RESUMO
Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here, we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore, gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks), and it was accompanied by epigenetic remodeling events. Finally, we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion, we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing.
RESUMO
B cells can differentiate into plasmablast and plasma cells, capable of producing antibodies for decades. Gene editing using zinc-finger nucleases (ZFN) enables the engineering of B cells capable of secreting sustained and high levels of therapeutic proteins. In this study, we established an advanced in vitro good manufacturing practice-compatible culturing system characterized by robust and consistent expansion rate, high viability, and efficient B cell differentiation. Using this process, an optimized B cell editing protocol was developed by combining ZFN/adeno-associated virus 6 technology to achieve site-specific insertion of the human factor IX R338L Padua into the silent TRAC locus. In vitro analysis revealed high levels of secreted human immunoglobulins and human factor IX-Padua. Following intravenous infusion in a mouse model, human plasma cells were detected in spleen and bone marrow, indicating successful and potentially long-term engraftment in vivo. Moreover, high levels of human immunoglobin and therapeutic levels of human factor IX-Padua were detected in mouse plasma, correlating with 15% of normal human factor IX activity. These data suggest that the proposed process promotes the production of functional and differentiated engineered B cells. In conclusion, this study represents an important step toward the development of a manufacturing platform for potential B cell-derived therapeutic products.
RESUMO
Transcription factor 4 (TCF4) is implicated in lymphoid cell differentiation and its expression predicts outcome in acute myeloid leukemia. Here, we investigated the role of TCF4 in myelopoiesis. Overexpression of TCF4 (TCF4OE) in umbilical cord blood (UCB) cells resulted in a twofold increase in erythroid colony forming units (CFU-Es), whereas knock-down (KD) of TCF4 (TCF4KD) caused a dramatic decrease in the number of erythroid colonies. In megakaryocyte CFUs (CFU-MKs), both TCF4KD and TCF4OE inhibited MK colony formation. TCF4 did not have an impact on granulocyte, macrophage, or granulocyte-macrophage colonies or on the proportion of MK-erythrocyte progenitors (MEPs) in culture. Because TCF4 affects erythroid/MK development and these lineages are affected in myelodysplastic syndrome (MDS), we studied the impact of TCF4 expression in this disease. MDS patients with high (≥median) TCF4 mRNA expression had higher hemoglobin (Hb) levels than MDS patients with low TCF4 expression (mean 9.0 vs. 8.55 g/dL, pâ¯=â¯0.02). Overall, TCF4 mRNA expression was lower in hematopoietic stem cells, common myeloid progenitors, and MEPs from MDS patients, but not in granulocyte-macrophage progenitors, compared with healthy controls. Therefore, in cell fractions with erythroid lineage potential, TCF4 is expressed less in MDS patients than in healthy controls. This correlates with the low overall Hb levels seen in MDS patients compared with healthy individuals and is consistent with the positive impact of TCF4 on erythroid development while not having impact on white colonies. These results indicate a role for TCF4 as a novel factor in erythroid-megakaryocytic differentiation.
Assuntos
Diferenciação Celular , Células Precursoras Eritroides/metabolismo , Hemoglobinas , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Mielopoese , Fator de Transcrição 4 , Células Cultivadas , Células Precursoras Eritroides/patologia , Sangue Fetal/metabolismo , Regulação da Expressão Gênica , Hemoglobinas/biossíntese , Hemoglobinas/genética , Humanos , Megacariócitos/patologia , Síndromes Mielodisplásicas/patologia , Fator de Transcrição 4/genética , Fator de Transcrição 4/metabolismoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Biomarcadores Tumorais/genética , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda/genética , Fatores de Transcrição/genética , Estudos de Coortes , Terapia Combinada , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Fator de Transcrição 4 , Transplante AutólogoRESUMO
The gray platelet syndrome is a hereditary, usually autosomal recessive bleeding disorder caused by a deficiency of alpha granules in platelets. We detected a nonsense mutation in the gene encoding the transcription factor GFI1B (growth factor independent 1B) that causes autosomal dominant gray platelet syndrome. Both gray platelets and megakaryocytes had abnormal marker expression. In addition, the megakaryocytes had dysplastic features, and they were abnormally distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant GFI1B transcriptional activity in a dominant-negative manner. Our studies show that GFI1B, in addition to being causally related to the gray platelet syndrome, is key to megakaryocyte and platelet development.
Assuntos
Plaquetas/patologia , Síndrome da Plaqueta Cinza/genética , Megacariócitos/patologia , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Medula Óssea/patologia , Feminino , Genes Dominantes , Síndrome da Plaqueta Cinza/patologia , Humanos , Masculino , Linhagem , Células-Tronco , Trombocitopenia/genéticaRESUMO
The ubiquitin system plays an important role in trafficking of signaling receptors from the plasma membrane to lysosomes. Triad1 is a ubiquitin ligase that catalyzes the formation of poly-ubiquitin chains linked via lysine-48 as well as lysine-63 residues. We show that depletion of Triad1 affects the sorting of both growth hormone and epidermal growth factor. Triad1-depleted cells accumulate both ligands in endosomes. While fluid phase transport to the lysosomes is reduced in the absence of Triad1, growth hormone receptor can recycle back to the plasma membrane together with transferrin. Using immune electron microscopy we show that Triad1 depletion results in enlarged endosomes with enlarged and irregular shaped intraluminal vesicles. The endosomes display prominent clathrin coats and show increased levels of growth hormone label. We conclude that Triad1 is required for the proper function of multivesicular bodies.
RESUMO
Activation of the NF-κB pathway requires the formation of Met1-linked 'linear' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed 'Linear ubiquitin chain Determining Domain' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains.