Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
Mais filtros

Base de dados
País/Região como assunto
Tipo de documento
Intervalo de ano de publicação
1.
Hepatology ; 77(3): 802-815, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35976053

RESUMO

BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.


Assuntos
Capsídeo , Dependovirus , Animais , Camundongos , Humanos , Capsídeo/química , Capsídeo/metabolismo , Sorogrupo , Dependovirus/genética , Transdução Genética , Vetores Genéticos , Fígado/metabolismo , Peptídeos/análise , Peptídeos/genética , Peptídeos/metabolismo , Terapia Genética/métodos
2.
BMC Med ; 21(1): 360, 2023 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-37726769

RESUMO

BACKGROUND: Bone marrow (BM) transplantation is a life-saving therapy for hematological diseases, and the BM harbors also highly useful (progenitor) cell types for novel cell therapies manufacture. Yet, the BM collection technique is not standardized. METHODS: Benchmarking our collection efficiency to BM collections worldwide (N = 1248), we noted a great variability of total nucleated cell (TNC) yields in BM products (HPC-M) with superior performance of our center, where we have implemented a small volume aspirate policy. Thus, we next prospectively aimed to assess the impact of BM collection technique on HPC-M quality. For each BM collection (N = 20 donors), small volume (3 mL) and large volume (10 mL) BM aspirates were sampled at 3 time points and analyzed for cell composition. RESULTS: Compared to large volume aspirates, small volume aspirates concentrated more TNCs, immune cells, platelets, hematopoietic stem/progenitor cells, mesenchymal stromal cells (MSCs), and endothelial progenitors. Inversely, the hemoglobin concentration was higher in large volume aspirates indicating more hemoglobin loss. Manufacturing and dosing scenarios showed that small volume aspirates save up to 42% BM volume and 44% hemoglobin for HPC-M donors. Moreover, MSC production efficiency can be increased by more than 150%. CONCLUSIONS: We propose to consider small volume BM aspiration as standard technique for BM collection.


Assuntos
Medula Óssea , Células-Tronco Mesenquimais , Humanos , Células-Tronco , Terapia Baseada em Transplante de Células e Tecidos , Hemoglobinas
3.
Blood ; 131(9): 1022-1031, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29246900

RESUMO

Gene therapy for hemophilia B aims to ameliorate bleeding risk and provide endogenous factor IX (FIX) activity/synthesis through a single treatment, eliminating the requirement for FIX concentrate. AMT-060 combines an adeno-associated virus-5 (AAV5) vector with a liver-specific promoter driving expression of a codon-optimized wild-type human FIX gene. This multinational, open-label study included 10 adults with hemophilia B (FIX ≤2% of normal) and severe-bleeding phenotype. No participants tested positive for AAV5-neutralizing antibodies using a green-fluorescent protein-based assay, and all 10 were enrolled. A single dose of 5 × 1012 or 2 × 1013 genome copies of AMT-060/kilogram was administered to 5 participants each. In the low-dose cohort, mean endogenous FIX activity increased to 4.4 IU/dL. Annualized FIX use was reduced by 81%, and mean annualized spontaneous bleeding rate (ASBR) decreased from 9.8% to 4.6% (53%). In the higher-dose cohort, mean FIX activity increased to 6.9 IU/dL. Annualized FIX use decreased by 73%, and mean ASBR declined from 3.0 to 0.9 (70%). There was no reduction in traumatic bleeds. FIX activity was stable in both cohorts, and 8 of 9 participants receiving FIX at study entry stopped prophylaxis. Limited, asymptomatic, and transient alanine aminotransferase elevations in the low-dose (n = 1) and higher-dose (n = 2) cohorts were treated with prednisolone. No decrease in FIX activity or capsid-specific T-cell responses were detected during transaminase elevations. A single infusion of AMT-060 had a positive safety profile and resulted in stable and clinically important increases in FIX activity, a marked reduction in spontaneous bleeds and FIX concentrate use, without detectable cellular immune responses against capsids. This trial was registered at www.clinicaltrials.gov as #NCT02396342; EudraCT #2013-005579-42.


