Figure 4. Insertion Sites Clustered in Specific Gene Regions.

The results of screening for the presence and clonal contribution of highly prominent common-insertion-site (CIS) clones are shown for Patient 1 (Panel A) and Patient 2 (Panel B). At every time point analyzed, sequence counts for all retroviral insertion sites (RISs) contributing to an individual CIS derived from bone marrow cells and primary blood leukocytes are clustered and relate to the total sequence count at the respective time point. To estimate the overall contribution, the relative sequence counts in all CIS clones are related to sequence counts of non–CIS-related genes. (An overview of CIS clones that were detected at more than one time point is provided in Table 5 in the Supplementary Appendix.) CISs of second order are defined as integration sites detected in a window of 30 kb, those of third order in a window of 50 kb, and those of fourth order in a window of 100 kb. CIS clones of fifth and higher orders were further defined in a window of 200 kb. The sequence count is given as a proportion of total sequence counts in CIS genes, as compared with the total overall sequence count, showing the number of days after gene therapy (GT). The relative clonal contribution of vector-targeted gene loci that have been shown to induce malignant clonal expansion in gene-therapy studies involving patients with chronic granulomatous disease (CGD) and severe combined immunodeficiency with γc-chain defects (γc-SCID) is shown for Patient 1 (Panel C) and Patient 2 (Panel D). In both patients, the presence of LMO2, CCND2, and BMI1 (which have been shown to trigger malignant transformation of CD3+ T cells in patients with γc-SCID) was almost exclusively restricted to CD3+ T cells. Accordingly, the presence of MDS1/EVI1, PRDM16, and SETBP1 (which have been shown to trigger myeloid clonal expansion in patients with CGD) was largely found in granulocytes (G). Numbers in parentheses indicate the number of clones containing an insertion site within or close to the respective RefSeq gene. The label “Other” indicates the number of all less frequently encountered genetic locations that carry insertion sites in the respective sample analyzed. The MDS1 clone carries an integration site at the MDS1 locus at position 169071575 bp on chromosome 3, which substantially contributes to myeloid regeneration.