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Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus.

Yamada K, Tsukahara T, Yoshino K, Kojima K, Agawa H, Yamashita Y, Amano Y, Hatta M, Matsuzaki Y, Kurotori N, Wakui K, Fukushima Y, Osada R, Shiozawa T, Sakashita K, Koike K, Kumaki S, Tanaka N, Takeshita T - Retrovirology (2009)

Bottom Line: 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.Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan. koichiro@shinshu-u.ac.jp

ABSTRACT
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 x 10(4) 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.

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MLV vector integrations into the TNIK and LMO2 gene loci. (A) Schematic representation of MLV vector integration into a gene locus. MLV vector integrations were detected using nested PCR with a combination of 3' or 5' LTR-specific primers (3' L1L2 or 5' L1L2) and gene-specific primers (F or R). (B) MLV integration sites in the integration hotspot of the TNIK gene locus. Upper: Diagrammatic representation of the TNIK gene locus. Exons and the transcription start site are shown as Ex and +1, respectively. Lower: MLV vectors integrated into an approximately 2-kb region within the TNIK hotspot were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and TNIK-specific primers (F1F2 or R1R2), as described in (A). The numbers indicate the nucleotide distance from the TNIK-specific primers (F1F2 or R1R2). The PCR products were sequenced, and the locations of the integration sites were determined by use of the human BLAST program. The integration site within the 705-9 cell hotspot was identified in our previous study [11] (large black arrowhead). (C) MLV integration sites near exon 1 of the LMO2 gene in TPA-Mat cells. Upper: Diagrammatic representation of the LMO2 gene locus. Lower: MLV vectors integrated into an approximately. ± 3-kb region from the transcription start site of LMO2 were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and LMO2-specific primers (F3F4, F5F6, F7F8, R3R4, R5R6 or R7R8), as described in (B). The numbers indicate the nucleotide distance from the transcription start site. Pt4 and Pt5 indicate the therapeutic MLV vector integration sites in patients 4 and 5, respectively, who developed leukemia after the French X-SCID gene therapy trials. (D) MLV integration sites near exon 1 of the LMO2 gene in Jurkat-ecoR cells. (E) MLV integration sites near exon 1 of the LMO2 gene in HeLa cells. (F) MLV integration sites near exon 1 of the LMO2 gene in human CD34+ cells. Black and white arrowheads respectively denote the reverse and forward orientation, relative to transcription, of the integrated MLV vectors.
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Figure 1: MLV vector integrations into the TNIK and LMO2 gene loci. (A) Schematic representation of MLV vector integration into a gene locus. MLV vector integrations were detected using nested PCR with a combination of 3' or 5' LTR-specific primers (3' L1L2 or 5' L1L2) and gene-specific primers (F or R). (B) MLV integration sites in the integration hotspot of the TNIK gene locus. Upper: Diagrammatic representation of the TNIK gene locus. Exons and the transcription start site are shown as Ex and +1, respectively. Lower: MLV vectors integrated into an approximately 2-kb region within the TNIK hotspot were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and TNIK-specific primers (F1F2 or R1R2), as described in (A). The numbers indicate the nucleotide distance from the TNIK-specific primers (F1F2 or R1R2). The PCR products were sequenced, and the locations of the integration sites were determined by use of the human BLAST program. The integration site within the 705-9 cell hotspot was identified in our previous study [11] (large black arrowhead). (C) MLV integration sites near exon 1 of the LMO2 gene in TPA-Mat cells. Upper: Diagrammatic representation of the LMO2 gene locus. Lower: MLV vectors integrated into an approximately. ± 3-kb region from the transcription start site of LMO2 were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and LMO2-specific primers (F3F4, F5F6, F7F8, R3R4, R5R6 or R7R8), as described in (B). The numbers indicate the nucleotide distance from the transcription start site. Pt4 and Pt5 indicate the therapeutic MLV vector integration sites in patients 4 and 5, respectively, who developed leukemia after the French X-SCID gene therapy trials. (D) MLV integration sites near exon 1 of the LMO2 gene in Jurkat-ecoR cells. (E) MLV integration sites near exon 1 of the LMO2 gene in HeLa cells. (F) MLV integration sites near exon 1 of the LMO2 gene in human CD34+ cells. Black and white arrowheads respectively denote the reverse and forward orientation, relative to transcription, of the integrated MLV vectors.