Assuntos
Fator IX , Terapia Genética , Vetores Genéticos , Hemofilia B , Parvovirinae , Prednisolona/administração & dosagem , Adulto , Dependovirus , Fator IX/biossíntese , Fator IX/genética , Feminino , Hemofilia B/sangue , Hemofilia B/genética , Hemofilia B/terapia , Humanos , Masculino
4.
J Allergy Clin Immunol ; 138(1): 219-228.e9, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26853280

RESUMO

BACKGROUND: Defects in phagocytic nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) function cause chronic granulomatous disease (CGD), a primary immunodeficiency characterized by dysfunctional microbicidal activity and chronic inflammation. OBJECTIVE: We sought to study the effect of chronic inflammation on the hematopoietic compartment in patients and mice with X-linked chronic granulomatous disease (X-CGD). METHODS: We used immunostaining and functional analyses to study the hematopoietic compartment in patients with CGD. RESULTS: An analysis of bone marrow cells from patients and mice with X-CGD revealed a dysregulated hematopoiesis characterized by increased numbers of hematopoietic progenitor cells (HPCs) at the expense of repopulating hematopoietic stem cells (HSCs). In patients with X-CGD, there was a clear reduction in the proportion of HSCs in bone marrow and peripheral blood, and they were also more rapidly exhausted after in vitro culture. In mice with X-CGD, increased cycling of HSCs, expansion of HPCs, and impaired long-term engraftment capacity were found to be associated with high concentrations of proinflammatory cytokines, including IL-1ß. Treatment of wild-type mice with IL-1ß induced enhanced cell-cycle entry of HSCs, expansion of HPCs, and defects in long-term engraftment, mimicking the effects observed in mice with X-CGD. Inhibition of cytokine signaling in mice with X-CGD reduced HPC numbers but had only minor effects on the repopulating ability of HSCs. CONCLUSIONS: Persistent chronic inflammation in patients with CGD is associated with hematopoietic proliferative stress, leading to a decrease in the functional activity of HSCs. Our observations have clinical implications for the development of successful autologous cell therapy approaches.


Assuntos
Doença Granulomatosa Crônica/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Adolescente , Adulto , Animais , Biomarcadores , Estudos de Casos e Controles , Contagem de Células , Diferenciação Celular , Criança , Pré-Escolar , Ensaio de Unidades Formadoras de Colônias , Citocinas/metabolismo , Citocinas/farmacologia , Modelos Animais de Doenças , Sobrevivência de Enxerto , Doença Granulomatosa Crônica/etiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Fenótipo , Transdução de Sinais , Adulto Jovem
5.
Mol Ther ; 23(1): 63-70, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25189742

RESUMO

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.


Assuntos
Antígenos CD/genética , Terapia Genética/métodos , Glicoproteínas/genética , Células-Tronco Hematopoéticas/imunologia , Lentivirus/genética , Peptídeos/genética , Antígeno AC133 , Animais , Antígenos CD/imunologia , Antígenos CD34/genética , Antígenos CD34/imunologia , Expressão Gênica , Vetores Genéticos , Glicoproteínas/imunologia , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/imunologia , Doença Granulomatosa Crônica/patologia , Doença Granulomatosa Crônica/terapia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Peptídeos/imunologia , Cultura Primária de Células , Transdução Genética , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
6.
Mol Ther ; 21(3): 648-61, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23207695

RESUMO

Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91(phox)) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders.


