Insertional Transformation of Hematopoietic Cells by Self-inactivating Lentiviral and Gammaretroviral Vectors
Authors: Ute Modlich, Susana Navarro, Daniela Zychlinski, Tobias Maetzig, Sabine Knoess, Martijn H. Brugman, Axel Schambach, Sabine Charrier, Anne Galy, Adrian J. Thrasher, Juan Bueren, Christopher Baum
Journal: Molecular Therapy (2009)

Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer–promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott–Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention. Gene transfer vectors may cause clonal imbalance and even malignant cell transformation by insertional upregulation of proto-oncogenes. Lentiviral vectors (LV) with their preferred integration in transcribed genes are considered less genotoxic than gammaretroviral vectors (GV) with their preference for integration next to transcriptional start sites and regulatory gene regions. Using a sensitive cell culture assay and a series of self-inactivating (SIN) vectors, we found that the lentiviral insertion pattern was approximately threefold less likely than the gammaretroviral to trigger transformation of primary hematopoietic cells. However, lentivirally induced mutants also showed robust replating, in line with the selection for common insertion sites (CIS) in the first intron of the Evi1 proto-oncogene. This potent proto-oncogene thus represents a CIS for both GV and LV, despite major differences in their integration mechanisms. Altering the vectors' enhancer–promoter elements had a greater effect on safety than the retroviral insertion pattern. Clinical grade LV expressing the Wiskott–Aldrich syndrome (WAS) protein under control of its own promoter had no transforming potential. Mechanistic studies support the conclusion that enhancer-mediated gene activation is the major cause for insertional transformation of hematopoietic cells, opening rational strategies for risk prevention. IntroductionGene vectors based on retroviruses are widely used for stable genetic modification of somatic cells.1Kay MA Glorioso JC Naldini L Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics.Nat Med. 2001; 7: 33-40Crossref PubMed Scopus (1060) Google Scholar Improvements of retroviral vector technology have led to the design of self-inactivating (SIN) vectors in which the enhancer–promoter sequences of the long terminal repeats (LTRs) are deleted and instead the gene of interest is expressed from an internal promoter. Another major progress was the conversion of complex lentiviruses, such as the human immunodeficiency virus type 1, into efficient gene vectors that combine advantages of the SIN design with the potential to stably transduce nondividing cells.2Naldini L Blömer U Gage FH Trono D Verma IM Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.Proc Natl Acad Sci USA. 1996; 93: 11382-11388Crossref PubMed Scopus (1261) Google Scholar However, of all retroviruses investigated to date, lentiviruses and derived vectors show the greatest preference to integrate within active transcription units. In contrast to gammaretroviral vectors (GV), lentiviral vectors (LV) have no preference to integrate in a 10 kilobase window surrounding the transcriptional start sites and also no preference to integrate near regulatory gene regions that coincide with DNAse 1 hypersensitive sites.3Wu X Li Y Crise B Burgess SM Transcription start regions in the human genome are favored targets for MLV integration.Science. 2003; 300: 1749-1751Crossref PubMed Scopus (1132) Google Scholar,4Lewinski MK Yamashita M Emerman M Ciuffi A Marshall H Crawford G et al.Retroviral DNA integration: viral and cellular determinants of target-site selection.PLoS Pathog. 2006; 2: e60Crossref PubMed Scopus (286) Google Scholar In freshly transduced primary human hematopoietic cells, insertions near proto-oncogenes are at least twice less likely for SIN-LV compared to LTR-driven GV.5Cattoglio C Facchini G Sartori D Antonelli A Miccio A Cassani B et al.Hot spots of retroviral integration in human CD34+ hematopoietic cells.Blood. 