Expansion of cell clones with integrated vectors in the HMGA2 third intron. (A) Map of integration sites detected in the HMGA2 locus, pooled over all the SCID-X1 patients. The green and red lines indicate the positions of vector integration sites. Forward indicates that the vector is oriented 5′ to 3′ relative to the chromosomal numbering system. Reverse indicates reverse orientation. (B) Longitudinal expansion of cell clones harboring integration events in HMGA2 in patient no.1 and patient no. 7. The x-axis shows the time after cell infusion, the y-axis shows the reconstructed percentage of all transduced cells contributed by cells harboring the HMGA2 integration site. Note the difference in the y-axis scale compared with Figure 3. (C) Structure of the major chimeric HMGA2-vector message. The major message (splice acceptor site at bp 1992 of the vector) was found in both patients no. 1 and no. 7. An alternative splice acceptor site at bp 2002 was found in patient no. 7. (D) Amplification strategy for determining the chimeric HMGA2-vector message structure using reverse-transcription PCR. The time points were 75 months (patient no. 1) and 56 months (patient no. 7). The bands marked “major” and “minor” HMGA2-vector message formed on the ethidium-stained gel were excised and subjected to Sanger DNA sequencing. Sequence analysis established that slower mobility bands corresponded exclusively to the chimeric HMGA2-vector forms. The mobility of the forms marked normal messages matched bands seen after amplification of control samples (data not shown). (E) Deduced structure of a minor form of the chimeric HMGA2-vector message found in lesser abundance in patient no. 1 only.