Assuntos
Alpharetrovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Doença Granulomatosa Crônica/terapia , Splicing de RNA , Animais , Células da Medula Óssea , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Granulócitos , Doença Granulomatosa Crônica/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética , Transgenes
7.
Mol Ther ; 19(1): 28-35, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21045810

RESUMO

The potential of gene therapy as a curative treatment for monogenetic disorders has been clearly demonstrated in a series of recent Phase I/II clinical trials. Among primary immunodeficiencies, gene transfer into hematopoietic stem (HSC)/progenitor cells has resulted in the long-term correction of immune and metabolic defects in treated patients. In most cases, successes were augmented by a recognized biological selection for successfully treated cells in vivo, perhaps even to some extent at the HSC level. In contrast, similar achievements have not turned into reality for immunodeficiencies in which gene-transduced cells lack selective advantages in vivo. This is the case for chronic granulomatous disease (CGD), a primary immunodeficiency, characterized by deficient antimicrobial activity in phagocytic cells. Several attempts to correct CGD by gene transfer in combination with bone marrow conditioning have resulted in low-level long-term engraftment and transient clinical benefits despite high levels of gene marking and high numbers of reinfused cells. This review summarizes the data from clinical trials for CGD and provides some insights into treatment options that may lead to a successful application of gene therapy for CGD.


Assuntos
Terapia Genética/métodos , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Animais , Transplante de Medula Óssea , Humanos , Condicionamento Pré-Transplante
8.
J Clin Invest ; 132(24)2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36326824

RESUMO

BACKGROUNDResults of many randomized trials on COVID-19 convalescent plasma (CCP) have been reported, but information on long-term outcome after CCP treatment is limited. The objectives of this extended observation of the randomized CAPSID trial are to assess long-term outcome and disease burden in patients initially treated with or without CCP.METHODSOf 105 randomized patients, 50 participated in the extended observation. Quality of life (QoL) was assessed by questionnaires and a structured interview. CCP donors (n = 113) with asymptomatic to moderate COVID-19 were included as a reference group.RESULTSThe median follow-up of patients was 396 days, and the estimated 1-year survival was 78.7% in the CCP group and 60.2% in the control (P = 0.08). The subgroup treated with a higher cumulative amount of neutralizing antibodies showed a better 1-year survival compared with the control group (91.5% versus 60.2%, P = 0.01). Medical events and QoL assessments showed a consistent trend for better results in the CCP group without reaching statistical significance. There was no difference in the increase in neutralizing antibodies after vaccination between the CCP and control groups.CONCLUSIONThe trial demonstrated a trend toward better outcome in the CCP group without reaching statistical significance. A predefined subgroup analysis showed a significantly better outcome (long-term survival, time to discharge from ICU, and time to hospital discharge) among those who received a higher amount of neutralizing antibodies compared with the control group. A substantial long-term disease burden remains after severe COVID-19.Trial registrationEudraCT 2020-001310-38 and ClinicalTrials.gov NCT04433910.FundingBundesministerium für Gesundheit (German Federal Ministry of Health).


Assuntos
COVID-19 , Humanos , COVID-19/terapia , COVID-19/etiologia , SARS-CoV-2 , Qualidade de Vida , Capsídeo , Seguimentos , Imunização Passiva/efeitos adversos , Anticorpos Neutralizantes , Anticorpos Antivirais
9.
Materials (Basel) ; 13(14)2020 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-32668732

RESUMO

Treatment of large bone defects is one of the great challenges in contemporary orthopedic and traumatic surgery. Grafts are necessary to support bone healing. A well-established allograft is demineralized bone matrix (DBM) prepared from donated human bone tissue. In this study, a fibrous demineralized bone matrix (f-DBM) with a high surface-to-volume ratio has been analyzed for toxicity and immunogenicity. f-DBM was transplanted to a 5-mm, plate-stabilized, femoral critical-size-bone-defect in Sprague-Dawley (SD)-rats. Healthy animals were used as controls. After two months histology, hematological analyses, immunogenicity as well as serum biochemistry were performed. Evaluation of free radical release and hematological and biochemical analyses showed no significant differences between the control group and recipients of f-DBM. Histologically, there was no evidence of damage to liver and kidney and good bone healing was observed in the f-DBM group. Reactivity against human HLA class I and class II antigens was detected with mostly low fluorescence values both in the serum of untreated and treated animals, reflecting rather a background reaction. Taken together, these results provide evidence for no systemic toxicity and the first proof of no basic immunogenic reaction to bone allograft and no sensitization of the recipient.

