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Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients
Steven J. Howe, … , H. Bobby Gaspar, Adrian J. Thrasher
Steven J. Howe, … , H. Bobby Gaspar, Adrian J. Thrasher
Published August 7, 2008
Citation Information: J Clin Invest. 2008;118(9):3143-3150. https://doi.org/10.1172/JCI35798.
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Research Article Article has an altmetric score of 55

Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients

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Abstract

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-β region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

Authors

Steven J. Howe, Marc R. Mansour, Kerstin Schwarzwaelder, Cynthia Bartholomae, Michael Hubank, Helena Kempski, Martijn H. Brugman, Karin Pike-Overzet, Stephen J. Chatters, Dick de Ridder, Kimberly C. Gilmour, Stuart Adams, Susannah I. Thornhill, Kathryn L. Parsley, Frank J.T. Staal, Rosemary E. Gale, David C. Linch, Jinhua Bayford, Lucie Brown, Michelle Quaye, Christine Kinnon, Philip Ancliff, David K. Webb, Manfred Schmidt, Christof von Kalle, H. Bobby Gaspar, Adrian J. Thrasher

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Figure 2

Gene expression levels and retroviral insertion site.

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FISH analysis reveals a chromosomal rearrangement.
Metaphase cells showi...
(A) Microarray analysis was used to compare levels of gene expression in the patient’s cells to those found in thymocytes or a panel of leukemias. DP1 and DP2 represent arrays performed on 2 different developmental stages of normal human CD4+CD8+ thymocytes. The y axis shows the fold change in expression, with values above 1 representing an increase and those below 1 a decrease. When compared with thymocytes and other leukemias, LMO2, NOTCH1, HES1, STIL, TAL1, and CMPK gene expression is upregulated. In contrast, expression levels of tumor suppressor genes p14(ARF1) and p16(INK4a) were reduced. γc mRNA was not overexpressed relative to thymocytes (consistent with surface expression data), although it was in comparison with the heterogeneous group of leukemias. (B) The vector insertion site is 35087 bp upstream of the LMO2 transcription start site in the opposite orientation (red X). LAM-PCR analysis (inset) of 10 ng and 1 ng of d717 PBMC DNA identified 1 dominant clone. M, 100-bp ladder; –C, 100 ng nontransduced DNA. When using limiting amounts of DNA, the internal control is out-competed by the LMO2 amplicon. The genomic locus and expression levels of genes surrounding LMO2 are shown in comparison with a dataset of arrays performed on other childhood leukemias or DP1 and DP2 human thymocytes. (C) Sequencing of denaturing HPLC analysis revealed a R1599P substitution in the HD-N domain of NOTCH1 in the leukemic cells (d717) but not before. No other NOTCH1 mutations were found.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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