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  • 1
    Call number: M040:66
    Keywords: Evolution
    Pages: 334 p. : ill.
    Edition: 3. Aufl.
    ISBN: 978-3-492-05271-9
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    M040:66 departmental collection or stack – please contact the library
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  • 2
    Keywords: CELLS ; CELL ; COMBINATION ; Germany ; THERAPY ; DISEASE ; DISEASES ; GENE ; gene therapy ; gene transfer ; GENE-TRANSFER ; ACTIVATION ; BIOLOGY ; treatment ; bone marrow ; BONE-MARROW ; TRIAL ; NUMBER ; MUTATION ; MUTATIONS ; STEM-CELLS ; INDIVIDUALS ; CD34(+) CELLS ; HEMATOPOIETIC-CELLS ; GENE-THERAPY ; hematopoietic stem cells ; HOST-DEFENSE ; NEUTROPHILS ; cord blood ; INTEGRATION ; ADULT ; ADULTS ; RE ; HEMATOPOIESIS ; stem cells ; SEVERE COMBINED IMMUNODEFICIENCY ; analysis ; hematopoietic stem cell ; BONE ; DEFECT ; stem cell ; STEM-CELL ; RETROVIRAL INTEGRATION ; ASPERGILLUS-FUMIGATUS ; GP91(PHOX) ; LIPOSOMAL BUSULFAN ; NADPH OXIDASE ACTIVITY
    Abstract: Gene transfer into hematopoietic stem cells has been used successfully for correcting lymphoid but not myeloid immunodeficiencies. Here we report on two adults who received gene therapy after nonmyeloablative bone marrow conditioning for the treatment of X-linked chronic granulomatous disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes resulting from mutations in gp91(phox). We detected substantial gene transfer in both individuals' neutrophils that lead to a large number of functionally corrected phagocytes and notable clinical improvement. Large-scale retroviral integration site-distribution analysis showed activating insertions in MDS1-EVI1, PRDM16 or SETBP1 that had influenced regulation of long-term hematopoiesis by expanding gene-corrected myelopoiesis three- to four-fold in both individuals. Although insertional influences have probably reinforced the therapeutic efficacy in this trial, our results suggest that gene therapy in combination with bone marrow conditioning can be successfully used to treat inherited diseases affecting the myeloid compartment such as CGD
    Type of Publication: Journal article published
    PubMed ID: 16582916
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  • 3
    Keywords: IN-VIVO ; DIFFERENTIATION ; MICE ; SELECTION ; INTEGRATION SITES ; PERIPHERAL-BLOOD ; VECTOR INTEGRATION ; ENGRAFTMENT ; IPS CELLS ; SCID-X1 GENE-THERAPY
    Abstract: A patient with beta(E) /beta(0) -thalassemia major was converted to transfusion-independence 4.5 years ago by lentiviral gene transfer in hematopoietic stem cells while showing a myeloid-biased cell clone. Induced pluripotent stem cells (iPSCs) are a potential alternative source of hematopoietic stem cells. If fetal to adult globin class, switching does not occur in vivo in iPSC-derived erythroid cells, beta-globin gene transfer would be unnecessary. To investigate both vector integration skewing and the potential use of iPSCs for the treatment of thalassemia, we derived iPSCs from the thalassemia gene therapy patient and compared iPSC-derived hematopoietic cells to their natural isogenic somatic counterparts. In NSG immunodeficient mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that the gene transfer is likely necessary for iPSC-based therapy of the beta-hemoglobinopathies. Lentivector integration occurred in regions of low and high genotoxicity. Surprisingly, common integration sites (CIS) were identified across those iPSCs and cells retrieved from isogenic and nonisogenic gene therapy patients with beta-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from nonrandom lentiviral integration rather than oncogenic in vivo selection. These findings bring the use of iPSCs closer to practicality and further clarify our interpretation of genome-wide lentivector integration. Stem Cells 2013;31:1785-1794.
    Type of Publication: Journal article published
    PubMed ID: 23712774
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  • 4
    Keywords: CLINICAL-TRIAL ; INTEGRATION SITES ; GENE-THERAPY ; HEMATOPOIETIC STEM-CELLS ; X-LINKED ADRENOLEUKODYSTROPHY ; TRANSGENE EXPRESSION ; JOINT PUBLICATION ; REVISED GUIDES ; NACAD GROUPS ; ENHANCER BLOCKING
    Abstract: A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a self-inactivating lentiviral vector (HPV569) containing an engineered beta-globin gene (beta(A-T87Q)-globin) in a subject with beta-thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 beta-thalassemic mice were transplanted with vector-or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with beta-thalassemia major and sickle cell disease.