2007; 110: 1770-1778Crossref PubMed Scopus (218) Google ScholarConsidering the complex organization of individual genes within the genome, the relatively global perspective of bioinformatic "integrome" studies can hardly predict the functional consequences of vector insertions. Illustrating this issue, in the few cases of cell transformation observed to date when using LTR-driven GV, the activation of cellular proto-oncogenes (LMO2, MDS-EVI1, CYCLIND2, or BMI1) occurred from many types of genomic locations: distant upstream or downstream, upstream promoter-proximal, introns, and across gene borders.6Ott 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 (974) Google Scholar,7Howe SJ Mansour MR Schwarzwaelder K Bartholomae C Hubank M Kempski H et al.Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.J Clin Invest. 2008; 118: 3143-3150Crossref PubMed Scopus (919) Google Scholar,8Hacein-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 (1363) Google Scholar Randomly isolated cell clones transduced by SIN-LV showed that the likelihood to activate a neighboring cellular gene largely depends upon the type of the vector's internal promoter, and similar observations were made with SIN-GV.9Hargrove PW Kepes S Hanawa H Obenauer JC Pei D Cheng C et al.Globin lentiviral vector insertions can perturb the expression of endogenous genes in beta-thalassemic hematopoietic cells.Mol Ther. 2008; 16: 525-533Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Recently, two model systems that used pretransformed target cells showed evidence for insertional transformation by LV containing strong enhancer–promoter sequences located in the LTRs,10Montini E Cesana D Schmidt M Sanvito F Bartholomae CC Ranzani M et al.The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.J Clin Invest. 2009; 119: 964-975Crossref PubMed Scopus (455) Google Scholar,11Bokhoven M Stephen SL Knight S Gevers EF Robinson IC Takeuchi Y et al.Insertional gene activation by lentiviral and gammaretroviral vectors.J Virol. 2009; 83: 283-294Crossref PubMed Scopus (104) Google Scholar whereas SIN-LV containing the same enhancer–promoter sequences as an internal promoter did not accelerate tumorigenesis. Based upon the comparison of LTR-proficient LV and GV, evidence was obtained that the GV insertion pattern induces a 10-fold increased risk of cell transformation.10Montini E Cesana D Schmidt M Sanvito F Bartholomae CC Ranzani M et al.The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.J Clin Invest. 2009; 119: 964-975Crossref PubMed Scopus (455) Google Scholar The actual risk induced by SIN-LV containing strong internal enhancer–promoter sequences remained unknown.We have previously used a simple cell culture model to quantify the risk of hematopoietic cell transformation by GV dependent on their design. This assay uses dose-escalated gene transfer into primary murine hematopoietic cells isolated from untreated adult C57BL6 mice, and thus operates in a genetic background lacking a pre-existing transforming lesion. Cell transformation is detected by culturing cells post-transduction under myeloid differentiation conditions, such that a replating step in limiting dilution suppresses residual self-renewal capacity unless insertional upregulation of cellular proto-oncogenes occurs. Animal experiments with a tumor end point are not required, and a quantification of transforming events is greatly facilitated when compared with presently available murine in vivo models. This approach, here referred to as in vitro immortalization (IVIM) assay, is relatively specific for upregulation of Evi1 or its close relative Prdm16 (refs. 12Du Y Jenkins NA Copeland NG Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells.Blood. 