10.
Dtsch Arztebl Int ; 116(47): 791-798, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31847949

RESUMO

BACKGROUND: Approximately 4550 persons were under treatment for hemophilia in Germany in 2017. The condition is currently treated with intravenous supplementa- tion of the missing clotting factor, either prophylactically or as needed. Newer treat- ment options rely on novel mechanisms of action. METHODS: This review is based on pertinent publications retrieved by a selective search in MEDLINE/PubMed, as well as on expert opinions and the recommenda- tions of specialty societies. RESULTS: Randomized controlled trials have shown that, in children aged 30 months to 6 years, prophylactic clotting-factor supplementation yields a markedly lower an- nual rate of hemorrhage than supplementation as needed: 3.27 (standard deviation [SD] 6.24) for the former vs. 17.69 (SD 9.25) for the latter. A similar large effect was seen in patients aged 12 to 50 years, with hemorrhage rates of 1.9 (SD 4.1) vs. 28.7 (SD 18.8). Clotting-factor preparations with longer half-lives make it possible to lessen the frequency of administration and to prevent subtherapeutic factor levels. A number of alternatives to clotting-factor supplementation have recently been approved or are currently being clinically tested. These new drugs are injected sub- cutaneously and have a longer half-life, possibly enabling better protection against bleeding than the current standard treatment. A further advantage of some of these drugs is that they can be given even in the presence of inhibitors to factor VIII. In addition, initial (phase I) clinical trials of gene therapy have been performed suc- cessfully for both hemophilia A and hemophilia B. CONCLUSION: Now that new alternatives to classic supplementation therapy are be- coming available, pertinent treatment algorithms for patients with hemophilia will have to be developed. It is still unclear to what extent the new drugs might supplant clotting factor supplementation as the first line of treatment.


Assuntos
Fatores de Coagulação Sanguínea/uso terapêutico , Hemofilia A/terapia , Hemofilia B/terapia , Ensaios Clínicos Fase I como Assunto , Alemanha , Hemorragia/prevenção & controle , Humanos , Ensaios Clínicos Controlados Aleatórios como Assunto
11.
Mol Ther Nucleic Acids ; 10: 1-8, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29499925

RESUMO

The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)-a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

12.
J Leukoc Biol ; 79(6): 1306-13, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16565323

RESUMO

The Wnt-signaling pathway plays a critical role in directing cell fate during embryogenesis. Several lines of evidence also suggest a role in inflammatory processes. Here, we analyzed whether Wnt signaling plays a role in leukocyte inflammatory responses. Monocytes from healthy donors expressed different Frizzled receptors, which are ligands for the Wnt molecules. Activation of the Wnt/beta-catenin pathway by LiCl or Wnt3a increased beta-catenin protein levels in monocytes but not in granulocytes. It is interesting that the activation of Wnt/beta-catenin signaling via Wnt3a in monocytes resulted in a decrease in migration through an endothelial layer (human dermal microvascular endothelial cell-1). Further experiments revealed that the decrease in transendothelial migration was associated with specific monocyte adherence to endothelial cells after Wnt exposure. The specificity was verified by a lack of Wnt3a-induced adhesion to fibronectin, laminin, or collagen compared with endothelial interaction. Analysis of the distribution of beta-catenin revealed a Wnt3a-induced increase of beta-catenin in the cytoplasm. Wnt3a exposure did not result in any activation of the classical Wnt-target gene c-myc or a Wnt-target gene involved in cell adhesion (Connexin43). Our study implicates for the first time a role of canonical Wnt signaling in inflammatory processes in monocytes.


