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  • 1
    Online Resource
    Online Resource
    Automated Retroviral Insertion Site Sequence Analysis and Mapping Tool Followed by Database Analysis.
    American Society of Hematology ; 2005
    In:  Blood Vol. 106, No. 11 ( 2005-11-16), p. 5528-5528
    Details
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 5528-5528
    Abstract: Increasing use of retroviral vector-mediated gene transfer and recent reports on insertional mutagenesis in mice and humans created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time consuming manual data processing and compilation, are thus commonly applied in gene therapy laboratories. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. The obtained sequences from the integration site analysis are judged to be authentic only if the matching part of the genomic query sequence is surrounded by the 5′LTR-sequence on the one side and the adapter-sequence on the other side. Therefore we developed an Integrationseq tool. In this task, different methods for converting the ABI sequence trace files to high quality sequences and for recognizing and deleting the LTR and adaptor parts of the isolated clones were implemented. If neither a primer nor a LTR could be found, the sequence is discarded. If the LTR is found on the complementary strand, the integration sequence is reversed. The remaining sequence between primer and LTR positions are taken as the n integration sequence and written to a sequence output file. We validated the Integrationseq tool using 259 trace files originating from integration site analysis (LM-PCR). Sequences can be trimmed by IntegrationSeq, leading to an increased yield of valid integration sequence detection, which has shown to be more sensitive (100%) than conventional analysis (94.3%) and 15 times faster than conventional analysis, while the specifities are equal (both 100%). Valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap runs 50 times faster than conventional methods and retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units and further parameters like the distance from the transcription start site to the integration. Further information, e.g. data about CpG-Islands, LINEs or SINEs, and their distances to the integration is also displayed. Output files generated by the task were found to be 99.8% identical with results retrieved by conventional mapping with the Ensembl alignment tool. Using both tools, IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V106.11.5528.5528
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
    Online Resource
    Online Resource
    A ComParison of Retroviral Integration Sites in Donor T-Lymphocytes In Vivo and In Vitro.
    American Society of Hematology ; 2005
    In:  Blood Vol. 106, No. 11 ( 2005-11-16), p. 1403-1403
    Details
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 1403-1403
    Abstract: Genetically modified T-Lymphocytes (TLCs) have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused TLCs have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Yet, reports on insertional mutagenesis in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. We investigated retroviral integration sites in vitro and in vivo. Therefore, donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN for a clinical HSV-Tk study were examined. TLCs of four different donors as well as whole blood samples of two patients transplanted with donor TLCs were analyzed either using highly sensitive and specific ligation-mediated PCR (LM-PCR). A total of 114 retroviral integration sites were detected in vitro. 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Current analyses are focusing at the in vivo pattern of retroviral integration in DNA of TLCs obtained from transplanted patient’s TLCs. Therefore we developed a new high sensitive PCR method (HS-PCR), an improved LM-PCR to even detect minimal quantities of transduced DNA.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V106.11.1403.1403
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
    Online Resource
    Online Resource
    769. Genes Encoding Receptors, Signal Transducers and Transcription Regulators Are Preferred Targets of Retroviral Vector Integration in T-Lymphocytes In Vitro and In Vivo
    Elsevier BV ; 2006
    In:  Molecular Therapy Vol. 13 ( 2006), p. S297-S298
    Details
    In: Molecular Therapy, Elsevier BV, Vol. 13 ( 2006), p. S297-S298
    Type of Medium: Online Resource
    ISSN: 1525-0016
    URL: Article
    DOI: 10.1016/j.ymthe.2006.08.854
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2006
    detail.hit.zdb_id: 2001818-6
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  • 4
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    Online Resource
    Lentiviral vector integration sites in human NOD/SCID repopulating cells
    Wiley ; 2006
    In:  The Journal of Gene Medicine Vol. 8, No. 10 ( 2006-10), p. 1197-1207
    Details
    In: The Journal of Gene Medicine, Wiley, Vol. 8, No. 10 ( 2006-10), p. 1197-1207
