Figure 3. Comparative analysis of vector integration and gene expression.

(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.