Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo
Kerstin Schwarzwaelder, … , Adrian J. Thrasher, Christof von Kalle
Kerstin Schwarzwaelder, … , Adrian J. Thrasher, Christof von Kalle
Published August 1, 2007
Citation Information: J Clin Invest. 2007;117(8):2241-2249. https://doi.org/10.1172/JCI31661.
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Research Article

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo

Abstract

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3+ T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34+ progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34+ progenitors during transduction. In contrast to those in CD34+ cells, RISs recovered from engrafted CD3+ T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.

Authors

Kerstin Schwarzwaelder, Steven J. Howe, Manfred Schmidt, Martijn H. Brugman, Annette Deichmann, Hanno Glimm, Sonja Schmidt, Claudia Prinz, Manuela Wissler, Douglas J.S. King, Fang Zhang, Kathryn L. Parsley, Kimberly C. Gilmour, Joanna Sinclair, Jinhua Bayford, Rachel Peraj, Karin Pike-Overzet, Frank J.T. Staal, Dick de Ridder, Christine Kinnon, Ulrich Abel, Gerard Wagemaker, H. Bobby Gaspar, Adrian J. Thrasher, Christof von Kalle

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Figure 3

Comparative analysis of vector integration and gene expression.

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Comparative analysis of vector integration and gene expression. (A and B...
(A and B) MvA plots for all probesets and probesets closest to RISs in Pt1 and healthy donor. (A) RNA expression determined by Affymetrix U133A microarray of CD3/CD28-stimulated CD4+ cells of Pt1. All 22,283 probesets on the array are shown in blue. Of these, 3,173 were significantly different in Pt1 versus control (P < 0.05, adjusted Sidak step-up; red), corresponding to 1,549 upregulated and 1,624 downregulated genes. 96 probesets (65 upregulated and 16 downregulated) genes exceeded log2 fold change of 2. None of these were associated with RISs. (B) MvA plots for 200 probesets (blue) describing 134 genes closest to RISs in Pt1. Expression in 48 probesets was significantly different (red), corresponding to 17 upregulated and 19 downregulated genes. Most differences were marginal; only 5 of these probesets — describing FLJ10986 and SPTLC2 (upregulated), and ITGAL, PDCD4, and DPH5 (downregulated) — had log2 fold changes between 1.5 and 2. (C) Comparative analysis of gene expression in CD34+ cells stimulated under transduction conditions and RISs retrieved from engrafted CD3+ cells in 5 patients and (D) comparison of gene expression in engrafted CD4+ T cells and RISs retrieved from corresponding CD3+ population of Pt1. There was a significant correlation between gene expression and the number of integration events, as expected, although less pronounced. All genes on the array were organized into 10 bins according to expression levels, and the number of integrations was calculated for each category. The number of genes in each expression level category assuming uniform random distribution is shown by horizontal line.