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1.
Oncogene ; 2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-30093631

RESUMO

Chromosomal translocations represent frequent events in leukemia. In t(8;21)+ acute myeloid leukemia, RUNX1 is fused to nearly the entire ETO protein, which contains four conserved nervy homology regions, NHR1-4. Furthermore RUNX1/ETO interacts with ETO-homologous proteins via NHR2, thereby multiplying NHR domain contacts. As shown recently, RUNX1/ETO retains oncogenic activity upon either deletion of the NHR3 + 4 N-CoR/SMRT interaction domain or substitution of the NHR2 tetramer domain. Thus, we aimed to clarify the specificities of the NHR domains. A C-terminally NHR3 + 4 truncated RUNX1/ETO containing a heterologous, structurally highly related non-NHR2 tetramer interface translocated into the nucleus and bound to RUNX1 consensus motifs. However, it failed to interact with ETO-homologues, repress RUNX1 targets, and transform progenitors. Surprisingly, transforming capacity was fully restored by C-terminal fusion with ETO's NHR4 zinc-finger or the repressor domain 3 of N-CoR, while other repression domains failed. With an inducible protein assembly system, we further demonstrated that NHR4 domain activity is critically required early in the establishment of progenitor cultures expressing the NHR2 exchanged truncated RUNX1/ETO. Together, we can show that NHR2 and NHR4 domains can be replaced by heterologous protein domains conferring tetramerization and repressor functions, thus showing that the NHR2 and NHR4 domain structures do not have irreplaceable functions concerning RUNX1/ETO activity for the establishment of human CD34+ cell expansion. We could resemble the function of RUNX1/ETO through modular recomposition with protein domains from RUNX1, ETO, BCR and N-CoR without any NHR2 and NHR4 sequences. As most transcriptional repressor proteins do not comprise tetramerization domains, our results provide a possible explanation as to the reason that RUNX1 is recurrently found translocated to ETO family members, which all contain tetramer together with transcriptional repressor moieties.

2.
Blood ; 132(3): 307-320, 2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-29724897

RESUMO

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.

3.
Hum Gene Ther Clin Dev ; 29(2): 69-79, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29664709

RESUMO

Chronic granulomatous disease (CGD) is a debilitating primary immunodeficiency affecting phagocyte function due to the absence of nicotinamide dinucleotide phosphate (NADPH) oxidase activity. The vast majority of CGD patients in the Western world have mutations within the X-linked CYBB gene encoding for gp91phox (NOX2), the redox center of the NADPH oxidase complex (XCGD). Current treatments of XCGD are not entirely satisfactory, and prior attempts at autologous gene therapy using gammaretrovirus vectors did not provide long-term curative effects. A new strategy was developed based on the use of the lentiviral vector G1XCGD expressing high levels of the gp91phox transgene in myeloid cells. As a requisite for a clinical trial approval, standardized non-clinical studies were conducted in vitro and in mice in order to evaluate the pharmacodynamics and biosafety of the vector and the biodistribution of G1XCGD-transduced cells. Transduced CD34+ cells derived from XCGD patients engrafted and differentiated similarly to their non-transduced counterparts in xenograft mouse models and generated therapeutically relevant levels of NADPH activity in myeloid cells expressing gp91phox. Expression of functional gp91phox in hematopoietic cells did not affect their homing properties, which engrafted at high levels in mice. Extensive in vitro and in vivo genotoxicity studies found no evidence for adverse mutagenesis related to vector treatment. These studies paved the way for the approval of clinical trials in Europe and in the United States for the treatment of XCGD patients with G1XCGD gene-modified autologous hematopoietic cells.

4.
Nucleic Acids Res ; 46(8): e48, 2018 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-29420816

RESUMO

Splicing is an essential and highly regulated process in mammalian cells. We developed a synthetic riboswitch that efficiently controls alternative splicing of a cassette exon in response to the small molecule ligand tetracycline. The riboswitch was designed to control the accessibility of the 3' splice site by placing the latter inside the closing stem of a conformationally controlled tetracycline aptamer. In the presence of tetracycline, the cassette exon is skipped, whereas it is included in the ligand's absence. The design allows for an easy, context-independent integration of the regulatory device into any gene of interest. Portability of the device was shown through its functionality in four different systems: a synthetic minigene, a reporter gene and two endogenous genes. Furthermore, riboswitch functionality to control cellular signaling cascades was demonstrated by using it to specifically induce cell death through the conditionally controlled expression of CD20, which is a target in cancer therapy.