2005; 106: 3932-3939Crossref PubMed Scopus (160) Google Scholar,13Zychlinski 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 (240) Google Scholar,14Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (274) Google Scholar), both of which have been shown to be clinically relevant as inducers of clonal imbalance in the context of a gene therapy trial to treat patients suffering from an X-linked form of chronic granulomatous disease.6Ott 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 (974) Google Scholar Activating rearrangements in the human EVI1 gene are a major driving force of acute myeloid leukemia,15Nucifora G Laricchia-Robbio L Senyuk V EVI1 and hematopoietic disorders: history and perspectives.Gene. 2006; 368: 1-11Crossref PubMed Scopus (84) Google Scholar underscoring the potential clinical relevance of the IVIM assay. As described earlier, the IVIM assay is able to quantify the incidence of mutants on the basis of the initial number of transduced cells and the clonal characterization of the mutants that show robust replating after limiting dilution. We show that the risk of insertional transformation by SIN-LV, just as previously reported for SIN-GV, depends upon the type of the internal promoter. Owing to the high sensitivity of the assay, we describe Evi1 as the first transforming common insertion sites (CIS) shared by SIN-GV and SIN-LV, and formally define the enhancer as the driving force of transformation in this model. SIN-LV designed to correct a genetic disease using a cellular promoter were unable to trigger cell transformation in this model.ResultsLower incidence of transformation induced by SIN-LV compared to SIN-GVTo address whether the clinically relevant SIN-LV may transform primary cells by insertional mutagenesis even in the absence of pre-established transforming lesions, we here performed a direct head-to-head comparison with GV, using the IVIM assay (experimental outline in Supplementary Figure S1).13Zychlinski 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 (240) Google Scholar,14Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (274) Google Scholar As a positive control, we chose LTR-driven GV (SF91.eGFP.pre) with the enhancer–promoter of spleen focus-forming virus (SFFV), which exhibits high activity in hematopoietic cells and is known to trigger leukemia by insertional mutagenesis in mice.16Baum C Itoh K Meyer J Laker C Ito Y Ostertag W The potent enhancer activity of the polycythemic strain of spleen focus-forming virus in hematopoietic cells is governed by a binding site for Sp1 in the upstream control region and by a unique enhancer core motif, creating an exclusive target for PEBP/CBF.J Virol. 1997; 71: 6323-6331Crossref PubMed Google Scholar,17Modlich 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 To study the impact of vector backbone, we tested SIN-GV (GV.SF.eGFP.pre) and SIN-LV (LV.SF.eGFP and LV.SF.eGFP.pre) containing the same internal enhancer–promoter of SFFV (Figure 1a).When exposing 100,000 cells per transduction in the IVIM assay, GV vectors with the SFFV enhancer–promoter induced replating in all experiments, and Southern blots revealed a maximum of two molecularly distinct clones per 100,000 transduced cells for SIN-GV.13Zychlinski 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 (240) Google Scholar,14Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (274) Google Scholar The average incidence of independent mutants obtained with this vector was thus 1–2 × 10−5. LV with a similar design and the same internal enhancer–promoter (SFFV) reduced the incidence of mutants to 5 × 10−6: replating clones occurred in every second assay, which resulted in a significant (approximately threefold) reduction of immortalizing events as compared to similarly designed SIN-GV (Fisher's exact test P = 0.0058) (Figure 1b). Importantly, a single insertion was sufficient for transformation by SIN-LV according to quantitative PCR detecting vector integrations (Table 1, clone S1).Table 1Insertion sites recovered from lentiviral immortalized clones transduced with the vector LV.SF.eGFP.pre (T1, T3, T5, T8, and S1) or LV.SF.eGFP (su1-1-1, su1-1-2, su1-1-3, and su1-2-2)CloneLocationGene IDGene symbolChromosomeDistance to transcriptional start siteOrientationDescriptionSu1-1-1123277522232358/56619EG232358/Clec4e619,171/-37,633F/FC-type lectin domain family 4, member eSu1-1-12783957100039652LOC100039652Y−2,121FUndefinedSu1-1-1149560299436240/245671EG436240/Klf8X6,968/-112,289F/RForkhead box protein R2/Kruppel-like factor 8Su1-1-185296511668848/72416LOC668848/Lrpprc1745,291/-106,385F/FLeucine-rich PPR-motif containingSu1-1-13535403679221Hdac912−140,297RHistone deacetylase 9Su1-1-135490247708444921508M14Rik1269,278FUndefinedSu1-1-110502182718479Pak1730,378RP21 (CDKN1A)-activated kinase 1Su1-1-131273821235135/235134Tmem45b/Nfrkb9−1,998/80,023F/FTransmembrane protein 45b/nuclear factor related to kB binding proteinSu 1-1-2149560210436240/245671EG436240/Klf8X6,968/-112,289F/RForkhead box protein R2/Kruppel-like factor 8Su 1-1-28979492023897Hax137,688FHCLS1-associated X-1Su 1-1-235490247708444921508M14Rik1269,278FUndefinedSu 1-1-3144779029230895/21938Vps13d/Tnfrsf1b45,869/57,744R/RVacuolar protein sorting 13 D (yeast)/tumor necrosis factor receptor superfamily member 1bSu 1-1-335490428708444921508M14Rik1269,097FUndefinedSu 1-2-241780680100042493/16157LOC100042493/Il11ra146,476/71,449R/FInterleukin-11 receptor, a chain 1Su PGK198502989100042886LOC100042886268,111RUndefinedS13000120014013Evi13−104,614REcotropic viral integration site 1T385088565629794LOC6297941472,424FUndefinedT3149974890242773Slc45a1451,393RSolute carrier family 45, member 1T312975760867596/119235830405N20Rik/Neurod410−26,708/-40,355R/RRIKEN cDNA 5830405N20 gene/neurogenic differentiation 4T33001189114013Evi13−115,305REcotropic viral integration site 1T59144371526905Eif2s3xX14,275REukaryotic translation initiation factor 2, subunit 3, structural gene X-linkedT585088427629794LOC6297941472,562FUndefinedT512975763767596/119235830405N20Rik/Neurod410−26,708/-40,355R/RRIKEN cDNA 5830405N20 gene/neurogenic differentiation 4T5149974890242773Slc45a1451,393RSolute carrier family 45, member 1T53001189114013Evi13−115,305REcotropic viral integration site 1T51418929314048Eya11110,987FEyes absent 1 homologue (Drosophila)T145957914383644LOC3836441−10,714RUndefinedT11633725967448Plxdc2259,310RPlexin domain containing 2T111113243111906Atbf18−106,113FAT motif binding factor 1T17645022359093Pcbp310−25,690FPoly(rC) binding protein 3T16609045173668Ttc21b24,223FTetratricopeptide repeat domain 21BT19982952818679Phka1X10,009FPhosphorylase kinase alpha 1T1111620859629967LOC62996711−31,229RZinc-finger protein 93T112707167811606Agt821,929FAngiotensinogen (serpin peptidase inhibitor, clade A, member 8)T820266790208618Etl4255,250REnhancer trap locus 4T83001082514013Evi13−114,239REcotropic viral integration site 1T83365530819027Sypl1216,475RSynaptophysin-like proteinT867588105433715LOC4337154−3,781FUndefinedT874820773100043883LOC1000438831717,238RUndefinedT850013507240283Dmxl11820,840RDmx-like 1T82125993819720Trim2713−11,890FTripartite motif protein 27T86670641070918Nsun7555,046FNOL1/NOP2/Sun domain family, member 7 Open table in a new tab Lower fitness of mutants induced by SIN-LVNext to the incidence of replating mutants, the assay also reports their "fitness" or self-renewal capacity. As mutants expand before the replating step, the frequency of positive wells in limiting dilution analysis reflects the self-renewing capacity of the clone.13Zychlinski 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 (240) Google Scholar,14Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (274) Google Scholar Assuming a positive correlation of the number of insertions with the risk of activating mutations, we correct the replating efficiency by the post-transduction copy number to calculate a replating index.13Zychlinski 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 (240) Google Scholar,14Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (274) Google Scholar To determine the average replating index of the mutants, we ignored assays with a negative result in the replating step (because no mutants were detectable in these cases). Interestingly, LV-induced mutants tended to have a lower replating index than GV harboring identical internal SFFV enhancer–promoters (approximately fourfold reduction, Figure 1b and Table 2). The post-transcriptional regulatory element derived from the woodchuck hepatitis virus, frequently used to boost titers and transgene expression in LV,18Zufferey R Donello JE Trono D Hope TJ Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.J Virol. 