent protein) reporter gene. These cells were transplanted into lethally irradiated C57Bl/6 mice. In fully reconstituted animals, GFP expression was observed in 8.0% of peripheral blood mononuclear cells for 20 weeks posttransplantation. Lineage analysis demonstrated that a similar percentage (approximately 8.0%) of GFP-positive cells was detected in peripheral blood B cells, T cells, granulocytes and monocytes, bone marrow erythroid precursor cells, splenic B cells, and thymic T cells. In secondary transplant recipients, up to 20% of some lineages expressed GFP. Our results suggest that quiescent, hematopoietic stem cells are efficiently transduced by lentiviral vectors without impairing self-renewal and normal lineage specification in vivo. Efficient gene delivery into murine stem cells with lentiviral vectors will allow direct tests of genetic therapies in mouse models of hematopoietic diseases such as sickle cell anemia and thalassemia, in which corrected cells may have a selective survival advantage.

PLoS ONE, 2013

To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first ... more To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4 + cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4 + epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6β4-epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4 + epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs) serotypes, AAV2 and AAV8, capable of transducing α6β4 + cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4 + epithelial cells significantly rescued defects in Cltransport. Therefore, targeting the α6β4 + epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.

Journal of Biological Chemistry, 2004

A new recombinant, human anti-sickling ␤-globin polypeptide designated ␤ AS3 (␤Gly 16 3 Asp/␤Glu ... more A new recombinant, human anti-sickling ␤-globin polypeptide designated ␤ AS3 (␤Gly 16 3 Asp/␤Glu 22 3 Ala/␤Thr 87 3 Gln) was designed to increase affinity for ␣-globin. The amino acid substitutions at ␤22 and ␤87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The ␤16 substitution confers the recombinant ␤-globin subunit (␤ AS3) with a competitive advantage over ␤ S for interaction with the ␣-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (␣ 2 ␤ AS3 2) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that ␤ AS3 has a distinct advantage over ␤ S for dimerization with ␣-globin. When equal amounts of ␤ Sand ␤ AS3-globin monomers compete for limiting ␣-globin chains up to 82% of the tetramers formed is HbAS3. Knockout transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knockout transgenic sickle mice the ␤ AS3 polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of ␤ AS3-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit ␣-thalassemia. Therefore, constructs expressing the ␤ AS3-globin gene may be suitable for future clinical trials for sickle cell disease.