- Short report
- Open Access
Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2locus
- Koichiro Yamada†1,
- Tomonori Tsukahara†1,
- Kazuhisa Yoshino†1,
- Katsuhiko Kojima1,
- Hideyuki Agawa1,
- Yuki Yamashita1,
- Yuji Amano1,
- Mariko Hatta1,
- Yasunori Matsuzaki1,
- Naoki Kurotori1,
- Keiko Wakui2,
- Yoshimitsu Fukushima2,
- Ryosuke Osada3,
- Tanri Shiozawa3,
- Kazuo Sakashita4,
- Kenichi Koike4,
- Satoru Kumaki5,
- Nobuyuki Tanaka6 and
- Toshikazu Takeshita1Email author
© Yamada et al; licensee BioMed Central Ltd. 2009
- Received: 16 February 2009
- Accepted: 2 September 2009
- Published: 2 September 2009
Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 104 cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34+ cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.
- Transcription Start Site
- Integration Site
- Green Fluorescent Protein Fluorescence
- Vector Integration
- Infection Efficiency
The IL2RG gene encodes the interleukin-2 receptor γ chain (IL-2Rγ) , and mutations in this gene cause X-linked severe combined immunodeficiency (X-SCID) . Gene therapy trials for X-SCID have achieved remarkably successful outcomes [3–5] but have also been associated with leukemogenesis in some patients. Analyses of leukemic cell clones from these patients revealed that the murine leukemia virus (MLV) vector had integrated proximal to the promoter of an oncogene involved in T-cell acute lymphoblastic leukemia, L I M - o nly protein 2 (LMO2), resulting in aberrant expression. These findings suggest that retroviral vector integration near the LMO2 promoter is the most likely cause of leukemogenesis in these cases [6–8]. Several oncogenes, including LMO2, have very recently been reported to be target genes for vector integration in two patients that developed leukemia following retroviral-mediated gene therapy [9, 10]. Accordingly, a determination of the frequency of vector integration near the transcription start site (TSS) of LMO2 would be important for understanding the mechanism of the LMO2 insertional mutagenesis observed in the leukemic cell clones. The frequency of vector integrations near the TSS of the LMO2 locus has not been previously described. In the present study, we have detected a region where vectors integrated with high frequency near the TSS of the LMO2 locus in two T cell lines and human cord blood-derived CD34+ cells, and we have subsequently determined the frequency of this vector integration in TPA-Mat and CD34+ cells.
We previously identified 340 integration sites and 15 integration hotspots (defined as ≥ 3 integration sites within a 100-kb region) for MLV vector integration in infected human T cell line clones . A hotspot in intron 2 of the T RAF2- and N CK-interacting k inase (TNIK) gene had three integration sites within 3.5-kb, indicating that this hotspot is an appropriate locus for estimating the integration frequency. We selected clone 705-9, which has an integrated vector in the hotspot region of the TNIK locus . We investigated the sensitivity of the PCR techniques utilized in this study. One copy of the junction sequence between the virus gene and the TNIK gene was amplified from DNA harvested from 705-9 cells, in the presence of 1 μg (1.5 × 105 cells) of genomic DNA from parental TPA-Mat-ecoR cells. A nested PCR using a 3' LTR-specific primer and a TNIK-specific primer showed that one copy of the integrated vector was detectable as a 1.5-kb PCR product (data not shown), demonstrating the sensitivity of this assay.
Subsequently, we examined whether vector integration would affect LMO2 expression in TPA-Mat-ecoR cells. Endogenous LMO2 mRNA was not detected after vector infection (100%, based on GFP fluorescence) in TPA-Mat-ecoR cells (Figure 2A). We then prepared a series of luciferase reporter gene constructs containing the region between -3020 and +147 of the LMO2 promoter region. The construct pGL3lmo2 (3020) containing the region (-3020 ~+147) was virtually silent compared with the pGL3-basic construct containing a SV40 promoter only (Figure 2B). The insertion of the MLV LTR into a site (-1798) within the HIR, where forward or reverse orientation of the inserted vector was observed (Additional file 3), resulted in significant increases in reporter gene activity. A similar result was obtained by the insertion into another site (-2965), which is an integration site reported in the leukemia patient. Consequently, these results suggest that vector integration at -1798 within the HIR may increase transcriptional activity of the LMO2 gene, similar to the report for vector integration at -2965 in the leukemia patient .