    Type of Publication: Journal article published
    PubMed ID: 25429463
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  • 5
    Keywords: proliferation ; carcinoma ; CELL ; Germany ; KINASE ; PATHWAY ; PATHWAYS ; COMMON ; FOLLOW-UP ; LUNG-CANCER ; RISK ; CLONING ; PROTEIN ; cell line ; DIFFERENTIATION ; LINES ; PATIENT ; ACTIVATION ; DNA ; primary ; DOMAIN ; CELL-LINES ; SEQUENCE ; FREQUENCY ; polymorphism ; FREQUENCIES ; NUMBER ; MUTATION ; METASTASIS ; CELL-LINE ; LINE ; MELANOMA ; TUMOR-SUPPRESSOR GENE ; RESECTION ; MUTATIONS ; ONCOGENE ; CARCINOMAS ; POLYMERASE-CHAIN-REACTION ; SERINE/THREONINE KINASE ; PREDICTION ; CHAIN-REACTION ; B-RAF ; BRAF ; Ras ; HUMAN-MALIGNANT MELANOMA ; GEL-ELECTROPHORESIS ; SEGMENT ; CHAIN ; SUBSET ; P53 GENE ; HUMAN CUTANEOUS MELANOMA ; HUMAN MELANOCYTES ; polymerase chain reaction ; SIGNALING MECHANISMS ; single-strand conformation polymorphism ; SKIN CANCERS
    Abstract: Downstream of Ras, the serine/threonine kinase Braf has recently been reported to be mutated, among other carcinomas, in a majority of melanoma cell lines with a preponderance of mutations within the kinase domain including the activating V599E transition. We therefore investigated a representative number of 50 primary melanoma resection specimens for the presence of mutations within the activation segment (exon 15) of the B-raf kinase domain. Applying polymerase chain reaction and single-strand conformation polymorphism gel electrophoresis, followed by DNA cloning and sequencing, we found 19 cases (38%) to harbor somatic B-raf exon 15 mutations. With respect to the B-raf protein sequence, the V599E mutation was predicted in 63% of these positive melanomas, followed in frequency by the V599K transition (31%). Detection of B-raf exon 15 mutations or prediction of the activating mutation V599E were not statistically associated with the risk for subsequent metastasis in the follow-up of patients. Altogether, the B-raf oncogene is affected in a substantial subset of melanoma resection specimens. As B-raf alterations possibly affect melanocyte-specific pathways controlling proliferation and differentiation, activation of this oncogene may contribute to the development of melanoma. Copyright (C) 2004 S. Karger AG, Basel
    Type of Publication: Journal article published
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  • 6
    Keywords: CANCER ; CELLS ; EXPRESSION ; CELL ; Germany ; CDNA ; GENE ; GENE-EXPRESSION ; GENES ; HYBRIDIZATION ; PROTEIN ; PROTEINS ; TISSUE ; COMPLEX ; COMPLEXES ; primary ; TISSUES ; tumour ; SUPPRESSION ; immunohistochemistry ; gene expression ; HEAT-SHOCK ; STRESS ; metastases ; MELANOMA ; DAMAGE ; RESECTION ; DEGRADATION ; FRAGMENTS ; MALIGNANT-MELANOMA ; CUTANEOUS MELANOMA ; C-MYC ; DIFFERENTIAL EXPRESSION ; protein expression ; MAJOR HISTOCOMPATIBILITY COMPLEX ; HYPOXIA ; CELL CARCINOMA ; assembly ; MELANOMA-CELLS ; CANDIDATE GENES ; heat shock cognate protein gene (HSP73) ; HLA-DR EXPRESSION ; II ANTIGEN-EXPRESSION ; LIBRARIES ; major histocompatibility complex (HLA-DR) gene ; METABOLIC STRESS ; PH ; SUBTRACTIVE HYBRIDIZATION ; subtractive suppression hybridization
    Abstract: Seeking to identify melanoma-associated genes by comparing gene expression in uncultured primary melanoma specimens with those in nevi, from which melanomas often are known to arise, we applied subtractive suppression hybridization. Generating a subtracted library of candidate genes up-regulated in primary melanomas, this library contained cDNA fragments of the genes encoding heat shock cognate protein (HSP73) and major histocompatibility complex (HLA-DR) which were overexpressed in further 19 independent melanoma resection specimens on cDNA Southern blots when compared to 19 acquired melanocytic nevi. Upon immunohistochemistry, HSP73 protein expression was detected in the cytoplasm of melanoma cells in primary tumours and metastases. In primary melanomas, the proportion of HSP73 protein expressing cells correlated with tumour thickness according to Breslow which was statistically significant. HSP73 immunostaining was stronger in melanoma metastases when compared with acquired melanocytic nevi which was statistically significant. In addition to melanoma, gastric and uterus cancer tissues exhibited higher HSP73 mRNA expression on a matched tumour/normal cDNA array than their normal counterparts which was statistically significant. Participating in the regulation of folding, assembly and degradation of proteins and protecting cellular proteins from the damage caused by cellular stress like hypoxia or changes in cellular pH, elevated HSP73 expression possibly confers proliferative advantage on melanoma cells