1999; 73: 2886-2892Crossref PubMed Google Scholar had no significant effect (compare LV.SF.eGFP and LV.SF.eGFP.pre in Figure 1b and Table 2). Irrespective of the backbone (LV and GV), SIN vectors induced a significantly lower replating index than the LTR-driven GV (GV SIN versus GV LTR P = 0.03, LV SIN versus GV LTR P = 0.011 Wilcoxon two-sample test, Figure 1b).Table 2Replating activity as a function of vector designVectorNumber of replating clones (100 cells/well)Number of replating clones (10 cells/well)Mean copy number per cellReplating frequencyReplating index (frequency/copy number)LV.WASP.WAS0n.d.2.52000n.d.5.15000n.d.3.12000n.d.0.8000Median2.82LV.PGK.eGFP0n.d.6.03000n.d.7.24002n.d.6.130.000210.000030n.d.8.09000n.d.9.00000n.d.8.00000n.d.20.07000n.d.19.8000Median8.04LV.VAV.cGreen0n.d.4.40000n.d.6.13003n.d.2.350.000320.000140n.d.3.01000n.d.2.84000n.d.2.13000n.d.2.35000n.d.7.9500Median2.93LV.SF.eGFP95n.d.8.230.045450.005530n.d.8.37000n.d.9.00000n.d.8.000023n.d.7.000.002740.000397n.d.3.000.000760.000250n.d.15.21000n.d.16.9200Median8.300.002740.00039LV.SF.eGFP.pre44n.d.16.000.006130.000380n.d.22.00007n.d.2.000.000760.0003813n.d.4.000.001460.0003658311.000.008420.000770015.000027n.d.0.990.003300.003340n.d.1.4600Median7.500.003300.00038GV.SF.eGFP.pre962916.400.043400.00265aReference data as described earlier.14841616.650.020000.00120aReference data as described earlier.1413420.920.001300.00006aReference data as described earlier.14963022.000.044000.00200aReference data as described earlier.149225.500.001100.00004aReference data as described earlier.14924126.600.041670.00157aReference data as described earlier.1470n.d.3.600.013060.0036396n.d.8.600.045750.005325n.d.4.500.000530.0001291n.d.11.600.029550.0025510n.d.1.440.001100.0007635n.d.3.210.004530.00141Median14.000.016530.00149SF91.eGFP.pre96n.d.7.000.045750.0004870n.d.2.780.012990.0046743n.d.4.640.005950.0012885n.d.2.260.021740.0096388n.d.5.230.025000.0047844n.d.5.500.006130.0011275n.d.5.500.015150.0027564n.d.6.660.010990.00165Median5.360.014070.00220Abbreviation: n.d., not determined.a Reference data as described earlier.14Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (274) Google Scholar Open table in a new tab CIS in the Evi1 proto-oncogene shared by SIN-LV and SIN-GVTo investigate whether the clones induced by LV shared CIS with the mutants induced by GV, we analyzed integration sites by ligation-mediated PCR. CIS in independent clones indicate selection for transformation.19Uren AG Kool J Berns A van Lohuizen M Retroviral insertional mutagenesis: past, present and future.Oncogene. 2005; 24: 7656-7672Crossref PubMed Scopus (212) Google Scholar In three of the six lentiviral clones examined, insertions occurred in a relatively small region of 11 kilobases (9%) of the first intron of the Evi1 proto-oncogene (Figure 2a). All insertions within this region retrieved from immortal clones were in reverse orientation, irrespective of the vector type used (Figure 2a). To our knowledge, this is the first demonstration of a shared transforming CIS for these only remotely related retroviruses (murine leukemia virus–based SIN-GV and human immunodeficiency virus type 1–based SIN-LV).Figure 2Evi1 is a common insertion site for both gammaretroviral and lentiviral vectors. (a) Insertion sites identified in Evi1: in three of six independent lentiviral clones analyzed, we detected one insertion site in the first intron of Evi1 (long arrows with asterisks). The insertions were found within a region comprising about 11 kilobases in which most of the gammaretroviral vector (GV) insertions were detected so far. Previously reported insertion sites detected in in vitro immortalization clones are indicated as short hatched arrows (GV.SF.eGFP.pre) and short arrows (SF91.eGFP.pre).14Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (274) Google Scholar GV insertions recovered in leukemias and dominant clones in vivo in the mouse are indicated below the horizontal line.37Kustikova 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 (b) Northern blot analysis showing strong upregulation of Evi1 transcript in all clones analyzed compared to the expression levels in mock-treated cells (left lane). (c) Evi1 expression levels in immortalized clones that were obtained and expanded in this study. All clones show strong upregulation of Evi1 mRNA when compared to the level in mock cells (grown for 2 weeks as mass culture).