5.
Oncotarget ; 8(16): 26169-26184, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28412732

RESUMO

Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.


Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias da Vesícula Biliar , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Bibliotecas de Moléculas Pequenas , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Sci Rep ; 6: 31995, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27573788

RESUMO

UPF1 is a key player in nonsense mediated mRNA decay (NMD) but also involved in posttranscriptional gene regulation. In this study we found that UPF1 regulates the expression of genes with functions in inflammation and myeloid cell differentiation via hnRNP E2. The majority of the UPF1-regulated genes identified in monocytic cells contain a binding site for hnRNP E2 within 5' UTR located introns with hnRNP E2 acting here as splicing regulator. We found that miRNA-328 which is significantly induced during monocytic cell differentiation acts independently from its gene silencing function as RNA decoy for hnRNP E2. One representative gene controlled by the hnRNP E2/miRNA-328 balance is S100A9 which plays an important role in cell differentiation and oxidative stress response of monocytes. Induction of miRNA-328 expression during cell differentiation antagonizes the blockade by hnRNP E2 which results in the upregulation of CD11b expression and ROS production in monocytic cells. Taken together, our data indicate that upregulation of miR-328 is responsible for the induction of hnRNP E2 target genes during myeloid cell differentiation.


Assuntos
Calgranulina B/metabolismo , MicroRNAs/metabolismo , Monócitos/metabolismo , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Antígeno CD11b/metabolismo , Adesão Celular , Diferenciação Celular , Linhagem Celular , Movimento Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Monócitos/citologia , Proteômica , Interferência de RNA , Proteínas de Ligação a RNA/genética , Espécies Reativas de Oxigênio/metabolismo
8.
Rev. chil. cir ; 68(3): 254-257, jun. 2016. ilus
Artigo em Espanhol | LILACS | ID: lil-787083

RESUMO

Objetivo: Dar a conocer una rara presentación de una patología frecuente. Caso clínico: Presentamos el caso de un paciente de 27 años de edad con clínica de apendicitis aguda con antecedentes de haberse realizado 9 años atrás una apendicectomía, encontrándose un apéndice inflamado, en posición retrocecal, ascendente y subseroso. En la segunda apendicectomía se encontró un nuevo apéndice sano en la unión de las 3 tenias. El estudio histológico posterior confirmó que ambas piezas quirúrgicas eran apéndices, uno inflamado y el otro normal.


Aim: To report a rare presentation of a frequent pathology. Case report: We report a patient of 27 years old with symptoms of acute appendicitis and medical records of an appendectomyy 9 years before where surgical findings were an inflamed appendix retrocecal ascendant and subserosa. In our procedure, we found a new one at the junction of the three tapeworms. Histological examination later confirmed that both surgical specimens were appendices, one swollen and the other normal.


Assuntos
Humanos , Masculino , Adulto , Apendicectomia , Apendicite/cirurgia , Apêndice/anormalidades , Apêndice/cirurgia , Reoperação , Doenças Raras
9.
PLoS Genet ; 12(3): e1005946, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26990877