Mentions: To estimate the integration frequency in a human T cell line, TPA-Mat-ecoR cells expressing the ecotropic mouse receptor were infected with an ecotropic MLV vector that encoded green fluorescent protein (GFP); the infection efficiency (29 - 46%, based on GFP fluorescence) was similar to that measured in patients in the gene therapy trials [12]. At 48 hours post-infection, genomic DNA was isolated from the cells. This acute infection system is suitable for analyzing the distribution of initial vector integration. The combinations of LTR- and TNIK- or LMO2-specific primers (Additional file 1) used for the PCR reactions are shown in Figure 1A. All resulting PCR products from 186 PCR amplifications (1 μg DNA was used for each PCR amplification, and 186 μg of sample DNA correspond to approximately 2.8 × 107 cells) carried out using LTR- and TNIK-specific primers were cloned and sequenced, and 55 integration sites were mapped within the human genome (Figure 1B and Additional files 2 and 3). For 1613 PCR amplifications (1613 μg of sample DNA corresponds to approximately 2.4 × 108 cells) using LTR- and LMO2-specific primers, 65 integration sites were unevenly distributed in an approximately ± 3-kb region surrounding exon 1 of LMO2 in the human genome (Figure 1C and Additional files 2 and 3). We found a high incidence region (HIR) of vector integration in the region upstream of -1740, near the LMO2 promoter (Figure 1C). A HIR was also observed around the 705-9 cell integration site of the TNIK locus. To confirm the HIR location in the LMO2 locus, we performed additional PCR assays in the region upstream of -3000. Since no integration into the region upstream of -3001 was detected with the indicated primers (Figure 1C and Additional files 2 and 3), we suggest that the HIR in the TPA-Mat-ecoR cells ranges from -1740 (the downstream edge) to -3001 (the upstream edge). We observed multiple-hit integrations that included two or three vector integrations at the same nucleotide position within this HIR of LMO2, as described below. In contrast, no integration was detected downstream (1 ~1500) of exon 1 (0/270) and only a few integrations were found from 1500 to 3000 (3/270). Subsequent analysis using the same primer sets in a second T cell line, Jurkat-ecoR (infection efficiency: 28-36%, based on GFP fluorescence) identified a HIR (-1801 to -2968) in a similar region as the TPA-Mat-ecoR cells. No other integration sites near the LMO2 promoter were detected in Jurkat-ecoR cells (Figure 1D and Additional files 2 and 3).


Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus.

Yamada K, Tsukahara T, Yoshino K, Kojima K, Agawa H, Yamashita Y, Amano Y, Hatta M, Matsuzaki Y, Kurotori N, Wakui K, Fukushima Y, Osada R, Shiozawa T, Sakashita K, Koike K, Kumaki S, Tanaka N, Takeshita T - Retrovirology (2009)