    Type of Publication: Journal article published
    PubMed ID: 15254721
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  • 7
    Keywords: CELLS ; EXPRESSION ; proliferation ; SURVIVAL ; tumor ; BLOOD ; CELL ; Germany ; IN-VIVO ; KINASE ; THERAPY ; VIVO ; FOLLOW-UP ; SITE ; SITES ; GENE ; GENE-EXPRESSION ; GENES ; PROTEIN ; PROTEINS ; transcription ; METABOLISM ; TRANSDUCTION ; gene transfer ; GENE-TRANSFER ; PATIENT ; DONOR ; TRANSPLANTATION ; CONTRAST ; SUFFICIENT ; treatment ; FREQUENCY ; FREQUENCIES ; TARGET ; CELL-SURVIVAL ; PATTERNS ; gene expression ; VECTOR ; LYMPHOCYTES ; HUMAN GENOME ; REGION ; REGIONS ; PROGENITOR CELLS ; IMMUNITY ; T-LYMPHOCYTES ; T lymphocyte ; CHILDREN ; PERIPHERAL-BLOOD ; T lymphocytes ; INTEGRATION ; PATTERN ; SEVERE COMBINED IMMUNODEFICIENCY ; LEVEL ; LONG ; progenitor ; EVENTS ; USA ; in vivo ; progenitor cell ; TRANSDUCED CELLS ; RECONSTITUTION ; RETROVIRAL GENE MARKING ; RETROVIRAL INTEGRATION ; MEDICINE ; VECTOR INTEGRATION ; PROGENITOR-CELL ; SCID-X1 ; ADA
    Abstract: We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and I healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+)T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation
    Type of Publication: Journal article published
    PubMed ID: 17671654
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  • 8
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    Human Gene Therapy 25 (6), 475-481 
    Keywords: HUMAN GENOME ; CD34(+) CELLS ; HEMATOPOIETIC STEM-CELLS ; POLYMERASE CHAIN-REACTION ; SEVERE COMBINED IMMUNODEFICIENCY ; CHRONIC GRANULOMATOUS-DISEASE ; RETROVIRAL INTEGRATION ; LIGATION MEDIATED PCR ; SCID-X1 GENE-THERAPY ; ENZYMATIC AMPLIFICATION
    Type of Publication: Journal article published
    PubMed ID: 24950086
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  • 9
    Keywords: IN-VIVO ; THERAPY ; ACTIVATION ; GENOME-WIDE ANALYSIS ; SEVERE COMBINED IMMUNODEFICIENCY ; RETROVIRAL DNA-INTEGRATION ; VECTOR INTEGRATION ; BETA-THALASSEMIA ; VIRUS TYPE-1 ; CONSEQUENT
    Abstract: Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory towards safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34+ cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that approximately 10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis.Molecular Therapy (2014); doi:10.1038/mt.2014.246.
    Type of Publication: Journal article published
    PubMed ID: 25523760
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  • 10
    Abstract: Integrating viral gene transfer vectors are commonly used gene delivery tools in clinical gene therapy trials providing stable integration and continuous gene expression of the transgene in the treated host cell. However, integration of the reverse-transcribed vector DNA into the host genome is a potentially mutagenic event that may directly contribute to unwanted side effects. A comprehensive and accurate analysis of the integration site (IS) repertoire is indispensable to study clonality in transduced cells obtained from patients undergoing gene therapy and to identify potential in vivo selection of affected cell clones. To date, next-generation sequencing (NGS) of vector-genome junctions allows sophisticated studies on the integration repertoire in vitro and in vivo. We have explored the use of the Illumina MiSeq Personal Sequencer platform to sequence vector ISs amplified by non-restrictive linear amplification-mediated PCR (nrLAM-PCR) and LAM-PCR. MiSeq-based high-quality IS sequence retrieval is accomplished by the introduction of a double-barcode strategy that substantially minimizes the frequency of IS sequence collisions compared to the conventionally used single-barcode protocol. Here, we present an updated protocol of (nr)LAM-PCR for the analysis of lentiviral IS using a double-barcode system and followed by deep sequencing using the MiSeq device.
    Type of Publication: Journal article published
    PubMed ID: 27317177
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