    Abstract: Recent observations of insertional mutagenesis in preclinical and clinical settings emphasize the relevance of investigating comprehensively the spectrum of integration sites targeted by specific vectors. Methods We followed the engraftment of lentivirally transduced human cord blood (CB) progenitor cells after transplantation into NOD/SCID mice using a self‐inactivating HIV‐1‐derived vector expressing the enhanced green fluorescent protein (EGFP). Results The mean of transduction of CD34 + CB cells was 41%, as deduced from the percentage of EGFP + cells before transplantation. At 3 weeks post‐transplantation, the average of EGFP + cells in the human cell population was 65 ± 8%, and increased to 75 ± 10% at 12 weeks post‐transplantation. In order to determine the proviral integration sites in human NOD/SCID repopulating cells (SRCs) we used the ligation‐mediated polymerase chain reaction (LM‐PCR) technique. Sixty‐eight percent of the integrations were found to be located in RefSeq genes, most of them in intron regions. Twenty percent of these integrations occurred within a distance of 10 kb from the transcription start site; a percentage that is significantly lower compared to that observed in cells transduced by gammaretroviral vectors. Sixty‐two percent of integrations occurred in genes with a biological function in cell metabolism, and four integrations were located in genes with a role in tumorigenesis. Conclusions These investigations indicate that integration of lentiviral vectors in human repopulating cells capable of engrafting NOD/SCID mice preferentially occur in coding regions of the human genome. Nevertheless, the clustering of integrations at the transcriptional start is not as high as that observed for gammaretroviral vectors. Copyright © 2006 John Wiley & Sons, Ltd.
    Type of Medium: Online Resource
    ISSN: 1099-498X , 1521-2254
    URL: Issue
    URL: Article
    DOI: 10.1002/jgm.v8:10
    DOI: 10.1002/jgm.958
    Language: English
    Publisher: Wiley
    Publication Date: 2006
    detail.hit.zdb_id: 2002203-7
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    Insertion of Retroviral Vectors in NOD/SCID Repopulating Human Peripheral Blood Progenitor Cells Occurs Preferentially in the Vicinity of Transcription Start Regions and in Introns
    Elsevier BV ; 2004
    In:  Molecular Therapy Vol. 10, No. 5 ( 2004-11), p. 874-881
    Details
    In: Molecular Therapy, Elsevier BV, Vol. 10, No. 5 ( 2004-11), p. 874-881
    Type of Medium: Online Resource
    ISSN: 1525-0016
    URL: Article
    DOI: 10.1016/j.ymthe.2004.08.001
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2004
    detail.hit.zdb_id: 2001818-6
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  • 6
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    Online Resource
    Collaborative RISC-Score Database: Creation of an International Database for Retroviral Integration Analysis.
    American Society of Hematology ; 2004
    In:  Blood Vol. 104, No. 11 ( 2004-11-16), p. 2110-2110
    Details
    In: Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 2110-2110
    Abstract: Increasing use of hematopoietic stem cells for retroviral vector-mediated gene therapy and recent reports on leukemogenesis in mice and humans have created intense interest to characterize vector integrations on the genomic level. As techniques to determine insertion sites are more commonly applied in gene therapy laboratories there is a need to systematically collect and analyze the data arising from such studies in a vector insertion database. This will allow determining factors responsible for preferential integration of various vector types in specific chromosomal regions, genes or gene sections. The information derived from a vector insertion data base will be useful to recognize more “dangerous” vector types and may provide useful information for vector design. We have set up an automatic sequence analysis tool (ensuring quality criteria e.g. verification of LTR- and adapter sequence, score 〉 40, e-value 〉 10e-40, hit RefSeq, next RefSeq etc.) which simplifies data input enormously while ensuring high quality standards. Our group is establishing the "collaborative RISC (retroviral insertion estimation into chromosome) -Score Database (CRSD)"- assessment project, based on the M-CHIPS (Multi-Conditional Hybridisation Intensity Processing System) microarray data warehouse and analysis software (K. Fellenberg et al. 2001, 2002). The data obtained from the sequence analysis tool were automatically fed in the data base. A total of 287 retroviral vector integration sites were isolated and sequence analysis was performed with the above describe analysis tool. In human bone marrow repopulating cells they occurred with significantly increased frequency into chromosomes 17 and 19 (n=189). Analysis of targeted RefSeq genes showed a favored integration (48%) within the first intron. In comparison, retroviral vector integrations in T-cells (n=98) showed