FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2007294672> ?p ?o ?g. }

• W2007294672 endingPage "1547" @default.
• W2007294672 startingPage "1537" @default.
• W2007294672 abstract "In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient γ-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of ~10−4. Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with >50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk <10−6). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore, our study offers perspectives for refinement of animal experiments in the assessment of vector-related genotoxicity. In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient γ-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of ~10−4. Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with >50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk <10−6). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore, our study offers perspectives for refinement of animal experiments in the assessment of vector-related genotoxicity. Multipotent stem cells support organ development in utero and form a regenerative reserve in the ageing organism. In most organs with a high cell turnover, somatic stem cells represent a rare cell population that is characterized by a largely open chromatin structure allowing the execution of multiple genetic programmes, active maintenance of chromosome telomeres over multiple cell divisions, and the capacity to repopulate specialized niches that support self-renewal.1Niwa H Open conformation chromatin and pluripotency.Genes Dev. 2007; 21: 2671-2676Crossref PubMed Scopus (79) Google Scholar,2Lansdorp PM Role of telomerase in hematopoietic stem cells.Ann N Y Acad Sci. 2005; 1044: 220-227Crossref PubMed Scopus (33) Google Scholar,3Martinez-Agosto JA Mikkola HK Hartenstein V Banerjee U The hematopoietic stem cell and its niche: a comparative view.Genes Dev. 2007; 21: 3044-3060Crossref PubMed Scopus (174) Google Scholar Organ-resident multipotent cells such as hematopoietic stem cells (HSCs) are a preferred resource for novel approaches in regenerative medicine. Gene-modified HSCs can lead to sustained therapeutic effects in patients suffering from severe genetic disorders of blood cell function.4Hacein-Bey-Abina S Garrigue A Wang GP Soulier J Lim A Morillon E et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J Clin Invest. 2008; 118: 3132-3142Crossref PubMed Scopus (1395) Google Scholar,5Aiuti A Cattaneo F Galimberti S Benninghoff U Cassani B Callegaro L et al.Gene therapy for immunodeficiency due to adenosine deaminase deficiency.N Engl J Med. 2009; 360: 447-458Crossref PubMed Scopus (815) Google Scholar However, transformation of hematopoietic cells by semi-randomly integrating gene vectors has been identified as a dose-limiting toxicity of gene therapy in both animal studies and clinical trials.4Hacein-Bey-Abina S Garrigue A Wang GP Soulier J Lim A Morillon E et al.Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1.J Clin Invest. 2008; 118: 3132-3142Crossref PubMed Scopus (1395) Google Scholar,6Li Z Dullmann J Schiedlmeier B Schmidt M von Kalle C Meyer J et al.Murine leukemia induced by retroviral gene marking.Science. 2002; 296: 497Crossref PubMed Scopus (572) Google Scholar,7Kustikova O Fehse B Modlich U Yang M Düllmann J Kamino K et al.Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.Science. 2005; 308: 1171-1174Crossref PubMed Scopus (304) Google Scholar,8Ott MG Schmidt M Schwarzwaelder K Stein S Siler U Koehl U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (983) Google Scholar,9Seggewiss R Pittaluga S Adler RL Guenaga FJ Ferguson C Pilz IH et al.Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque.Blood. 2006; 107: 3865-3867Crossref PubMed Scopus (110) Google Scholar Similar to the situation observed in murine and avian tumors caused by replicating retroviruses, upregulation of crucial proto-oncogenes by semi-random vector insertion in their chromosomal neighborhood has been identified as a causal event. The risk of insertional cell transformation by nonreplicating gene vectors depends on the number of insertions acquired per cell, the insertion properties, and the cargo of the vector.10Modlich U Kustikova OS Schmidt M Rudolph C Meyer J Li Z et al.Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.Blood. 2005; 105: 4235-4246Crossref PubMed Scopus (159) Google Scholar,11Montini E Cesana D Schmidt M Sanvito F Ponzoni M Bartholomae C et al.Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration.Nat Biotechnol. 