12 results on '"Anastacia Loberg"'
Search Results
2. Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential
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Johannes Christiaan Maria van der Loo, Mehdi Keddache, Paritha Arumugam, Michael A. Goodman, Dennis D. Hickstein, Danielle Lynn, Phillip J. Dexheimer, Anastacia Loberg, Mohammed Nasimuzzaman, Devin Pillis, David W. Russell, Punam Malik, and Thomas R. Bauer
- Subjects
0301 basic medicine ,viruses ,Immunology ,Genetic Vectors ,Insulator (genetics) ,Microbiology ,Proto-Oncogene Mas ,Virus ,Viral vector ,03 medical and health sciences ,Mice ,Gene Delivery ,Retrovirus ,Transduction, Genetic ,Virology ,CRISPR ,Animals ,Transgenes ,Enhancer ,Cells, Cultured ,Gammaretrovirus ,Adaptor Proteins, Signal Transducing ,biology ,Mutagenicity Tests ,Terminal Repeat Sequences ,Genetic Therapy ,LIM Domain Proteins ,biology.organism_classification ,Hematopoietic Stem Cells ,Long terminal repeat ,Mutagenesis, Insertional ,030104 developmental biology ,Insect Science ,Spumavirus ,Insulator Elements ,CRISPR-Cas Systems - Abstract
Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required. IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy.
- Published
- 2017
3. Activated Transcription Factor 3 in Association with Histone Deacetylase 6 Negatively Regulates MicroRNA 199a2 Transcription by Chromatin Remodeling and Reduces Endothelin-1 Expression
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Chen Li, Yu Zhou, Vijay K. Kalra, Punam Malik, Stanley M. Tahara, and Anastacia Loberg
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0301 basic medicine ,Proteomics ,Transcription, Genetic ,Down-Regulation ,Transcription coregulator ,Anemia, Sickle Cell ,Biology ,Histone Deacetylase 6 ,Histone Deacetylases ,Cell Line ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Sp3 transcription factor ,Animals ,Humans ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Lung ,Sp1 transcription factor ,Histone deacetylase 5 ,Activating Transcription Factor 3 ,Endothelin-1 ,Pioneer factor ,Membrane Proteins ,Cell Biology ,Articles ,Chromatin Assembly and Disassembly ,HDAC4 ,Activating transcription factor 2 ,Cell biology ,Disease Models, Animal ,MicroRNAs ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein - Abstract
Previous studies showed that high levels of placenta growth factor (PlGF) correlated with increased plasma levels of endothelin-1 (ET-1), a potent vasoconstrictor, in sickle cell disease (SCD). PlGF-mediated transcription of the ET-1 gene occurs by activation of hypoxia inducible factor 1α (HIF-1α) and posttranscriptionally by microRNA 199a2 (miR-199a2), which targets the 3' untranslated region (UTR) of HIF-1α mRNA. However, relatively less is known about how PlGF represses the expression of miR-199a2 located in the DNM3 opposite strand (DNM3os) transcription unit. Here, we show that PlGF induces the expression of activated transcription factor 3 (ATF3), which, in association with accessory proteins (c-Jun dimerization protein 2 [JDP2], ATF2, and histone deacetylase 6 [HDAC6]), as determined by proteomic analysis, binds to the DNM3os promoter. Furthermore, we show that association of HDAC6 with ATF3 at its binding site in this promoter was correlated with repression of miR-199a2 transcription, as shown by DNM3os transcription reporter and chromatin immunoprecipitation (ChIP) assays. Tubacin, an inhibitor of HDAC6, antagonized PlGF-mediated repression of DNM3os/pre-miR-199a2 transcription with a concomitant reduction in ET-1 levels in cultured endothelial cells. Analysis of lung tissues from Berkeley sickle (BK-SS) mice showed increased levels of ATF3 and increased expression of ET-1. Delivery of tubacin to BK-SS mice significantly attenuated plasma ET-1 and PlGF levels. Our studies demonstrated that ATF3 in conjunction with HDAC6 acts as a transcriptional repressor of the DNM3os/miR-199a2 locus.
