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  • insertional mutagenesis  (3)
  • IN-VIVO  (2)
  • CELL  (1)
  • CELLS  (1)
Keywords
  • 1
    Keywords: CELLS ; tumor ; BLOOD ; CELL ; Germany ; human ; IN-VIVO ; MODEL ; THERAPY ; FOLLOW-UP ; QUANTIFICATION ; LONG-TERM ; GENE ; gene therapy ; TIME ; TRANSDUCTION ; gene transfer ; GENE-TRANSFER ; TRANSPLANTATION ; BIOLOGY ; BREAST-CANCER ; NO ; resistance ; VECTOR ; IMMUNODEFICIENT MICE ; STEM-CELLS ; PROGENITOR CELLS ; POLYMERASE-CHAIN-REACTION ; CHAIN-REACTION ; HEMATOPOIETIC-CELLS ; PERIPHERAL-BLOOD ; MULTIDRUG-RESISTANCE ; RESISTANCE MDR-1 GENE ; retroviral vector ; insertional mutagenesis ; LACKING ; multidrug resistance ; CHAIN ; ONCOLOGY ; THERAPIES ; polymerase chain reaction ; P-GLYCOPROTEIN ; BONE-MARROW-CELLS ; stem cells ; LEVEL ; USA ; progenitor cell ; microbiology ; STEM ; PROGENITOR-CELL ; rhesus macaque ; biotechnology ; CD34+ ; DOMINANCE ; long-term follow-up ; multidrug resistance 1
    Abstract: Previous murine studies have suggested that retroviral multidrug resistance 1 ( MDR1) gene transfer may be associated with a myeloproliferative disorder. Analyses at a clonal level and prolonged long-term follow-up in a model with more direct relevance to human biology were lacking. In this study, we analyzed the contribution of individual CD34selected peripheral blood progenitor cells to long-term rhesus macaque hematopoiesis after transduction with a retroviral vector either expressing the multidrug resistance 1 gene ( HaMDR1 vector) or expressing the neomycin resistance ( NeoR) gene ( G1Na vector). We found a total of 122 contributing clones from 8 weeks up to 4 years after transplantation. One hundred two clones contained the G1Na vector, whereas only 20 clones contained the HaMDR1 vector. Here, we show for the first time realtime polymerase chain reaction based quantification of individual transduced cell clones constituting 0.0008% +/- 0.0003% to 0.0041% +/- 0.00032% of primate peripheral blood cells. No clonal dominance was observed
    Type of Publication: Journal article published
    PubMed ID: 17615269
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  • 2
    Keywords: THERAPY ; ACTIVATION ; REPLICATION ; insertional mutagenesis ; HUMAN-IMMUNODEFICIENCY-VIRUS ; INTEGRATION ; REPOPULATING CELLS ; MARKING ; SCID-X1 ; SITE ANALYSIS
    Abstract: Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat-driven gamma-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus-long terminal repeat-driven gamma-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of linear amplification-mediated PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for linear amplification-mediated PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with gamma-retroviral long terminal repeat vectors.
    Type of Publication: Journal article published
    PubMed ID: 22989760
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  • 3
    Keywords: IN-VIVO ; PATHWAY ; THERAPY ; MICE ; PATTERNS ; HEMATOPOIETIC-CELLS ; MARROW-REPOPULATING CELLS ; GREEN FLUORESCENT PROTEIN ; insertional mutagenesis ; IN-VIVO SELECTION ; O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE ; retroviral vector integration ; CHRONIC GRANULOMATOUS-DISEASE ; LARGE-ANIMAL MODEL ; RESISTANCE GENE-THERAPY
    Abstract: Gene transfer of mutant O-6-methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs. However, insertional mutagenesis, high regenerative stress associated with selection, and the genotoxic potential of alkylating drugs represent considerable risk factors for clinical applications of this approach. Therefore, we investigated the long-term effect of MGMT(P140K) gene transfer followed by repetitive, dose-intensive treatment with alkylating agents in a murine serial bone marrow transplant model and assessed clonality of hematopoiesis up to tertiary recipients. The substantial selection pressure resulted in almost completely transduced hematopoiesis in all cohorts. Ligation-mediated PCR and next-generation sequencing identified several repopulating clones carrying vector insertions in distinct genomic regions that were similar to 9 kb of size (common integration sites). Beside polyclonal reconstitution in the majority of the mice, we also detected monoclonal or oligoclonal re-population patterns with HSC clones showing vector insertions in the Usp10 or Tubb3 gene. Interestingly, neither Usp10, Tubb3, nor any of the genes located in common integration sites have been linked to clonal expansion in previous preclinical or clinical gene therapy trials. However, a considerable number of these genes are involved in DNA damage response and cell fate decision pathways following cytostatic drug application. Thus, in summary, our study advocates ligation-mediated PCR and next generation sequencing as an effective and reliable method to identify gene products associated with clonal survival in specific experimental settings such as chemoselection using alkylating agents
    Type of Publication: Journal article published
    PubMed ID: 21319998
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