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1.
N Engl J Med ; 385(21): 1929-1940, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34788506

RESUMO

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. METHODS: We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. RESULTS: We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. CONCLUSIONS: The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.).


Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Iduronidase/metabolismo , Mucopolissacaridose I/terapia , Pré-Escolar , Feminino , Seguimentos , Vetores Genéticos , Glicosaminoglicanos/urina , Humanos , Iduronidase/deficiência , Iduronidase/genética , Lactente , Lentivirus , Masculino , Mucopolissacaridose I/metabolismo , Mutação , Transplante de Células-Tronco , Transplante Autólogo
2.
Circulation ; 125(23): 2892-903, 2012 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-22580331

RESUMO

BACKGROUND: Macrophage activation plays a crucial role in regulating adipose tissue inflammation and is a major contributor to the pathogenesis of obesity-associated cardiovascular diseases. On various types of stimuli, macrophages respond with either classic (M1) or alternative (M2) activation. M1- and M2-mediated signaling pathways and corresponding cytokine production profiles are not completely understood. The discovery of microRNAs provides a new opportunity to understand this complicated but crucial network for macrophage activation and adipose tissue function. METHODS AND RESULTS: We have examined the activity of microRNA-223 (miR-223) and its role in controlling macrophage functions in adipose tissue inflammation and systemic insulin resistance. miR-223(-/-) mice on a high-fat diet exhibited an increased severity of systemic insulin resistance compared with wild-type mice that was accompanied by a marked increase in adipose tissue inflammation. The specific regulatory effects of miR-223 in myeloid cell-mediated regulation of adipose tissue inflammation and insulin resistance were then confirmed by transplantation analysis. Moreover, using bone marrow-derived macrophages, we demonstrated that miR-223 is a novel regulator of macrophage polarization, which suppresses classic proinflammatory pathways and enhances the alternative antiinflammatory responses. In addition, we identified Pknox1 as a genuine miR-223 target gene and an essential regulator for macrophage polarization. CONCLUSION: For the first time, this study demonstrates that miR-223 acts to inhibit Pknox1, suppressing proinflammatory activation of macrophages; thus, it is a crucial regulator of macrophage polarization and protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance.


Assuntos
Adipócitos/imunologia , Tecido Adiposo/imunologia , Inflamação/imunologia , Macrófagos/imunologia , MicroRNAs/imunologia , Obesidade/imunologia , Adipócitos/citologia , Tecido Adiposo/citologia , Animais , Transplante de Medula Óssea , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Polaridade Celular/genética , Polaridade Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Gorduras na Dieta/farmacologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Proteínas de Homeodomínio/metabolismo , Imunofenotipagem , Resistência à Insulina/imunologia , Macrófagos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Células Mieloides/citologia , Células Mieloides/imunologia , Valor Preditivo dos Testes , RNA Interferente Pequeno/genética
4.
Stem Cell Reports ; 16(9): 2336-2350, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34450041

RESUMO

Activation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.


Assuntos
Hipóxia Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Receptores Notch/metabolismo , Antígenos CD34/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Biologia Computacional/métodos , Humanos , Anotação de Sequência Molecular , Receptores Notch/genética , Transdução de Sinais , Transcriptoma
5.
Exp Hematol ; 73: 1-6.e6, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30986494

RESUMO

A causal link between hematopoietic stem/progenitor cell (HSPC) dysfunction and DNA damage accrual has been proposed. Clinically relevant strategies to maintain genome integrity in these cells are needed. Here we report that eltrombopag, a small molecule agonist of the thrombopoietin (TPO) receptor used in the clinic, promotes DNA double-strand break (DSB) repair in human HSPCs. We found that eltrombopag specifically activates the classic nonhomologous end-joining (C-NHEJ) DNA repair mechanism, a pathway known to support genome integrity. Eltrombopag-mediated DNA repair results in enhanced genome stability, survival, and function of primary human HSPCs, as demonstrated in karyotyping analyses, colony-forming unit assays and after transplantation in immunodeficient NSG mice. Eltrombopag may offer a new therapeutic modality to protect human HSPCs against genome insults.


Assuntos
Benzoatos/farmacologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Hidrazinas/farmacologia , Pirazóis/farmacologia , Células Cultivadas , Humanos , Receptores de Trombopoetina/metabolismo
6.
J Vis Exp ; (76)2013 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-23851980

RESUMO

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.


Assuntos
Técnicas Citológicas/métodos , Macrófagos/citologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos
7.
Mol Cancer Res ; 11(8): 912-22, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23604034

RESUMO

UNLABELLED: The microRNA miR-150, a critical regulator of hematopoiesis, is downregulated in mixed-lineage leukemia (MLL). In this study, miR-150 acts as a potent leukemic tumor suppressor by blocking the oncogenic properties of leukemic cells. By using MLL-AF9-transformed cells, we demonstrate that ectopic expression of miR-150 inhibits blast colony formation, cell growth, and increases apoptosis in vitro. More importantly, ectopic expression of miR-150 in MLL-AF9-transformed cells completely blocked the development of myeloid leukemia in transplanted mice. Furthermore, gene expression profiling revealed that miR-150 altered the expression levels of more than 30 "stem cell signature" genes and many others that are involved in critical cancer pathways. In addition to the known miR-150 target Myb, we also identified Cbl and Egr2 as bona fide targets and shRNA-mediated suppression of these genes recapitulated the pro-apoptotic effects observed in leukemic cells with miR-150 ectopic expression. In conclusion, we demonstrate that miR-150 is a potent leukemic tumor suppressor that regulates multiple oncogenes. IMPLICATIONS: These data establish new, key players for the development of therapeutic strategies to treat MLL-AF9-related leukemia.


Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Oncogenes , Animais , Apoptose/genética , Apoptose/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Células HEK293 , Humanos , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
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