an entirely different chromosomal distribution pattern while the percentage of the targeted RefSeq genes was similar (46%). Further, more than 1200 sequences were submitted to the data base, originating from different vectors (SF-MDR-, MoLV-based TK/neoR-Mo3TIN-, Moloney-MGMT-, Harvey-based Neo-, Harvey-based MDR-, and lentiviral GFP-SIN-vectors) and different transduced cells (mouse hematopoietic cells, mouse fibroblasts, rhesus hematopoietic cells, human hematopoietic cells, human T-cells). The set-up and internal structure of the data base will be presented. Collaborations have been forged to include further groups and vector types. Bioinformatical analysis will allow recognizing even complex vector integration patterns and will broaden our understanding for the determinants of vector integration into the genome. This in turn can lead to the construction of "favorable" vectors and help to reduce the genotoxicity of retroviral or lentiviral vector-mediated gene transfer.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V104.11.2110.2110
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2004
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 7
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    Online Resource
    Genes Involved in Acute Leukemias Are Favored Targets of HIV Vector Integration
    American Society of Hematology ; 2007
    In:  Blood Vol. 110, No. 11 ( 2007-11-16), p. 3738-3738
    Details
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 3738-3738
    Abstract: Insertional mutagenesis and development of leukemia following retroviral gene therapy has created intense interest in assessing the safety of viral vectors for further gene therapy trials. Using the gtsg.org database we analyzed more than 14,900 different viral integration sites of ASLV, FIV, FV, HIV, MLV and SIV based vectors in terms of insertions into fragile sites, cancer genes, transcription factor binding sites, CpG islands, and repetitive elements (SINE, LINE, LTR elements). When we compared these data with our newly generated random set, containing 1,000,000 random integrations, we discovered that the gene density on fragile sites strongly correlates to the HIV vector insertion frequency. Furthermore, we report a up to a five fold increased frequency of HIV, MLV and SIV insertions in cancer genes. The majority of cancer genes preferentially hit by HIV viruses were found associated to acute leukemias, while MLV and SIV vector insertion sites are seen more evenly spread over the cancer gene repertoire. When analyzing different cell entities, it turned out that CD34+ hematopoetic stem cells had highest rates of intragenic insertions and hosted significantly more HIV and FV insertions in cancer genes than other cell types, such as HeLa, T cells, 293T cells, macrophages, fibroblasts, or SupT1 cells.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V110.11.3738.3738
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 8
    Online Resource
    Online Resource
    Profiling Retroviral Integration Sites in Donor T-Lymphocytes for Suicide Gene Therapy.
    American Society of Hematology ; 2004
    In:  Blood Vol. 104, No. 11 ( 2004-11-16), p. 1744-1744
    Details
    In: Blood, American Society of Hematology, Vol. 104, No. 11 ( 2004-11-16), p. 1744-1744
    Abstract: Retroviral vectors encoding the herpes simplex thymidine kinase gene (HSV-Tk) have been employed to render T-lymphocytes (TLCs) sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused T cells have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Recent reports on insertional “genotoxicity” in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. Here we investigated retroviral integration sites in donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN of four donors in a clinical HSV-Tk study. A total of 114 retroviral integration sites were detected using highly sensitive and specific ligation-mediated PCR (LM-PCR). 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). The zinc ion binding group makes up 4 (resp. 6, minding that GTF2HII contains a C2H2 type and KIAA0427 a C-x8-C-x5-C-x3-H-type zinc finger) of them. Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Hit genes are currently examined more systematically in terms of function, e.g. involvement in signal transduction and transcription promoting processes. RISC (Retroviral Insertion estimate of Chromosomal Integration) scores and integration specific data will be submitted to a data warehouse, the collaborative RISC score database (CRSD). Such a systematic data collection similar to the IBMTR or EBMT databases will allow to recognize factors contributing to the safety, optimal transgene expression and persistence of transduced cells in the setting of allogenic matched donor transplantation.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V104.11.1744.1744
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2004
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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