Assuntos
Endotélio Vascular/citologia , Monócitos/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Núcleo Celular/química , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Citoplasma/química , Receptores Frizzled/biossíntese , Receptores Frizzled/genética , Receptores Frizzled/fisiologia , Granulócitos/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Humanos , Cloreto de Lítio/farmacologia , Camundongos , Monócitos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Wnt/genética , Proteínas Wnt/farmacologia , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/biossíntese , beta Catenina/genética
13.
Cancer Res ; 65(21): 9643-50, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16266983

RESUMO

Up to 30% of patients with acute myeloid leukemia (AML) harbor internal tandem duplications (ITD) within the FLT3 gene, encoding a receptor tyrosine kinase. These mutations induce constitutive tyrosine kinase activity in the absence of the natural Flt3 ligand and confer growth factor independence, increased proliferation, and survival to myeloid precursor cells. The signaling pathways and downstream nuclear targets mediating leukemic transformation are only partly identified. Here, we show that the presence of Flt3-ITD constitutively activates Akt (PKB), a key serine-threonine kinase within the phosphatidylinositol 3-kinase pathway. Constitutive activation of Akt phosphorylated and inhibited the transcription factor Foxo3a. Restored Foxo3a activity reversed Flt3-ITD-mediated growth properties and dominant-negative Akt prevented Flt3-ITD-mediated cytokine independence. Conditional Akt activation targeted to the cell membrane induced cytokine-independent survival, cell cycle progression, and proliferation. Importantly, Akt activation was sufficient to cause in vitro transformation of 32D myeloid progenitor cells and in vivo promoted the development of a leukemia-like myeloid disease. Akt phosphorylation was found in myeloid blasts of 86% of AML patients, suggesting an important role in leukemogenesis. In summary, Akt is necessary for increased survival, proliferation, and leukemic transformation by Flt3-ITD, possibly by inactivation of Foxo transcription factors. These findings indicate that Akt and Foxo transcription factors are attractive targets for therapeutic intervention in AML.


Assuntos
Transformação Celular Neoplásica/metabolismo , Leucemia Mieloide/enzimologia , Leucemia Mieloide/patologia , Células Mieloides/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo , Doença Aguda , Animais , Ciclo Celular , Processos de Crescimento Celular , Transformação Celular Neoplásica/genética , Ativação Enzimática , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Células Mieloides/enzimologia , Células Mieloides/metabolismo , Fosforilação , Sequências de Repetição em Tandem , Transcrição Gênica , Tirosina Quinase 3 Semelhante a fms/genética
14.
Cancer Res ; 65(5): 1778-82, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15753374

RESUMO

Development of distant metastasis after tumor resection is the leading cause of death in early-stage non-small cell lung cancer (NSCLC). Receptor tyrosine kinases (RTK) are involved in tumorigenesis but only few RTKs have been systematically studied in NSCLC. Here, we provide quantitative real-time reverse transcription-PCR expression data of all RTKs (n=56) in primary tumors of 70 patients with early-stage (I-IIIA) NSCLC. Overall, 33 RTKs were expressed in at least 25% of the patients. Several RTKs were significantly expressed higher in tumors that ultimately metastasized. The hazard risk for metastasis development in stage I/II disease was increased at least 3-fold for tumors with high expression levels of insulin receptor, neurotrophic tyrosine receptor kinase 1, epidermal growth factor receptor, ERBB2, ERBB3, platelet-derived growth factor receptor beta, fibroblast growth factor receptor 1, or leukocyte tyrosine kinase. Relative risks were reduced 3-fold by expression of EPHB6 or DKFZ1. Three members of the epidermal growth factor receptor family were associated with a high risk of metastasis, emphasizing the validity of our data. High ERBB3 expression was significantly associated with decreased survival. Taken together, our genome-wide RTK expression map uncovered the previously unknown value of several RTKs as potential markers for prognosis and metastasis prediction in early-stage NSCLC. The identified RTKs represent promising novel candidates for further functional analyses.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Receptores Proteína Tirosina Quinases/genética , Primers do DNA/química , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
15.
Mol Cancer Ther ; 5(12): 3105-12, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17172413