- Published
- 2016
4. Placenta growth factor augments airway hyperresponsiveness via leukotrienes and IL-13
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Vijay K. Kalra, Timothy D. Le Cras, Nancy Ratner, Shiva Kumar Shanmukhappa, Eric B. Brandt, Gurjit K. Khurana Hershey, Devin Pillis, Peter Carmeliet, Bart Jonck, Anastacia Loberg, Tilat A. Rizvi, Paritha Arumugam, Swati Tiwari, Marsha Wills-Karp, Marthe Sandrine Eiymo Mwa Mpollo, Mark Lindsey, and Punam Malik
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medicine.medical_specialty ,Leukotrienes ,Inflammation ,Anemia, Sickle Cell ,Pregnancy Proteins ,Mice ,Th2 Cells ,Internal medicine ,medicine ,Eosinophilia ,Animals ,Hydroxyurea ,Respiratory system ,Asthma ,STAT6 ,Placenta Growth Factor ,Mice, Knockout ,Leukotriene ,Interleukin-13 ,business.industry ,General Medicine ,Zileuton ,respiratory system ,medicine.disease ,respiratory tract diseases ,Disease Models, Animal ,Endocrinology ,Immunology ,Interleukin 13 ,medicine.symptom ,business ,medicine.drug ,Research Article - Abstract
Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.
- Published
- 2014
5. Reactive Oxygen Species Produced by NADPH Oxidase Contribute to Cardiac Pathology in a Mouse Model of Sickle Cell Disease
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Yi Zheng, Anastacia Loberg, Theodosia A. Kalfa, Punam Malik, Satheesh Chonat, Diamantis G. Konstantinidis, Iñigo Valiente-Alandi, Burns C. Blaxall, Georgios E Christakopoulos, Amanda M Schleper, Jeanne James, and Nihal Bakeer
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Cardiac function curve ,medicine.medical_specialty ,Pathology ,NADPH oxidase ,biology ,Reticulocytosis ,Immunology ,Cardiomyopathy ,Diastole ,Cell Biology ,Hematology ,medicine.disease_cause ,medicine.disease ,Biochemistry ,Endocrinology ,Internal medicine ,biology.protein ,medicine ,P22phox ,medicine.symptom ,Isovolumic relaxation time ,Oxidative stress - Abstract
Cardiopulmonary complications are an important contributor to morbidity and mortality in patients with sickle cell anemia (SCA). We have recently discovered that patients with SCA develop cardiomyopathy with restrictive pattern, characterized by diastolic dysfunction with progressive left atrial dilation, superimposed on the anemia-associated hyperdynamic physiology causing hypertrophied, dilated ventricles (Niss et al, JACC: Cardiovascular Imaging, 2016). Using a longitudinal systematic analysis of mouse models of SCA versus mice with iron-deficiency anemia, we found that this unique cardiomyopathy is indeed not due to chronic anemia and is associated with upregulation of genes related to oxidative stress pathways (Bakeer et al, PNAS, in press). These findings along with the known fact of increased inflammation and oxidative stress in SCA led us to postulate that NADPH oxidase (Nox)-mediated reactive oxygen species (ROS) may be an important pathogenic mechanism underlying cardiomyopathy in SCA and consequently mice lacking Nox-mediated ROS might be relatively protected from cardiac dysfunction. To investigate the role of NADPH oxidase in SCA cardiovascular pathophysiology, we bred the knock-in humanized SCA murine model Hbbtm2(HBG1,HBB*)Tow (where mouse α- and β-globin genes have been replaced by the human α- and βS globin genes; commonly known as UAB mice and herein called SS mice) with mice knocked out for the p22phox subunit of NADPH oxidase (a common subunit for all mouse Nox isoforms). We evaluated the phenotype of SS;p22phox-/- mice in blood and determined their cardiac function as compared to SS (p22phox+/+) littermate control mice. Hemolysis and ensuing reticulocytosis did not appear significantly improved in the SS;p22phox-/- mice; red blood cell (RBC) ROS was also stable likely due to the major contribution of mitochondrial ROS in the reticulocytes. Baseline ROS levels in the neutrophils of SS;p22phox-/-mice were similar to the levels in SS mice, but inducible ROS was almost eliminated as expected with deficiency of the p22phox subunit and inactivation of all NADPH oxidase isoforms. Starting at 8 weeks of age, cardiac structure and function were assessed on age-matched SS and SS;p22phox-/- mice by serial echocardiography. We studied 3 timepoints: 8-15 weeks of age, 16-24 weeks, and 25-34 weeks. SS mice developed progressively increased left atrial dimension (LAd) starting at 16 weeks of age while SS;p22phox-/- mice had stable LAd, with values