Multipotent transduced progenitors capable of self-renewal contribute to hematopoiesis. (A) LAM-PCR analysis of LTC-IC–derived colonies. From 0.01 to 1 ng of DNA directly isolated from LTC-IC–derived colonies was analyzed, sequenced, and aligned to the human genome. Numbers denote months after reinfusion; CD3, T cells; LTC-IC, long-term culture-initiating cell–derived colonies; -C, water control. (B) Follow-up tracking of LTC-IC–derived myeloid insertion sites in T cells. Genomic flanking primers were designed for 6 individual LTC-IC–derived retroviral integration sites (Table 1). Three LTC-IC–derived insertion sites (P2: clones 1529 and 1770; P4: clone 7324) could be identified in highly purified T cells (1 to 200 ng) over time at time points of up to 8 months prior to the bone marrow aspiration from which the LTC-IC assays were performed, demonstrating that these clones generated mature lymphocytes a long time before generating LTC-ICs and indicating that human CD34⁺ cells with self-renewal capacity have been transduced and have long-term activity in this clinical study. Note that the additional bands in size below 100 bp detected in PCR tracking specific for clone 7324 have been sequenced and correspond to primer multimers. Arrows with numbers denote position and size of specific PCR amplicons in base pairs. Months indicates months after gene therapy; *, sequenced; °, after chemotherapy; °°, after allotransplantation; CD3, T cells; LTC-IC, long-term culture-initiating cells; -C, 1.0 μg nontransduced human leukocyte DNA was used as a negative control; first lane, 100-bp ladder.