2006; 24: 687-696Crossref PubMed Scopus (586) Google Scholar,12Zychlinski D Schambach A Modlich U Maetzig T Meyer J Grassman E et al.Physiological promoters reduce the genotoxic risk of integrating gene vectors.Mol Ther. 2008; 16: 718-725Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar The impact of the target cell type is less well understood, although this question is of key interest to identify the parameters that explain the context-dependence of insertional transformation. Underlining this issue, it was recently demonstrated that mature T-lymphocytes are largely refractory to malignant transformation even following retroviral vector–mediated expression of potent oncogenes.13Newrzela S Cornils K Li Z Baum C Brugman MH Hartmann M et al.Resistance of mature T cells to oncogene transformation.Blood. 2008; 112: 2278-2286Crossref PubMed Scopus (170) Google Scholar Current protocols used in model organisms and clinical trials typically target a mixed population composed of a minority of repopulating HSCs (<1%) and a majority (>99%) of more mature hematopoietic progenitor cells (HPCs) that lack the potential for long-term repopulation. To obtain deeper insight into the extent of clonal skewing resulting from insertional mutagenesis, recently several groups have studied insertion profiles in the progeny of CD34+ cells isolated before infusion and compared the results with insertion profiles after long-term repopulation in clinical studies.14Deichmann A Hacein-Bey-Abina S Schmidt M Garrigue A Brugman MH Hu J et al.Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.J Clin Invest. 2007; 117: 2225-2232Crossref PubMed Scopus (219) Google Scholar,15Schwarzwaelder K Howe SJ Schmidt M Brugman MH Deichmann A Glimm H et al.Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo.J Clin Invest. 2007; 117: 2241-2249Crossref PubMed Scopus (175) Google Scholar,16Aiuti A Cassani B Andolfi G Mirolo M Biasco L Recchia A et al.Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy.J Clin Invest. 2007; 117: 2233-2240Crossref PubMed Scopus (224) Google Scholar This approach assumes that insertional mutagenesis may trigger the acquisition of long-term repopulation potential in HPC, which represent the great majority of the CD34+ population. Following a similar concept, targeting highly purified HSCs has been proposed to prevent insertional adverse events.17Mostoslavsky G Kotton DN Fabian AJ Gray JT Lee JS Mulligan RC Efficiency of transduction of highly purified murine hematopoietic stem cells by lentiviral and oncoretroviral vectors under conditions of minimal in vitro manipulation.Mol Ther. 2005; 11: 932-940Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar Several studies provided evidence that HPCs, although originally lacking the potential for long-term engraftment and self-renewal, can be transformed by retroviral gene transfer of certain oncogenes, further supporting the hypothesis that progenitor cells may contribute to the origin of transformed clones after insertional mutagenesis.18Cozzio A Passegué E Ayton PM Karsunky H Cleary ML Weissman IL Similar MLL-associated leukemias arising from self-renewing stem cells and short-lived myeloid progenitors.Genes Dev. 2003; 17: 3029-3035Crossref PubMed Scopus (568) Google Scholar,19Huntly BJ Shigematsu H Deguchi K Lee BH Mizuno S Duclos N et al.MOZ-TIF2, but not BCR-ABL, confers properties of leukemic stem cells to committed murine hematopoietic progenitors.Cancer Cell. 2004; 6: 587-596Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar,20Krivtsov AV Twomey D Feng Z Stubbs MC Wang Y Faber J et al.Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9.Nature. 2006; 442: 818-822Crossref PubMed Scopus (1163) Google Scholar Here, we addressed this important question in a murine model of competitive bone marrow (BM) transplantation using typical transduction conditions for γ-retroviral vectors but sorted cell populations as the starting material. In the same model, we also used serial clonal tracking to investigate the potential induction of clonal restriction following lentiviral vector transduction. To identify the origin of insertional mutants, we used syngenic C57BL/6J mice in which donor and host cells are distinguished by a chimerism in the common leukocyte antigen (CD45.2+ donor cells). We enriched repopulating hematopoietic cells, including the most primitive HSCs, in the LSK population: lineage marker negative, Sca1+, and c-Kit+.21Okada S Nakauchi H Nagayoshi K Nishikawa S Miura Y Suda T In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells.Blood. 