RESUMO

In acute myeloid leukemia (AML), receptor tyrosine kinase ligands promote growth and survival and contribute to AML-associated marrow neoangiogenesis. We have tested simultaneous inhibition of vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor receptor signaling by novel indolinone derivatives using 14 myeloid, including 11 human leukemic, cell lines. Compounds inhibited colony formation of all cell lines in a dose-dependent fashion. Inhibitory concentrations for 50% of the colony formation/survival (IC50) for BIBF1000 were <100 nmol/L for 3 of 11,

Assuntos
Indóis/farmacologia , Leucemia Mieloide/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Doença Aguda , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citarabina/farmacologia , Humanos , Leucemia Mieloide/sangue , Leucemia Mieloide/patologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/sangue , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/sangue , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Oncogene ; 23(57): 9162-72, 2004 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-15516979

RESUMO

Microarray analyses were performed to identify target genes that are shared by the acute myeloid leukemia (AML) translocation products PML-RARalpha, PLZF-RARalpha and AML1-ETO in inducibly transfected U937 cell lines. The cytoplasmic serine and threonine kinase MNK1 was identified as one of the target genes. At the protein level, MNK1 was significantly induced by each of the three fusion proteins. Protein half-life analyses showed that PML-RARalpha enhanced MNK1 protein stability in U937 cells and ATRA exposure decreased MNK1 half-life in NB4 cells. EIF4E, the main MNK1 substrate, plays a role in the pathogenesis of a variety of cancers. Upon MNK1 overexpression, eIF4E phosphorylation increased as a sign of functional activation. Interestingly, MNK1 protein expression decreased during myeloid differentiation. Inhibition of MNK1 activity by a specific inhibitor (CGP57380) enhanced differentiation of HL60 and 32D cells, further suggesting a role for MNK1 in the myeloid differentiation. In addition, kinase dead mutants of MNK1 significantly impaired proliferation of 32D cells. Immunohistochemistry of primary AML bone marrow biopsies showed strong cytoplasmic MNK1 expression in 25 of 99 AML specimens (25%). MNK1 expression was associated with high levels of c-myc expression. Taken together, we identified MNK1 as a target gene of several leukemogenic fusion proteins in AML. MNK1 plays a role in myeloid differentiation. These data suggest a role for MNK1 in the AML fusion protein-associated differentiation block.


Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética
18.
Crit Rev Oncol Hematol ; 56(2): 195-221, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16236521

RESUMO

The description of the molecular pathogenesis of acute myeloid leukemias (AML) has seen dramatic progress over the last years. Two major types of genetic events have been described that are crucial for leukemic transformation: alterations in myeloid transcription factors governing hematopoietic differentiation and activating mutations of signal transduction intermediates. These processes are highly interdependent, since the molecular events changing the transcriptional control in hematopoietic progenitor cells modify the composition of signal transduction molecules available for growth factor receptors, while the activating mutations in signal transduction molecules induce alterations in the activity and expression of several transcription factors that are crucial for normal myeloid differentiation. The purpose of this article is to review the current literature describing these genetic events, their biological consequences and their clinical implications. As the article will show, the recent description of several critical transforming mutations in AML may soon give rise to more efficient and less toxic molecularly targeted therapies of this deadly disease.