RESUMO

A network of lineage-specific transcription factors and microRNAs tightly regulates differentiation of hematopoietic stem cells along the distinct lineages. Deregulation of this regulatory network contributes to impaired lineage fidelity and leukemogenesis. We found that the hematopoietic master regulator RUNX1 controls the expression of certain microRNAs, of importance during erythroid/megakaryocytic differentiation. In particular, we show that the erythorid miR144/451 cluster is epigenetically repressed by RUNX1 during megakaryopoiesis. Furthermore, the leukemogenic RUNX1/ETO fusion protein transcriptionally represses the miR144/451 pre-microRNA. Thus RUNX1/ETO contributes to increased expression of miR451 target genes and interferes with normal gene expression during differentiation. Furthermore, we observed that inhibition of RUNX1/ETO in Kasumi1 cells and in RUNX1/ETO positive primary acute myeloid leukemia patient samples leads to up-regulation of miR144/451. RUNX1 thus emerges as a key regulator of a microRNA network, driving differentiation at the megakaryocytic/erythroid branching point. The network is disturbed by the leukemogenic RUNX1/ETO fusion product.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/genética , MicroRNAs/biossíntese , Proteínas de Fusão Oncogênica/genética , Diferenciação Celular/genética , Linhagem da Célula , Subunidade alfa 2 de Fator de Ligação ao Core/biossíntese , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes/genética , Humanos , Leucemia Mieloide Aguda/patologia , Megacariócitos/citologia , MicroRNAs/genética , Proteínas de Fusão Oncogênica/biossíntese
10.
Elife ; 52016 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-27021569

RESUMO

Mammalian oocytes are arrested in the dictyate stage of meiotic prophase I for long periods of time, during which the high concentration of the p53 family member TAp63α sensitizes them to DNA damage-induced apoptosis. TAp63α is kept in an inactive and exclusively dimeric state but undergoes rapid phosphorylation-induced tetramerization and concomitant activation upon detection of DNA damage. Here we show that the TAp63α dimer is a kinetically trapped state. Activation follows a spring-loaded mechanism not requiring further translation of other cellular factors in oocytes and is associated with unfolding of the inhibitory structure that blocks the tetramerization interface. Using a combination of biophysical methods as well as cell and ovary culture experiments we explain how TAp63α is kept inactive in the absence of DNA damage but causes rapid oocyte elimination in response to a few DNA double strand breaks thereby acting as the key quality control factor in maternal reproduction.


Assuntos
Dano ao DNA , Oócitos/fisiologia , Fosfoproteínas/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Transativadores/metabolismo , Animais , Apoptose , Feminino , Camundongos , Fosforilação , Controle de Qualidade
11.
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
12.
Oncotarget ; 6(31): 31877-88, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26397134

RESUMO

Gallbladder cancer (GBC) is a highly malignant tumor characterized by a poor response to chemotherapy and radiotherapy. We evaluated the in vitro and in vivo antitumor efficacy of mTOR inhibitors, rapamycin and WYE-354. In vitro assays showed WYE-354 significantly reduced cell viability, migration and invasion and phospho-P70S6K expression in GBC cells. Mice harboring subcutaneous gallbladder tumors, treated with WYE-354 or rapamycin, exhibited a significant reduction in tumor mass. A short-term treatment with a higher dose of WYE-354 decreased the tumor size by 68.6% and 52.4%, in mice harboring G-415 or TGBC-2TKB tumors, respectively, compared to the control group. By contrast, treatment with a prolonged-low-dose regime of rapamycin almost abrogated tumor growth, exhibiting 92.7% and 97.1% reduction in tumor size, respectively, compared to control mice. These results were accompanied by a greater decrease in the phosphorylation status of P70S6K and a lower cell proliferation Ki67 index, compared to WYE-354 treated mice, suggesting a more effective mTOR pathway inhibition. These findings provide a proof of concept for the use of rapamycin or WYE-354 as potentially good candidates to be studied in clinical trials in GBC patients.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Vesícula Biliar/tratamento farmacológico , Guanina/análogos & derivados , Sirolimo/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimioterapia Combinada , Neoplasias da Vesícula Biliar/patologia , Guanina/farmacologia , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Biochem J ; 472(2): 225-37, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26417114

RESUMO

Understanding how glucose metabolism is finely regulated at molecular and cellular levels in the liver is critical for knowing its relationship to related pathologies, such as diabetes. In order to gain insight into the regulation of glucose metabolism, we studied the liver-expressed isoforms aldolase B and fructose-1,6-bisphosphatase-1 (FBPase-1), key enzymes in gluconeogenesis, analysing their cellular localization in hepatocytes under different metabolic conditions and their protein-protein interaction in vitro and in vivo. We observed that glucose, insulin, glucagon and adrenaline differentially modulate the intracellular distribution of aldolase B and FBPase-1. Interestingly, the in vitro protein-protein interaction analysis between aldolase B and FBPase-1 showed a specific and regulable interaction between them, whereas aldolase A (muscle isozyme) and FBPase-1 showed no interaction. The affinity of the aldolase B and FBPase-1 complex was modulated by intermediate metabolites, but only in the presence of K(+). We observed a decreased association constant in the presence of adenosine monophosphate, fructose-2,6-bisphosphate, fructose-6-phosphate and inhibitory concentrations of fructose-1,6-bisphosphate. Conversely, the association constant of the complex increased in the presence of dihydroxyacetone phosphate (DHAP) and non-inhibitory concentrations of fructose-1,6-bisphosphate. Notably, in vivo FRET studies confirmed the interaction between aldolase B and FBPase-1. Also, the co-expression of aldolase B and FBPase-1 in cultured cells suggested that FBPase-1 guides the cellular localization of aldolase B. Our results provide further evidence that metabolic conditions modulate aldolase B and FBPase-1 activity at the cellular level through the regulation of their interaction, suggesting that their association confers a catalytic advantage for both enzymes.


Assuntos
Metabolismo Energético , Frutose-Bifosfatase/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Gluconeogênese , Glicólise , Hepatócitos/metabolismo , Modelos Biológicos , Animais , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Frutose-Bifosfatase/química , Frutose-Bifosfatase/genética , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/genética , Células HeLa , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Masculino , Microscopia Confocal , Transporte Proteico , Ratos Wistar , Proteínas Recombinantes de Fusão/metabolismo
14.
Biomaterials ; 69: 191-200, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26295532

RESUMO

X-linked chronic granulomatous disease (X-CGD) is an inherited disorder of the immune system. It is characterized by a defect in the production of reactive oxygen species (ROS) in phagocytic cells due to mutations in the NOX2 locus, which encodes gp91phox. Because the success of retroviral gene therapy for X-CGD has been hampered by insertional activation of proto-oncogenes, targeting the insertion of a gp91phox transgene into potential safe harbor sites, such as AAVS1, may represent a valid alternative. To conceptually evaluate this strategy, we generated X-CGD patient-derived induced pluripotent stem cells (iPSCs), which recapitulate the cellular disease phenotype upon granulocytic differentiation. We examined AAVS1-specific zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for their efficacy to target the insertion of a myelo-specific gp91phox cassette to AAVS1. Probably due to their lower cytotoxicity, TALENs were more efficient than ZFNs in generating correctly targeted iPSC colonies, but all corrected iPSC clones showed no signs of mutations at the top-ten predicted off-target sites of both nucleases. Upon differentiation of the corrected X-CGD iPSCs, gp91phox mRNA levels were highly up-regulated and the derived granulocytes exhibited restored ROS production that induced neutrophil extracellular trap (NET) formation. In conclusion, we demonstrate that TALEN-mediated integration of a myelo-specific gp91phox transgene into AAVS1 of patient-derived iPSCs represents a safe and efficient way to generate autologous, functionally corrected granulocytes.


Assuntos
Terapia Genética , Granulócitos/metabolismo , Doença Granulomatosa Crônica/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Glicoproteínas de Membrana/genética , NADPH Oxidases/genética , Diferenciação Celular , Linhagem Celular , Desoxirribonucleases/genética , Engenharia Genética , Granulócitos/citologia , Doença Granulomatosa Crônica/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células Mieloides/citologia , NADPH Oxidase 2
15.
Mol Ther Methods Clin Dev ; 2: 14061, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26052530

