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1.
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
2.
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
4.
Cell Death Dis ; 13(3): 204, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-35246516

RESUMO

Specialized surveillance mechanisms are essential to maintain the genetic integrity of germ cells, which are not only the source of all somatic cells but also of the germ cells of the next generation. DNA damage and chromosomal aberrations are, therefore, not only detrimental for the individual but affect the entire species. In oocytes, the surveillance of the structural integrity of the DNA is maintained by the p53 family member TAp63α. The TAp63α protein is highly expressed in a closed and inactive state and gets activated to the open conformation upon the detection of DNA damage, in particular DNA double-strand breaks. To understand the cellular response to DNA damage that leads to the TAp63α triggered oocyte death we have investigated the RNA transcriptome of oocytes following irradiation at different time points. The analysis shows enhanced expression of pro-apoptotic and typical p53 target genes such as CDKn1a or Mdm2, concomitant with the activation of TAp63α. While DNA repair genes are not upregulated, inflammation-related genes become transcribed when apoptosis is initiated by activation of STAT transcription factors. Furthermore, comparison with the transcriptional profile of the ΔNp63α isoform from other studies shows only a minimal overlap, suggesting distinct regulatory programs of different p63 isoforms.


Assuntos
Transativadores , Proteína Supressora de Tumor p53 , Apoptose/genética , DNA/metabolismo , Oócitos/metabolismo , Fosfoproteínas/metabolismo , Isoformas de Proteínas/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Leukemia ; 33(7): 1700-1712, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30635626

RESUMO

The transcriptional regulator far upstream element binding protein 1 (FUBP1) acts as an oncoprotein in solid tumor entities and plays a role in the maintenance of hematopoietic stem cells. However, its potential function in leukemia is unknown. In murine models of chronic (CML) and acute myeloid leukemia (AML) induced by BCR-ABL1 and MLL-AF9, respectively, knockdown of Fubp1 resulted in prolonged survival, decreased numbers of CML progenitor cells, decreased cell cycle activity and increased apoptosis. Knockdown of FUBP1 in CML and AML cell lines recapitulated these findings and revealed enhanced DNA damage compared to leukemia cells expressing wild type FUBP1 levels. FUBP1 was more highly expressed in human CML compared to normal bone marrow cells and its expression correlated with disease progression. In AML, higher FUBP1 expression in patient leukemia cells was observed with a trend toward correlation with shorter overall survival. Treatment of mice with AML with irinotecan, known to inhibit topoisomerase I and FUBP1, significantly prolonged survival alone or in combination with cytarabine. In summary, our data suggest that FUBP1 acts as cell cycle regulator and apoptosis inhibitor in leukemia. We demonstrated that FUBP1 might play a role in DNA repair, and its inhibition may improve outcome in leukemia patients.


Assuntos
Apoptose , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide Aguda/patologia , Proteínas de Ligação a RNA/metabolismo , Animais , Transplante de Medula Óssea , Ciclo Celular , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Humanos , Irinotecano/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Inibidores da Topoisomerase I/farmacologia , Células Tumorais Cultivadas
6.
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.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Doença Granulomatosa Crônica/genética , NADPH Oxidase 2/genética , NADPH Oxidases/genética , Animais , Ensaios Clínicos como Assunto , Regulação da Expressão Gênica/efeitos dos fármacos , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Doença Granulomatosa Crônica/patologia , Doença Granulomatosa Crônica/terapia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Xenoenxertos , Humanos , Lentivirus/genética , Camundongos , NADPH Oxidase 2/administração & dosagem
7.
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
8.
Hum Gene Ther Clin Dev ; 24(2): 86-98, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23845071

RESUMO

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.


Assuntos
Gammaretrovirus/genética , Vetores Genéticos/metabolismo , Doença Granulomatosa Crônica/terapia , Animais , Aspergillus fumigatus/patogenicidade , Células Cultivadas , Metilação de DNA , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Terapia Genética , Vetores Genéticos/genética , Humanos , Pneumopatias/microbiologia , Pneumopatias/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fes/genética , Superóxidos/metabolismo
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