Assuntos
Regulação Leucêmica da Expressão Gênica/genética , Hematopoese/genética , Leucemia Mieloide Aguda/genética , Mutação , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas de Neoplasias/metabolismo
19.
Clin Cancer Res ; 10(4): 1241-9, 2004 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-14977821

RESUMO

Novel high-throughput analyses in molecular biology allow sensitive and rapid identification of disease-related genes and drug targets. We have used quantitative real-time reverse transcription-PCR reactions (n = 23000) to analyze expression of all human receptor tyrosine kinases (n = 56) in malignant tumors (n = 313) of different origins and normal control samples (n = 58). The different tumor types expressed very different numbers of receptor tyrosine kinases: whereas brain tumors and testicular cancer expressed 50 receptor tyrosine kinases, acute myeloid leukemia (AML) samples expressed only 20 different ones. Specimens of similar tumor origin exhibited characteristic receptor tyrosine kinase expression patterns and were grouped together in hierarchical cluster analyses. When we focused on specific tumor entities, receptor tyrosine kinases were identified that were disease and/or stage specific. Leukemic blasts from AML bone marrow samples differed significantly in receptor tyrosine kinase expression compared with normal bone marrow and purified CD34+ cells. Among the differentially expressed receptor tyrosine kinases, we found FLT3, c-kit, CSF1 receptor, EPHB6, leukocyte tyrosine kinase, and ptk7 to be highly overexpressed in AML samples. Whereas expression changes of some of these were associated with altered differentiation patterns (e.g., CSF1 receptor), others, such as FLT3, were genuinely overexpressed in leukemic blasts. These data and the associated database (http://medweb.uni-muenster.de/institute/meda/research/) provide a comprehensive view of receptor tyrosine kinase expression in human cancer. This information can assist in the definition of novel drug targets.


Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Genoma , Neoplasias/genética , Proteínas Tirosina Quinases/genética , Antígenos CD34/biossíntese , Primers do DNA/farmacologia , DNA Complementar/metabolismo , Regulação para Baixo , Humanos , Proteínas de Membrana/metabolismo , Mutação , Neoplasias/metabolismo , Prognóstico , Proteínas Tirosina Quinases/metabolismo , RNA/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Regulação para Cima
20.
Curr Gene Ther ; 15(4): 416-27, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25981636

RESUMO

We report on a series of sequential events leading to long-term survival and cure of pediatric X-linked chronic granulomatous disease (X-CGD) patients after gamma-retroviral gene therapy (GT) and rescue HSCT. Due to therapyrefractory life-threatening infections requiring hematopoietic stem cell transplantation (HSCT) but absence of HLAidentical donors, we treated 2 boys with X-CGD by GT. Following GT both children completely resolved invasive Aspergillus nidulans infections. However, one child developed dual insertional activation of ecotropic viral integration site 1 (EVI1) and signal transducer and activator of transcription 3 (STAT3) genes, leading to myelodysplastic syndrome (MDS) with monosomy 7. Despite resistance to mismatched allo-HSCT with standard myeloablative conditioning, secondary intensified rescue allo-HSCT resulted in 100 % donor chimerism and disappearance of MDS. The other child did not develop MDS despite expansion of a clone with a single insertion in the myelodysplasia syndrome 1 (MDS1) gene and was cured by early standard allo-HSCT. The slowly developing dominance of clones harboring integrations in MDS1-EVI1 may guide clinical intervention strategies, i.e. early rescue allo-HSCT, prior to malignant transformation. GT was essential for both children to survive and to clear therapy-refractory infections, and future GT with safer lentiviral self-inactivated (SIN) vectors may offer a therapeutic alternative for X-CGD patients suffering from life-threatening infections and lacking HLA-identical HSC donors.


Assuntos
Terapia Genética/métodos , Doença Granulomatosa Crônica/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Aspergilose/terapia , Aspergillus nidulans/patogenicidade , Criança , Deleção Cromossômica , Cromossomos Humanos Par 7 , Proteínas de Ligação a DNA/genética , Gammaretrovirus/genética , Terapia Genética/efeitos adversos , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , Glicoproteínas de Membrana/genética , Síndromes Mielodisplásicas/etiologia , NADPH Oxidase 2 , NADPH Oxidases/genética , Proto-Oncogenes/genética , Fator de Transcrição STAT3/genética , Fatores de Transcrição/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA