Polyclonality of vector inserts in blood leukocyte lineages of P2 at 12 and 24 months after gene therapy. Retroviral vector insertions are assessed by the LAM-PCR DNA amplification method²² ; amplified bands of multiple sizes are then separated by gel electrophoresis, where the various bands correspond to different retroviral-genomic DNA junctions. Shown in this figure are 3 electrophoresis gels cropped to show the relevant adjacent lanes. The different gels are separated by a vertical white line. Gels A and B were run at the same time and amplifications of DNA from the various blood-cell lineages separated from a single blood sample from subject P2 were obtained at 12 months after gene therapy. Shown for each lineage are 2 to 3 independent amplifications for DNA from T (CD3⁺ lymphocytes), M (CD14⁺ monocytes), N (CD15⁺ neutrophils), B (CD19⁺ lymphocytes), and NK (CD56⁺CD3⁻) cells. Note that the unlabeled lane at the far right of gel B contained a sample of the same amplification of T as run in the left-most T lane in gel A to assure that gels A and B had run similarly. Also included in gel B are lanes containing the amplification of control (before gene therapy) blood cell DNA from P2 (C) and a water control (W). All gels contain a lan with molecular weight markers (MW), with the molecular weight indicated in base pairs (bp) at the left margin of gel A. The recurrent bright band at 227 bp (arrow at left) is derived from internal retroviral sequence. Polyclonality is demonstrated by bands of many sizes in T, B, and NK cells and neutrophils at 12 months after gene therapy. Gel C was run at a later time than gels A and B and contains amplifications of DNA from the various blood-cell lineages separated from a single blood sample from subject P2 obtained at 24 months after gene therapy. Shown in gel C are 2 independent LAM-PCR amplifications for the T and M lineages but only 1 amplification for N and NK lineages. Polyclonality of vector inserts is still observed at the 24-month time point.