1992; 80: 3044-3050PubMed Google Scholar,22Osawa M Nakamura K Nishi N Takahasi N Tokuomoto Y Inoue H et al.In vivo self-renewal of c-Kit+ Sca-1+ Lin(low/−) hemopoietic stem cells.J Immunol. 1996; 156: 3207-3214PubMed Google Scholar In parallel, we purified lineage marker negative, Sca1−, and c-Kit+ HPCs, hereafter referred to as LK cells (Figure 1a). The purity of the sorted subpopulations was >94%. Sorted cells were cultured in serum-free media supplemented with recombinant cytokines and transduced with γ-retroviral vectors encoding enhanced green fluorescent protein (EGFP) under control of retroviral long terminal repeat (LTR) enhancer–promoters (SF91EGFPpre*) (Table 1). This vector type is known to trigger insertional dominance or leukemias in murine models and in a clinical trial.7Kustikova O Fehse B Modlich U Yang M Düllmann J Kamino K et al.Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.Science. 2005; 308: 1171-1174Crossref PubMed Scopus (304) Google Scholar,8Ott MG Schmidt M Schwarzwaelder K Stein S Siler U Koehl U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (983) Google ScholarTable 1Overview of experimentsExperimentMouse IDDonor cells CD45.2+, sorted populationVectorDay of Day of transduction transplantationNo. of transplanted cells (input equivalents) per mouseType and number of competitor cellsNo. of miceObservation time, weeks1#1-5LSKNon.a.42.0 × 103CD45.1+; 3 × 105527#6-10LKNon.a.42.8 × 105CD45.1+; 3 × 105527#11-15LSKγ-Retroviral (SF91GFPpre)2-341.5 × 104CD45.1+; 3 × 105527#16-20LKγ-Retroviral (SF91GFPpre)2-342.8 × 105CD45.1+; 3 × 1055272A#21-25STHSCsNon.a.40.8 × 103CD45.1+; 3 × 105531#26-30LTHSCsγ-Retroviral (SF91GFPpre)340.9 × 102CD45.1+; 3 × 105531#31-35STHSCsγ-Retroviral (SF91GFPpre)2-340.8 × 103CD45.1+; 3 × 105531aFinal analysis for recipient ST#31 was performed at week 27.#36-40MPPγ-Retroviral (SF91GFPpre)2-341.7 × 104CD45.1+; 3 × 1055312B#41-45LTHSCsLentiviral (pRRL. PPT.SF.GFP.pre)010.9 × 102CD45.1+; 2 × 105532#46-50STHSCsLentiviral (pRRL. PPT.SF.GFP.pre)011.6 × 103CD45.1+; 2 × 105532Abbreviations: HSC, hematopoietic stem cell; LTHSC, long-term HSC; MPP, multipotent progenitor; n.a., not applicable; STHSC, short-term HSC. Host leukocytes were CD45.1+.a Final analysis for recipient ST#31 was performed at week 27. Open table in a new tab Abbreviations: HSC, hematopoietic stem cell; LTHSC, long-term HSC; MPP, multipotent progenitor; n.a., not applicable; STHSC, short-term HSC. Host leukocytes were CD45.1+. After preparatory experiments to establish transplant doses and transduction conditions, we achieved similar gene marking rates in LSK and LK cells (80 and 70% EGFP+, respectively) (Figure 1a). Reflecting the initial frequency of the populations, we transplanted 19× more EGFP+ LK than LSK cells (280,000 vs. 15,000 per recipient). The subpopulations were thus used in quantities that match standard experimental conditions in studies of insertional transformation and clonal dominance. Control animals received untransduced donor and host cells following the same scheme (experiment 1, Table 1). In a second experiment (experiment 2A, Table 1), we further split the LSK population into long-term repopulating HSCs (LTHSCs), short-term repopulating HSC (STHSCs), and multipotent progenitor (MPP) cells following marker definitions established for steady-state BM cells (Figure 1b).23Yang L Bryder D Adolfsson J Nygren J Månsson R Sigvardsson M et al.Identification of Lin−Sca1+kit+CD34+Flt3- short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients.Blood. 2005; 105: 2717-2723Crossref PubMed Scopus (321) Google Scholar We transduced all three fractions with the γ-retroviral vector (Supplementary Figure S1a). In another arm of this experiment (experiment 2B, Table 1), we transduced LTHSCs and STHSCs with a lentiviral vector (Supplementary Figure S1b). Therefore, a short culture time (<24 hours) in the presence of only two cytokines was used (Materials and Methods, Table 1). To monitor long-term repopulation, mice of all groups were prospectively observed for 7 months, with regular analyses of peripheral blood (PB) every 5–6 weeks and detailed final analysis of PB, BM, spleen, and in some cases liver. To detect insertional dominance, we monitored donor chimerism, EGFP expression in PB leukocytes and analyzed the “integrome” of transduced cells using a ligation-mediated PCR (LMPCR) procedure that focuses on dominant bands and neglects weak amplicons; previous studies have indicated that dominant bands correlate with dominant clones.24Kustikova OS Geiger H Li Z Brugman MH Chambers SM Shaw CA et al.Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways.Blood. 2007; 109: 1897-1907Crossref PubMed Scopus (78) Google Scholar Hereafter, we refer to this approach as dominant band insertion site analysis (DBISA). Following the known rules of hematopoietic reconstitution by sorted cell fractions, we expected that LK cells were not capable of long-term reconstitution unless being transformed by insertional mutagenesis. We found that neither transduced (EGFP+) nor untransduced (EGFP−) LK cells gave rise to significant levels of long-term repopulation (kinetics in Figure 2a; representative dot plots in Supplementary Figure S2). At early time points (PB, 5 weeks), EGFP+ LK progeny contributed only 0.31 ± 0.27% (mean ± 95% confidence interval) of the entire population with a further continuous decline until week 27 (0.04 ± 0.04%). Donor chimerism in PB and BM fell to 0.3 ± 0.1 and 0.8 ± 0.3%, respectively (Supplementary Table S1). MPP progeny (experiment 2A) showed somewhat better survival long-term, although it was also not capable of establishing significant levels of hematopoiesis. EGFP+ MPP progeny contributed 10.38 ± 2.11% of PB leukocytes at early time points (6 weeks post-transplantation) with a decline to 1.48 ± 0.62% at final analysis (31 weeks, Figure 2c). Donor chimerism analysis in PB, BM, and spleen reached only 3.3 ± 1.7, 1.7 ± 0.4, and 2.4 ± 0.3%, respectively (Supplementary Table S1). While γ-retroviral transduction was efficient (~70%, Supplementary Figure S1a), no evidence was obtained for preferential survival of EGFP+ cells within the MPP progeny (Supplementary Figure S2). LMPCR is more sensitive to detect transduced cells than flow cytometry but preferentially amplifies insertions from dominant clones.24Kustikova OS Geiger H Li Z Brugman MH Chambers SM Shaw CA et al.Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways.Blood. 2007; 109: 1897-1907Crossref PubMed Scopus (78) Google Scholar The pattern of γ-retroviral vector insertion sites (VISs) became reproducible when the starting material contained ~5 × 104 cells, corresponding to ~50 ng of clonal genomic DNA (Supplementary Figure S3a,b). In the LK progeny, authentic insertion sites could be recovered at early time points (week 11). In the MPP progeny, an oligoclonal pattern persisted until late time points (e.g., week 26, Supplementary Figure S4). DBISA revealed that one VIS in LK progeny and 7 out of 38 (18.4%) VISs in MPP progeny marked overt or suggested proto-oncogenes (Stat5b in LK; Fos, Bach2, D16Ertd472e, Notch1, Il4ra, Cd47, and BC031781 in MPP, Supplementary Tables S2 and S3). Despite such “suspicious” hits, some clones disappeared at later time points and none outcompeted competitor cells to contribute to >2% of hematopoiesis. Thus, we obtained no evidence that LK cells or MPP, or their transduced progeny, contribute to systemically relevant levels of clonal imbalance after retroviral vector–mediated insertional mutagenesis. Although transplanting almost 20-fold lower cell numbers in comparison with the LK conditions, the LSK progeny efficiently repopulated hosts in competition with cotransplanted, freshly isolated host-type cells (Figure 2a; engraftment analyses of individual animals in Supplementary Figure S2). γ-Retroviral marking rates for LSK EGFP+ progeny at week 27 in PB were 7–40%, accounting for 30–70% of donor-derived hematopoiesis (Figure 2a,b). Three mice showed a slight increase of the proportion of EGFP+ cells within the donor population after 16 weeks. Similar results were obtained in the progeny of γ-retrovirally transduced STHSCs, in marked contrast to the LK and MPP progeny (Figure 2c,d, Supplementary Figure S2). The progeny of STHSCs persisted at relatively high levels long-term even in the absence of γ-retroviral transduction (untransduced control group in Supplementary Figure S2). We cannot exclude that the culture conditions may have contributed to the long-term repopulation potential of STHSCs (see Discussion). Interestingly, γ-retroviral transduction of purified LTHSCs was inefficient with our experimental conditions and these cells showed a substantial loss of their competitive repopulation potential (Figure 2c,d and Supplementary Figure S2). Control experiments with lentiviral transduction conditions revealed that this was not due to an experimental artefact in the sorting of LTHSCs. To explore hematopoietic