similar to WT (Figure 1A). This difference became more pronounced with aging (P=0.02 at 25-34 weeks). Isovolumic relaxation time (IVRT), the time interval between closure of the aortic valve and opening of the mitral valve, was prolonged in the SS mice older than 16 weeks of age, consistent with diastolic dysfunction. In contrast, SS;p22phox-/- mice had no change in IVRT (Figure 1B). Also consistent with diastolic dysfunction, the ratio of transmitral E and e-(MV IVS E/e-) was increased in the SS group vs SS;p22phox-/-. Finally, the SS mice showed elevated left ventricular (LV) mass and decreased LV shortening fraction by 22-34 weeks whereas these parameters were preserved in the SS;p22phox-/-mice. Histopathology studies were performed to evaluate changes in the cardiac tissue. In the SS mice, H&E, picrosirius red and Masson trichrome staining showed not only significant LV hypertrophy and dilation but also significant interstitial fibrosis. Immunostaining for the extracellular matrix proteins collagen and fibronectin, showed extensive deposition of these proteins in the SS mouse heart. In contrast, SS; p22phox-/-mouse hearts were relatively spared. In conclusion, our data show that SS;p22phox-/-mice demonstrate better preserved diastolic and systolic heart function compared to SS mice, and decreased heart tissue damage. These findings suggest that therapeutic manuevers aimed at decreasing oxidative stress in SCA may be an effective strategy to counter SCA cardiomyopathy. Figure 1 Echocardiographic parameters evaluating diastolic function of the heart of SS vs SS;p22phox-/-mice. A. Left atrial dimension (LAd). B. Isovolumic relaxation time (IVRT). SS: Hbbtm2(HBG1,HBB*)Tow (UAB) with wild-type p22phox; SS;p22phox-/-: UAB mice with targetted deletion of p22phox.(statistical analysis was performed using Wilcoxon rank-sum test; JMP 9.0 SAS Institute, Cary, North Carolina) Figure 1. Echocardiographic parameters evaluating diastolic function of the heart of SS vs SS;p22phox-/-mice. A. Left atrial dimension (LAd). B. Isovolumic relaxation time (IVRT). SS: Hbbtm2(HBG1,HBB*)Tow (UAB) with wild-type p22phox; SS;p22phox-/-: UAB mice with targetted deletion of p22phox.(statistical analysis was performed using Wilcoxon rank-sum test; JMP 9.0 SAS Institute, Cary, North Carolina) Disclosures No relevant conflicts of interest to declare.
- Published
- 2016
6. Placenta Growth Factor Links the IL-13 Response and the Leukotriene Pathway to Augment Airway Hyper-Responsiveness
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Timothy D. LeCras, Shiva Kumar Shanmukhappa, Paritha Arumugam, Marthe-Sandrine Eiymo Mwa Mpollo, Gurjit K. Khurana Hershey, Punam Malik, Peter Carmeliet, Tilat A. Rizvi, Swati Tiwari, Marsha Wills-Karp, Mark Lindsey, Eric B. Brandt, Bart Jonckx, Nancy Ratner, Vijay K. Kalra, and Anastacia Loberg
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medicine.medical_specialty ,medicine.medical_treatment ,Immunology ,Inflammation ,Biochemistry ,Internal medicine ,medicine ,Eosinophilia ,Leukotriene ,Lung ,biology ,business.industry ,Cell Biology ,Hematology ,Zileuton ,respiratory system ,respiratory tract diseases ,Cytokine ,Endocrinology ,medicine.anatomical_structure ,Arachidonate 5-lipoxygenase ,Interleukin 13 ,biology.protein ,medicine.symptom ,business ,medicine.drug - Abstract
Airway hyper-responsiveness (AHR) affects 55-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the underlying mechanisms of this highly prevalent AHR in a hemoglobinopathy remain unknown. We hypothesized that Placenta Growth Factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates this AHR. We studied AHR in allergen-exposed PlGF-/- and SCD mice. Sickle mice genetically deficient in PlGF were not viable. PlGF augmented AHR, lung inflammation and blood/lung eosinophilia via IL-13 (a Th2-cytokine) and increased 5-lipoxygenase (a leukotriene synthetic enzyme) expression. AHR, or pulmonary inflammation and leukotriene levels were blunted in PlGF-/- mice or in PlGFWT mice treated with anti-PlGF-Ab, and was rescued by intratracheal administration of leukotrienes to PlGF-/- mice. Notably, Th2-mediated Stat-6 activation further increased PlGF expression from lung epithelium, eosinophils and macrophages, linking the PlGF-leukotriene pathway in a positive feedback loop. Th2 response is classically seen in asthma, and indeed, expression of PlGF and its downstream genes was increased in respiratory epithelial cells of asthma patients. Like patients with SCD, sickle hematopoietic chimeras developed increased AHR and had higher leukotriene levels; and both were abrogated by anti-PlGF-Ab or zileuton (5-lipoxygenase inhibitor) phenocopying the blunted AHR in PlGF-/- mice, and indicating that sickle erythroid-cells/RBC potentiate AHR. Overall, PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. Both zileuton, licensed for asthma and anti-PlGF-Ab, in clinical trials for cancer, could be promising therapies to reduce the pulmonary morbidity in SCD. Disclosures Jonckx: ThromboGenics: Employment.