We compared the integration pattern in the TPA-Mat-ecoR cells with the integration sites identified in patients (Pt) 4 and 5 who developed leukemia during the gene therapy trials for treatment of X-SCID [12, 17]. Vector integration into the position detected in Pt4 (Figure 1C) was rare in TPA-Mat-ecoR cells; differences in the integration frequencies between the upstream (-1 ~-3000; 60/533) and downstream (1 ~3000; 3/540) regions of exon 1 (Figure 1C) were observed. In contrast, the integration site (-2965) in Pt5 was located in the HIR (-1740 ~-3001) (Figure 1C). Since CD34+ hematopoietic stem cells have been infected with the MLV vector in the clinical trials, we investigated whether the HIR is found in human CD34+ hematopoietic stem cells. Using the same primer sets in umbilical cord blood CD34+ cells (infection efficiency: 14.7%, based on GFP fluorescence), we have identified an HIR (-1882 to -2971) (18/270) in a similar region as the TPA-Mat-ecoR and Jurkat-ecoR cells (Figure 1F, Additional files 2 and 3). Only a few integrations were found from 1 to -1500 (2/270) (Figure 1F, Additional files 2 and 3). Thus, analyzing the location of the HIR in hematopoietic stem cells in these patients will provide insights into leukemogenic integration sites and may have an impact on future gene therapy trials. The HIR is also a suitable region for analyzing the molecular mechanism of vector integration with target-site preferences [14, 18]. On the other hand, results showing retroviral integration sites 35 kb upstream  and 10.6 kb downstream  of the TSS were reported in the patients, and sites 36.3 kb, 69.2 kb, 68.0 kb, 68.3 kb and 0.9 kb upstream of the LMO2 TSS were detected in a murine leukemia model . This indicates that integrations in the sites or regions which are far from the TSS are closely associated in LMO2-related leukemogenesis. Analysis of the differences and similarities between the HIR near the TSS and the regions far from the TSS will therefore facilitate the elucidation of LMO2-related leukemogenesis in the future and may identify additional HIRs that may exist far from the TSS.
In conclusion, the identification of the HIR near exon 1 of the LMO2 locus in the T cell lines and human CD34+ cells may partially explain the mechanism responsible for the LMO2-insertional mutagenesis observed in leukemic cell clones. It may help us to better understand vector-induced leukemogenesis.
We thank Dr. T. Kitamura (University of Tokyo, Tokyo, Japan) for providing pMXs and gagpol-IRES-brs; Dr. T. Kafri (University of North Carolina, Chapel Hill, NC, USA) for providing pcDNA VSV-G, Dr. H. Sakai (Shinshu University, Nagano, Japan) for help with statistical analyses, and Dr. T. Kikuchi (Shinshu University, Nagano, Japan) for critical discussion. We also thank the Instrumental Analysis Research Center for Human and Environmental Science at Shinshu University for technical assistance with DNA sequencing and flow cytometric analyses. This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Young Scientists (B 19790335) (T. Tsukahara) and the Human Resource Development Plan for Cancer (T. Takeshita).
- Takeshita T, Asao H, Ohtani K, Ishii N, Kumaki S, Tanaka N, Munakata H, Nakamura M, Sugamura K: Cloning of the gamma chain of the human IL-2 receptor. Science. 1992, 257: 379-382. 10.1126/science.1631559.View ArticlePubMedGoogle Scholar
- Noguchi M, Yi H, Rosenblatt HM, Filipovich AH, Adelstein S, Modi WS, McBride OW, Leonard WJ: Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell. 1993, 73: 147-157. 10.1016/0092-8674(93)90167-O.View ArticlePubMedGoogle Scholar
- Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, et al: Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science. 2000, 288: 669-672. 10.1126/science.288.5466.669.View ArticlePubMedGoogle Scholar
- Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP, Thrasher AJ, Wulffraat N, Sorensen R, Dupuis-Girod S, et al: Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med. 2002, 346: 1185-1193. 10.1056/NEJMoa012616.View ArticlePubMedGoogle Scholar