reconstitution after transducing STHSCs and LTHSCs using human immunodeficiency virus-1-based lentiviral vectors with their improved ability to transduce nondividing cells, we devised a short-term transduction protocol in a minimal cytokine cocktail, similar to earlier reports (see Material and methods and Table 1 for details).17Mostoslavsky G Kotton DN Fabian AJ Gray JT Lee JS Mulligan RC Efficiency of transduction of highly purified murine hematopoietic stem cells by lentiviral and oncoretroviral vectors under conditions of minimal in vitro manipulation.Mol Ther. 2005; 11: 932-940Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar Of note, the lentiviral vector contained the same retroviral enhancer–promoter as the γ-retroviral vector, albeit as a single element located between self-inactivating (SIN) LTR. Considering that SIN vectors with an internal retroviral enhancer–promoter may trigger insertional transformation,12Zychlinski D Schambach A Modlich U Maetzig T Meyer J Grassman E et al.Physiological promoters reduce the genotoxic risk of integrating gene vectors.Mol Ther. 2008; 16: 718-725Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar this vector was chosen to allow for potential upregulation of a proto-oncogene in case of a semi-random insertion event in its vicinity. However, the lentiviral vector design is expected to lower the risk of insertional gene activation because the number of active enhancers is reduced to one, and direct gene activation by the 3′-LTR is not possible. Compared to the results achieved with the prolonged γ-retroviral transduction protocol, we observed a significantly better engraftment of transduced cells with the lentiviral conditions (Wilcoxon rank sum test P < 0.05), although interanimal variability was still pronounced (Figures 2e,f and 3a,b; Supplementary Figure S2 and Supplementary Table S1). Surprisingly, STHSCs again showed superior long-term repopulation than LTHSCs (see Discussion). Within individual animals, marking levels were relatively stable, suggesting no major silencing of expression. Some animals tended to increase marking levels after week 12 (Figure 2e,f), as previously observed using the γ-retroviral conditions (Figure 2 b,d). To address whether a bias toward insertions in proto-oncogenes or related signaling genes had occurred in the progeny of sorted cell fractions, we analyzed hematopoietic samples of all 50 mice (overview in Table 1) every 5–6 weeks by DBISA (band pattern of the five recipients of transduced LSK cells in Figure 4a). Clonal fluctuations occurred in all mice, with oligoclonal stabilization in four of the five recipients (#12–15, Figure 4a). STHSC progeny generated the same oligoclonal pattern with progressive reduction of diversity and long-term persistence (representative data in Figure 4b). The poor results achieved with γ-retroviral transduction of purified LTHSCs indicate that this pattern cannot be attributed to the potential contamination of STHSC cultures with a priori LTHSCs. As in our previous studies using DBISA,7Kustikova O Fehse B Modlich U Yang M Düllmann J Kamino K et al.Clonal dominance of hematopoietic stem cells triggered by retroviral gene marking.Science. 2005; 308: 1171-1174Crossref PubMed Scopus (304) Google Scholar,10Modlich U Kustikova OS Schmidt M Rudolph C Meyer J Li Z et al.Leukemias following retroviral transfer of multidrug resistance 1 (MDR1) are driven by combinatorial insertional mutagenesis.Blood. 2005; 105: 4235-4246Crossref PubMed Scopus (159) Google Scholar,24Kustikova OS Geiger H Li Z Brugman MH Chambers SM Shaw CA et al.Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways.Blood. 2007; 109: 1897-1907Crossref PubMed Scopus (78) Google Scholar we excised the most prominent amplicons, likely to reflect dominant clones,24Kustikova OS Geiger H Li Z Brugman MH Chambers SM Shaw CA et al.Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways.Blood. 2007; 109: 1897-1907Crossref PubMed Scopus (78) Google Scholar for sequence analysis. To address whether the pattern observed in the PB reflected the primary site of hematopoiesis, we analyzed samples from BM and spleen. In all cases the dominant bands chosen for insertion site sequencing were also found in at least one of these two major hematopoietic organs (Supplementary Figure S4b,c). The complete list of “dominant amplicons” recovered in LSK progeny (n = 52) and STHSC progeny (n = 49) by DBISA is provided in Supplementary Tables S2 and S3. Very similar to our earlier studies targeting a bulk population of lineage-negative cells,24Kustikova OS Geiger H Li Z Brugman MH Chambers SM Shaw CA et al.Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways.Blood. 2007; 109: 1897-1907Crossref PubMed Scopus (78) Google Scholar we found an over-representation of insertions within 150 kb of genes listed as common insertion sites in the retrovirally tagged cancer gene database (Supplementary Table S4).25Akagi K Suzuki T Stephens RM Jenkins NA Copeland NG RTCGD: retroviral tagged cancer gene database.Nucleic Acids Res. 2004; 32: D523-D527Crossref PubMed Google Scholar We found no difference in the distribution around the transcriptional start site between the IDDb, data sets derived from primitive cell populations (LSK and STHSCs) and data sets originating from cell populations that lack the potential for clonal dominance (LK and MPP) (P = 0.432, Wilcoxon rank sum test). Insertional pattern of cultured (untransplanted) γ-retrovirally transduced MPP and STHSCs revealed highly polyclonal situations (data not shown), as reported earlier.24Kustikova OS Geiger H Li Z Brugman MH Chambers SM Shaw CA et al.Retroviral vector insertion sites associated with dominant hematopoietic clones mark “stemness” pathways.Blood. 2007; 109: 1897-1907Crossref PubMed Scopus (78) Google Scholar Analysis of cultured cells also demonstrated the ability of γ-retroviral vectors to transduce the progeny of LTHSCs (data not shown). The lentiviral LMPCR protocol demonstrated an initial polyclonal pattern especially in the mice receiving transduced STHSCs, whereas mice receiving transduced LTHSCs showed a more oligoclonal pattern (Figure 4c,d). Based on the number of observed integrations, we estimate that about 10% of the initial LTHSC input (~90 cells per animal, Table 1) manifested as dominant clones. This suggests that many LTHSCs may have lost their homing and engraftment properties due to the induction of cell cycle activity in response to cytokines,26Bowie MB McKnight KD Kent DG McCaffrey L Hoodless PA Eaves CJ Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect.J Clin Invest. 2006; 116: 2808-2816Crossref PubMed Scopus (273) Google Scholar and that others did not become dominant as a result of the known cell-intrinsic heterogeneity. Towards the final analysis, a trend to oligoclonal dominance was also observed in the STHSC group, with consistent clonal representations in PB, BM, and spleen (Figure 4d; Supplementary Figure S5). Remarkably, this had no impact on overall marking levels and chimerism (Figure 2e,f; Supplementary Figure S1). In contrast, the LTHSC group showed a similar clonality pattern at the final analysis as at week 8 (Figure 4c), revealing the correctness of our conditions for cell sorting. To address whether the clonality pattern observed in mice receiving lentivirally transduced HSCs was the result of insertional mutagenesis, we sequenced the dominant amplicons, using the same criteria as above (Supplementary Table S5). Dominant amplicons of lentiviral insertions were not enriched for locations close to proto-oncogenes or other signaling genes (Supplementary Table S4) and instead showed a much more frequent insertion in repeat regions than γ-retroviral insertions. Thus, although the vector contained a strong internal retroviral promoter that is capable of long-distance enhancer interactions and proto-oncogene upregulation from a SIN vector,27Modlich U Schambach A Brugman MH Wicke DC Knoess S Li Z et al.Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16.Leukemia. 2008; 22: 1519-1528Crossref PubMed Scopus (86) Google Scholar “integrome” studies gave no evidence for clonal imbalance as a result of insertional hits in proto-oncogenes by lentiviral vectors. The absence of “suspicious” insertion events reveals a s" @default.
• W2007294672 created "2016-06-24" @default.
• W2007294672 creator A5003922989 @default.
• W2007294672 creator A5004798089 @default.
• W2007294672 creator A5012852870 @default.
• W2007294672 creator A5028771222 @default.
• W2007294672 creator A5032959249 @default.
• W2007294672 creator A5043855146 @default.
• W2007294672 creator A5047937141 @default.
• W2007294672 date "2009-09-01" @default.
• W2007294672 modified "2023-09-24" @default.
• W2007294672 title "Cell-intrinsic and Vector-related Properties Cooperate to Determine the Incidence and Consequences of Insertional Mutagenesis" @default.
• W2007294672 cites W1546094383 @default.
• W2007294672 cites W1557986998 @default.
• W2007294672 cites W1588840730 @default.