- Published
- 2015
7. A Precise Tool to Edit Human Hematopoietic Stem Cells at High Efficiency and Decipher and Optimize the Different Double Strand Break Repair Outcomes
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Jayavaradhan, Rajeswari, Pillis, Devin, McGraw, Sarah, Anastacia, Loberg, Andreassen, Paul R., and Malik, Punam
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- 2017
- Full Text
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8. Uncoupling Sickle Hematopoiesis Induced Angiotensin Receptor Signaling in Sickle Nephropathy
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Roy, Swarnava, Rai, Parul, Eiymo Mwa Mpollo, Marthe-Sandrine, Chang, Kyung-Hee, Rizvi, Tilat, Anastacia, Loberg, Aronow, Bruce J., Inagami, Tadashi, Witte, David, Cancelas, Jose A, and Malik, Punam
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- 2017
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9. Increased Oxidative Stress In Sickle Cell Disease Activates The Renin-Angiotensin-TGF-β Pathway To Mediate Sickle Nephropathy
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Roy, Swarnava, primary, Konstantinidis, Diamantis G., additional, Rizvi, Tilat, additional, Chang, Kyung-Hee, additional, Sundaram, Nambirajan, additional, Wourms, Michael, additional, Anastacia, Loberg, additional, Song, Lanxi, additional, Burke, Katie, additional, Inagami, Tadashi, additional, Ratner, Nancy, additional, Witte, David, additional, Kalfa, Theodosia A., additional, Cancelas, Jose A, additional, Shanmukhappa, Shiva Kumar, additional, and Malik, Punam, additional
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- 2013
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10. Diminished Multi-Organ Pathologies and Inflammation Associated With Sickle Cell Disease In Mice With Genetically Limited Prothrombin Levels
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Paritha Arumugam, Eric S. Mullins, Maureen A Shaw, Kumar Shanmukhappa, Anastacia Loberg, Tilat Rizvi, Janaka Wansapura, David Witte, Punam Malik, and Jay L. Degen
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Hemolytic anemia ,Kidney ,medicine.medical_specialty ,business.industry ,Immunology ,Organ dysfunction ,Cell Biology ,Hematology ,Smooth muscle hyperplasia ,medicine.disease ,Biochemistry ,Pulmonary hypertension ,Muscle hypertrophy ,Pathogenesis ,medicine.anatomical_structure ,Endocrinology ,Right ventricular hypertrophy ,Internal medicine ,medicine ,medicine.symptom ,business - Abstract
Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso-occlusions, chronic inflammation and an oxidative state, with progressive and widespread organ dysfunction and reduced life expectancy. Systemic coagulation system activation has been well documented in both SCD patients and animal models of SCD. However, the precise causal relationship, if any, between hemostatic factors and SCD pathogenesis has remained uncertain. Here, we test the hypothesis that thrombin-mediated proteolysis is a key mediator of the end-organ pathologies in SCD by evaluating the phenotypic consequences of imposed deficits in prothrombin in the well-characterized humanized murine model of SCD, Berkeley sickle mice (HbS mice). Prothrombin (fII) specific anti-sense oligonucleotide (ASO) gapmers (ISIS Pharmaceutics) were used to pharmacologically suppress circulating plasma fII levels to less than 10% of normal in cohorts of HbS mice starting at 3 weeks of age. Kaplan-Meier analysis revealed a major survival benefit in HbS mice when hepatic fII expression and circulating fII levels were selectively lowered, with ∼90% of fIILow mice surviving to 15 weeks of age as compared to just 60% of HbS mice treated with control ASO gapmer (P Arumugam P and Mullins E contributed equally. Malik P and Degen J are Co-corresponding authors. Disclosures: Mullins: Baxter : Consultancy.