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantitative reverse transcription–PCR and northern blots revealed strongly increased Evi1 expression starting from the second alternative exon 1b, but not from exon 1a. Interestingly, Mds-Evi1 fusion transcripts were downregulated compared to controls in all clones tested. Northern blots showed a common larger band irrespective of the type of vector used, matching the full-length Evi1 transcript starting from exon 1b (BCO76620). Shorter transcripts were also induced whose sizes differed depending on the vector used (Figure 2b). An alternative transcript terminating with exon 8 (AK051098) could be amplified from immortalized clones. Expression of this shorter transcript in murine lineage-negative cells was not transforming (n = 3), in contrast to the established transforming activity of the full-length Evi1 transcript.12Du Y Jenkins NA Copeland NG Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells.Blood. 2005; 106: 3932-3939Crossref PubMed Scopus (160) Google Scholar It remains to be determined whether the different splice pattern observed dependent on the type of integrated vector sequences contributed to the observed differences in cell fitness.Real-time reverse transcription–PCR indicated a >500-fold induction of Evi1 mRNA in clones induced by LV, similar to data obtained after insertional transformation with GV (Figure 2c).13Zychlinski 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 (240) Google Scholar,14Modlich U Bohne J Schmidt M von Kalle C Knöss S Schambach A et al.Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.Blood. 2006; 108: 2545-2553Crossref PubMed Scopus (274) Google Scholar This supports the conclusion that the lower incidence of mutants obtained with LV compared to GV is primarily related to the integration pattern.SIN-LV equipped with cellular promoters are less genotoxicIn a further series of experiments, we addressed whether the replacement of the SFFV enhancer–promoter by cellular promoters would reduce the transforming risk of LV insertions. We tested SIN-LV containing cellular constitutive promoters derived from the VAV and PGK genes. Replating cells could only be observed with a very low frequency, indicating a low incidence and also a low fitness of potential mutants (Figure 1b). Sustained growth was not observed. Insertion site analysis revealed untransduced cells or, in one clone, an insertion in a region of low gene density on chromosome 2 (Table 1), suggesting background replating activity. Thus, the choice of the promoter was more important to prevent the induction of mutants than the difference between the GV and LV insertion pattern.Furthermore, we assessed whether experimental conditions could be adapted to test clinical vectors expressing potentially therapeutic genes. The SIN-LV named WASP.WAS is a potentially therapeutic construct expressing the Wiskott–Aldrich syndrome (WAS) protein under control of a fragment of its own promoter. WAS patients suffer from a severe inherited immunodeficiency syndrome. WAS promoter native regulatory sequences are weak compared to other promoters such as phosphoglycerate kinase (PGK) or SFFV,20Charrier S Dupré L Scaramuzza S Jeanson-Leh L Blundell MP Danos O et al.Lentiviral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients.Gene Ther. 2007; 14: 415-428Crossref PubMed Scopus (102) Google Scholar but may preserve natural gene regulation. Preclinical murine studies did not reveal potential induction of leukemia in the long-term follow-up.21Marangoni F Bosticardo M Charrier S Draghici E Locci M Scaramuzza S et al.Evidence for long-term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models.Mol Ther. 2009; 17: 1073-1082Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar In the IVIM assay, up to five copies of this vector per cell did not induce replating, in contrast to the control SIN-LV containing the SFFV enhancer–promoter (Figure 1b).Vector enhancer but not promoter sequences explains the induction of mutants in the IVIM assayIn cell clones transduced by GV and LV, dysregulation of neighboring cellular genes depended on the nature of the vector's enhancer–promoter.22Maruggi