• W2007294672 cites W1969435169 @default.
• W2007294672 cites W1971062917 @default.
• W2007294672 cites W1971410311 @default.
• W2007294672 cites W1977278866 @default.
• W2007294672 cites W1979262175 @default.
• W2007294672 cites W1991478808 @default.
• W2007294672 cites W1992900585 @default.
• W2007294672 cites W1999009002 @default.
• W2007294672 cites W2009078306 @default.
• W2007294672 cites W2017598005 @default.
• W2007294672 cites W2025774463 @default.
• W2007294672 cites W2026990179 @default.
• W2007294672 cites W2034087089 @default.
• W2007294672 cites W2037256495 @default.
• W2007294672 cites W2039081870 @default.
• W2007294672 cites W2044697812 @default.
• W2007294672 cites W2045662960 @default.
• W2007294672 cites W2047608367 @default.
• W2007294672 cites W2049688202 @default.
• W2007294672 cites W2052350491 @default.
• W2007294672 cites W2055033089 @default.
• W2007294672 cites W2058895038 @default.
• W2007294672 cites W2065226637 @default.
• W2007294672 cites W2072521874 @default.
• W2007294672 cites W2079664984 @default.
• W2007294672 cites W2093537780 @default.
• W2007294672 cites W2097606337 @default.
• W2007294672 cites W2107046588 @default.
• W2007294672 cites W2108244937 @default.
• W2007294672 cites W2109336877 @default.
• W2007294672 cites W2110510042 @default.
• W2007294672 cites W2111521836 @default.
• W2007294672 cites W2111967568 @default.
• W2007294672 cites W2119324304 @default.
• W2007294672 cites W2120478862 @default.
• W2007294672 cites W2121444412 @default.
• W2007294672 cites W2123659322 @default.
• W2007294672 cites W2137650223 @default.
• W2007294672 cites W2141621225 @default.
• W2007294672 cites W2147376659 @default.
• W2007294672 cites W2149294465 @default.
• W2007294672 cites W2156125289 @default.
• W2007294672 cites W2156338907 @default.
• W2007294672 cites W2157144442 @default.
• W2007294672 cites W2157996490 @default.
• W2007294672 cites W2167523976 @default.
• W2007294672 cites W2418944682 @default.
• W2007294672 doi "https://doi.org/10.1038/mt.2009.134" @default.
• W2007294672 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2835258" @default.
• W2007294672 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19532134" @default.
• W2007294672 hasPublicationYear "2009" @default.
• W2007294672 type Work @default.
• W2007294672 sameAs 2007294672 @default.
• W2007294672 citedByCount "64" @default.
• W2007294672 countsByYear W20072946722012 @default.
• W2007294672 countsByYear W20072946722013 @default.
• W2007294672 countsByYear W20072946722014 @default.
• W2007294672 countsByYear W20072946722015 @default.
• W2007294672 countsByYear W20072946722016 @default.
• W2007294672 countsByYear W20072946722017 @default.
• W2007294672 countsByYear W20072946722018 @default.
• W2007294672 countsByYear W20072946722019 @default.
• W2007294672 countsByYear W20072946722020 @default.
• W2007294672 countsByYear W20072946722023 @default.
• W2007294672 crossrefType "journal-article" @default.
• W2007294672 hasAuthorship W2007294672A5003922989 @default.
• W2007294672 hasAuthorship W2007294672A5004798089 @default.
• W2007294672 hasAuthorship W2007294672A5012852870 @default.
• W2007294672 hasAuthorship W2007294672A5028771222 @default.
• W2007294672 hasAuthorship W2007294672A5032959249 @default.
• W2007294672 hasAuthorship W2007294672A5043855146 @default.
• W2007294672 hasAuthorship W2007294672A5047937141 @default.
• W2007294672 hasBestOaLocation W20072946721 @default.
• W2007294672 hasConcept C104317684 @default.
• W2007294672 hasConcept C107635520 @default.
• W2007294672 hasConcept C143065580 @default.
• W2007294672 hasConcept C16318435 @default.
• W2007294672 hasConcept C40767141 @default.
• W2007294672 hasConcept C501734568 @default.
• W2007294672 hasConcept C54355233 @default.
• W2007294672 hasConcept C70721500 @default.
• W2007294672 hasConcept C86803240 @default.
• W2007294672 hasConcept C92087593 @default.

• next