- Gaspar HB, Parsley KL, Howe S, King D, Gilmour KC, Sinclair J, Brouns G, Schmidt M, Von Kalle C, Barington T, et al: Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet. 2004, 364: 2181-2187. 10.1016/S0140-6736(04)17590-9.View ArticlePubMedGoogle Scholar
- McCormack MP, Rabbitts TH: Activation of the T-cell oncogene LMO2 after gene therapy for X-linked severe combined immunodeficiency. N Engl J Med. 2004, 350: 913-922. 10.1056/NEJMra032207.View ArticlePubMedGoogle Scholar
- Pike-Overzet K, de Ridder D, Weerkamp F, Baert MR, Verstegen MM, Brugman MH, Howe SJ, Reinders MJ, Thrasher AJ, Wagemaker G, et al: Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy. Leukemia. 2007, 21: 754-763.PubMedGoogle Scholar
- Pike-Overzet K, Burg van der M, Wagemaker G, van Dongen JJ, Staal FJ: New insights and unresolved issues regarding insertional mutagenesis in X-linked SCID gene therapy. Mol Ther. 2007, 15: 1910-1916. 10.1038/sj.mt.6300297.View ArticlePubMedGoogle Scholar
- Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, Clappier E, Caccavelli L, Delabesse E, Beldjord K, et al: Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest. 2008, 118: 3132-3142. 10.1172/JCI35700.PubMed CentralView ArticlePubMedGoogle Scholar
- Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H, Brugman MH, Pike-Overzet K, Chatters SJ, de Ridder D, et al: Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest. 2008, 118: 3143-3150. 10.1172/JCI35798.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsukahara T, Agawa H, Matsumoto S, Matsuda M, Ueno S, Yamashita Y, Yamada K, Tanaka N, Kojima K, Takeshita T: Murine leukemia virus vector integration favors promoter regions and regional hot spots in a human T-cell line. Biochem Biophys Res Commun. 2006, 345: 1099-1107. 10.1016/j.bbrc.2006.05.007.View ArticlePubMedGoogle Scholar
- Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E, et al: LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003, 302: 415-419. 10.1126/science.1088547.View ArticlePubMedGoogle Scholar
- Wu X, Li Y, Crise B, Burgess SM: Transcription start regions in the human genome are favored targets for MLV integration. Science. 2003, 300: 1749-1751. 10.1126/science.1083413.View ArticlePubMedGoogle Scholar
- Cattoglio C, Facchini G, Sartori D, Antonelli A, Miccio A, Cassani B, Schmidt M, von Kalle C, Howe S, Thrasher AJ, et al: Hot spots of retroviral integration in human CD34+ hematopoietic cells. Blood. 2007, 110: 1770-1778. 10.1182/blood-2007-01-068759.View ArticlePubMedGoogle Scholar
- Berry C, Hannenhalli S, Leipzig J, Bushman FD: Selection of target sites for mobile DNA integration in the human genome. PLoS Comput Biol. 2006, 2: e157-10.1371/journal.pcbi.0020157.PubMed CentralView ArticlePubMedGoogle Scholar
- Hammond SM, Crable SC, Anderson KP: Negative regulatory elements are present in the human LMO2 oncogene and may contribute to its expression in leukemia. Leuk Res. 2005, 29: 89-97. 10.1016/j.leukres.2004.05.013.View ArticlePubMedGoogle Scholar
- Deichmann A, Hacein-Bey-Abina S, Schmidt M, Garrigue A, Brugman MH, Hu J, Glimm H, Gyapay G, Prum B, Fraser CC, et al: Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy. J Clin Invest. 2007, 117: 2225-2232. 10.1172/JCI31659.PubMed CentralView ArticlePubMedGoogle Scholar
- Bushman F, Lewinski M, Ciuffi A, Barr S, Leipzig J, Hannenhalli S, Hoffmann C: Genome-wide analysis of retroviral DNA integration. Nat Rev Microbiol. 2005, 3: 848-858. 10.1038/nrmicro1263.View ArticlePubMedGoogle Scholar
- Dave UP, Akagi K, Tripathi R, Cleveland SM, Thompson MA, Yi M, Stephens R, Downing JR, Jenkins NA, Copeland NG: Murine leukemias with retroviral insertions at Lmo2 are predictive of the leukemias induced in SCID-X1 patients following retroviral gene therapy. PLoS Genet. 2009, 5: e1000491-10.1371/journal.pgen.1000491.PubMed CentralView ArticlePubMedGoogle Scholar
- Dave UP, Jenkins NA, Copeland NG: Gene therapy insertional mutagenesis insights. Science. 2004, 303: 333-10.1126/science.1091667.View ArticlePubMedGoogle Scholar
- Aiuti A, Cassani B, Andolfi G, Mirolo M, Biasco L, Recchia A, Urbinati F, Valacca C, Scaramuzza S, Aker M, et al: Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy. J Clin Invest. 2007, 117: 2233-2240. 10.1172/JCI31666.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.