RESUMO

Gene transfer to hematopoietic stem cells with integrating vectors not only allows sustained correction of monogenic diseases but also tracking of individual clones in vivo. Quantitative real-time PCR (qPCR) has been shown to be an accurate method to quantify individual stem cell clones, yet due to frequently limited amounts of target material (especially in clinical studies), it is not useful for large-scale analyses. To explore whether vector integration site (IS) recovery techniques may be suitable to describe clonal contributions if combined with next-generation sequencing techniques, we designed artificial ISs of different sizes which were mixed to simulate defined clonal situations in clinical settings. We subjected all mixes to either linear amplification-mediated PCR (LAM-PCR) or nonrestrictive LAM-PCR (nrLAM-PCR), both combined with 454 sequencing. We showed that nrLAM-PCR/454-detected clonality allows estimating qPCR-detected clonality in vitro. We then followed the kinetics of two clones detected in a patient enrolled in a clinical gene therapy trial using both, nrLAM-PCR/454 and qPCR and also saw nrLAM-PCR/454 to correlate to qPCR-measured clonal contributions. The method presented here displays a feasible high-throughput strategy to monitor clonality in clinical gene therapy trials is at hand.

16.
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
17.
Front Pharmacol ; 6: 21, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25729364

RESUMO

In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy.

18.
Nucleic Acids Res ; 43(3): 1577-92, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25605798

RESUMO

Epigenetic silencing of transgene expression represents a major obstacle for the efficient genetic modification of multipotent and pluripotent stem cells. We and others have demonstrated that a 1.5 kb methylation-free CpG island from the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) effectively prevents transgene silencing and variegation in cell lines, multipotent and pluripotent stem cells, and their differentiated progeny. However, the bidirectional promoter activity of this element may disturb expression of neighboring genes. Furthermore, the epigenetic basis underlying the anti-silencing effect of the UCOE on juxtaposed promoters has been only partially explored. In this study we removed the HNRPA2B1 moiety from the A2UCOE and demonstrate efficient anti-silencing properties also for a minimal 0.7 kb element containing merely the CBX3 promoter. This DNA element largely prevents silencing of viral and tissue-specific promoters in multipotent and pluripotent stem cells. The protective activity of CBX3 was associated with reduced promoter CpG-methylation, decreased levels of repressive and increased levels of active histone marks. Moreover, the anti-silencing effect of CBX3 was locally restricted and when linked to tissue-specific promoters did not activate transcription in off target cells. Thus, CBX3 is a highly attractive element for sustained, tissue-specific and copy-number dependent transgene expression in vitro and in vivo.


Assuntos
Cromatina/metabolismo , Epigênese Genética , Inativação Gênica , Células-Tronco Multipotentes/metabolismo , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Separação Celular , Imunoprecipitação da Cromatina , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Transgenes
19.
Mol Ther ; 23(2): 330-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25373520

RESUMO

Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.


Assuntos
Expressão Gênica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptor ErbB-2/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Evolução Clonal , Citotoxicidade Imunológica , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia , Lentivirus/genética , Teste de Cultura Mista de Linfócitos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Fenótipo , Transdução Genética , Ensaios Antitumorais Modelo de Xenoenxerto
20.
ACS Synth Biol ; 4(5): 526-34, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25265236

RESUMO

Robust synthetic devices are requisite for the construction of synthetic genetic circuits and important scientific and technological tools to control cellular processes. We developed tetracycline-dependent ribozymes, which can switch on gene expression up to 8.7-fold upon addition of tetracycline. A tetracycline aptamer was grafted onto the hammerhead ribozyme in such a way that ligand binding to the aptamers destroys a loop-loop interaction within the ribozyme thereby inhibiting ribozyme cleavage and allowing gene expression. The advantage of the presented regulatory system is its independence of any regulatory proteins. The stable integration of the ribozyme into the genome of HeLa cells indicates a low background activity in the absence of ligand. Furthermore, the ligand concentration required to robustly flip the switch does not affect cell viability and therefore allows a long-term application of the system. These properties turn the tetracycline-dependent ribozymes into a very promising tool for conditional gene expression in mammalian cells.


Assuntos
Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , RNA Catalítico/genética , Tetraciclina/farmacologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Células HeLa , Humanos , Ligantes , Mamíferos , Conformação de Ácido Nucleico , Riboswitch/efeitos dos fármacos , Riboswitch/genética
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