- Published
- 2013
11. Correction of Sickle Cell Anemia with γ-Globin Gene Delivered by Lentivirus Vector in the Setting of Myeloablative or Reduced Intensity Conditioning, and Establishing Critical Determinants for Successful Gene Therapy for Sickle Cell Disease
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Herbert J. Meiselman, Anastacia Loberg, Robert S. Franco, Tomoyasu Higashimoto, David P. Witte, Kristy Lauderback, Gabriel Estevez-Pagani, Ajay Perumbeti, Fabrizia Urbinati, Punam Malik, and Clinton H. Joiner
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Myeloid ,Anemia ,business.industry ,Genetic enhancement ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Sickle cell anemia ,Andrology ,Haematopoiesis ,medicine.anatomical_structure ,Fetal hemoglobin ,medicine ,Leukocytosis ,Bone marrow ,medicine.symptom ,business - Abstract
While genetic delivery of recombinant anti-sickling β-globin genes have been shown to correct murine sickle cell anemia (SCA), correction of SCA by delivery of a natural hemoglobin, fetal hemoglobin (HbF), the proportion of genetically modified hematopoietic stem cells (HSC), or amount of HbF necessary to correct the disease is unknown. We designed a lentivirus vector carrying γ-globin exons with β-globin regulatory elements and non-coding sequences, GbG. First, GbG or mock transduced Berkeley sickle HSC were transplanted using a myeloablative (lethal irradiation) transplant model, to acheive full donor chimerism. GbG mice showed high HbF expression (HbF 41 ± 5% measured by HPLC) that was sustained in primary (6 mo) and secondary (7.5 mo) transplant recipients, and resulted in effective correction of hematological and functional RBC parameters, and reduction of inflammation that results from sickle cell disease. We found significantly reduced irreversibly sickled cells (2.3 ± 0.7% in GbG versus 10.2 ± 0.3% in mock mice; p30% HbF/F-cell and >60% F-cells consistently corrected SCA. The mean HSC transduction (assessed by secondary HbF+ CFU-S at 6 months post transplant) was 50% and 30% in the myeloablative and reduced intensity transplant models, respectively, with 1–3 GbG copies/ cell. Furthermore, three GbG mice showed correction of SCA with 20% HSC transduction, a clinically achievable goal. Taken together, this study is the first demonstration of correction of SCA with gene therapy using γ-globin, and defines critical determinants for effective gene therapy of this disease. Mouse Model Hb (g/dl) RBC 106/ul) Reticulocyte (%) WBC (K/ul) *p
- Published
- 2008
12. Increased Oxidative Stress In Sickle Cell Disease Activates The Renin-Angiotensin-TGF-ß Pathway To Mediate Sickle Nephropathy
- Author
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Roy, Swarnava, Konstantinidis, Diamantis G., Rizvi, Tilat, Chang, Kyung-Hee, Sundaram, Nambirajan, Wourms, Michael, Anastacia, Loberg, Song, Lanxi, Burke, Katie, Inagami, Tadashi, Ratner, Nancy, Witte, David, Kalfa, Theodosia A., Cancelas, Jose A, Shanmukhappa, Shiva Kumar, and Malik, Punam
- Abstract
No relevant conflicts of interest to declare.
- Published
- 2013
- Full Text
- View/download PDF
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