MLV vector integrations into the TNIK and LMO2 gene loci. (A) Schematic representation of MLV vector integration into a gene locus. MLV vector integrations were detected using nested PCR with a combination of 3' or 5' LTR-specific primers (3' L1L2 or 5' L1L2) and gene-specific primers (F or R). (B) MLV integration sites in the integration hotspot of the TNIK gene locus. Upper: Diagrammatic representation of the TNIK gene locus. Exons and the transcription start site are shown as Ex and +1, respectively. Lower: MLV vectors integrated into an approximately 2-kb region within the TNIK hotspot were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and TNIK-specific primers (F1F2 or R1R2), as described in (A). The numbers indicate the nucleotide distance from the TNIK-specific primers (F1F2 or R1R2). The PCR products were sequenced, and the locations of the integration sites were determined by use of the human BLAST program. The integration site within the 705-9 cell hotspot was identified in our previous study [11] (large black arrowhead). (C) MLV integration sites near exon 1 of the LMO2 gene in TPA-Mat cells. Upper: Diagrammatic representation of the LMO2 gene locus. Lower: MLV vectors integrated into an approximately. ± 3-kb region from the transcription start site of LMO2 were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and LMO2-specific primers (F3F4, F5F6, F7F8, R3R4, R5R6 or R7R8), as described in (B). The numbers indicate the nucleotide distance from the transcription start site. Pt4 and Pt5 indicate the therapeutic MLV vector integration sites in patients 4 and 5, respectively, who developed leukemia after the French X-SCID gene therapy trials. (D) MLV integration sites near exon 1 of the LMO2 gene in Jurkat-ecoR cells. (E) MLV integration sites near exon 1 of the LMO2 gene in HeLa cells. (F) MLV integration sites near exon 1 of the LMO2 gene in human CD34+ cells. Black and white arrowheads respectively denote the reverse and forward orientation, relative to transcription, of the integrated MLV vectors.
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: MLV vector integrations into the TNIK and LMO2 gene loci. (A) Schematic representation of MLV vector integration into a gene locus. MLV vector integrations were detected using nested PCR with a combination of 3' or 5' LTR-specific primers (3' L1L2 or 5' L1L2) and gene-specific primers (F or R). (B) MLV integration sites in the integration hotspot of the TNIK gene locus. Upper: Diagrammatic representation of the TNIK gene locus. Exons and the transcription start site are shown as Ex and +1, respectively. Lower: MLV vectors integrated into an approximately 2-kb region within the TNIK hotspot were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and TNIK-specific primers (F1F2 or R1R2), as described in (A). The numbers indicate the nucleotide distance from the TNIK-specific primers (F1F2 or R1R2). The PCR products were sequenced, and the locations of the integration sites were determined by use of the human BLAST program. The integration site within the 705-9 cell hotspot was identified in our previous study [11] (large black arrowhead). (C) MLV integration sites near exon 1 of the LMO2 gene in TPA-Mat cells. Upper: Diagrammatic representation of the LMO2 gene locus. Lower: MLV vectors integrated into an approximately. ± 3-kb region from the transcription start site of LMO2 were detected by PCR with combinations of MLV vector-specific primers (3' L1L2 or 5' L1L2) and LMO2-specific primers (F3F4, F5F6, F7F8, R3R4, R5R6 or R7R8), as described in (B). The numbers indicate the nucleotide distance from the transcription start site. Pt4 and Pt5 indicate the therapeutic MLV vector integration sites in patients 4 and 5, respectively, who developed leukemia after the French X-SCID gene therapy trials. (D) MLV integration sites near exon 1 of the LMO2 gene in Jurkat-ecoR cells. (E) MLV integration sites near exon 1 of the LMO2 gene in HeLa cells. (F) MLV integration sites near exon 1 of the LMO2 gene in human CD34+ cells. Black and white arrowheads respectively denote the reverse and forward orientation, relative to transcription, of the integrated MLV vectors.
Mentions: To estimate the integration frequency in a human T cell line, TPA-Mat-ecoR cells expressing the ecotropic mouse receptor were infected with an ecotropic MLV vector that encoded green fluorescent protein (GFP); the infection efficiency (29 - 46%, based on GFP fluorescence) was similar to that measured in patients in the gene therapy trials [12]. At 48 hours post-infection, genomic DNA was isolated from the cells. This acute infection system is suitable for analyzing the distribution of initial vector integration. The combinations of LTR- and TNIK- or LMO2-specific primers (Additional file 1) used for the PCR reactions are shown in Figure 1A. All resulting PCR products from 186 PCR amplifications (1 μg DNA was used for each PCR amplification, and 186 μg of sample DNA correspond to approximately 2.8 × 107 cells) carried out using LTR- and TNIK-specific primers were cloned and sequenced, and 55 integration sites were mapped within the human genome (Figure 1B and Additional files 2 and 3). For 1613 PCR amplifications (1613 μg of sample DNA corresponds to approximately 2.4 × 108 cells) using LTR- and LMO2-specific primers, 65 integration sites were unevenly distributed in an approximately ± 3-kb region surrounding exon 1 of LMO2 in the human genome (Figure 1C and Additional files 2 and 3). We found a high incidence region (HIR) of vector integration in the region upstream of -1740, near the LMO2 promoter (Figure 1C). A HIR was also observed around the 705-9 cell integration site of the TNIK locus. To confirm the HIR location in the LMO2 locus, we performed additional PCR assays in the region upstream of -3000. Since no integration into the region upstream of -3001 was detected with the indicated primers (Figure 1C and Additional files 2 and 3), we suggest that the HIR in the TPA-Mat-ecoR cells ranges from -1740 (the downstream edge) to -3001 (the upstream edge). We observed multiple-hit integrations that included two or three vector integrations at the same nucleotide position within this HIR of LMO2, as described below. In contrast, no integration was detected downstream (1 ~1500) of exon 1 (0/270) and only a few integrations were found from 1500 to 3000 (3/270). Subsequent analysis using the same primer sets in a second T cell line, Jurkat-ecoR (infection efficiency: 28-36%, based on GFP fluorescence) identified a HIR (-1801 to -2968) in a similar region as the TPA-Mat-ecoR cells. No other integration sites near the LMO2 promoter were detected in Jurkat-ecoR cells (Figure 1D and Additional files 2 and 3).

Bottom Line: 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.Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan. koichiro@shinshu-u.ac.jp

ABSTRACT
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 x 10(4) 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.

Show MeSH
Related in: MedlinePlus