Multipotent transduced progenitors contribute to hematopoiesis. (A) Long-term γc expression on circulating CD15⁺ granulocytes. The proportions of transduced granulocytes are 0.19% for P2, 0.12% for P7, and 0.3% for P9 at 56, 33, and 26 months (M indicates months) after gene therapy, respectively. An isotype control ofγc is also shown (upper panel). (B) LAM-PCR analysis of CD14 and CD15 FACS-sorted myeloid cells. DNA samples directly isolated from sorted peripheral blood leukocytes (CD3: 2 to 20 ng; myeloid cells: 10 to 100 ng) were analyzed at different time points after treatment. Compared with the polyclonal pattern of insertion sites in the CD3 samples, oligoclonal insertion was observed in the myeloid cell populations. Individual myeloid LAM-PCR amplicons were sequenced to obtain the insertion site fusion sequences. The genomic component of all fusion sequences was aligned to the human genome (Table 1). Numbers denote months after reinfusion; CD3, T cells; CD14, monocytes; CD15, granulocytes; -C, water control. (C) Follow-up tracking of CD14- and CD15-derived myeloid insertion sites in T cells. The sequence information of 7 individual retroviral integration sites was used to design genomic flanking primers that were unique for each individual site. Follow-up tracking was performed on 1 to 200 ng of purified T cells at different time points after genetic correction and reinfusion. In highly purified T cells, 6 of the 7 myeloid insertion sites (P1: clones 5521, 5522, 5523, 3682, 3686; P4: clone 7318) could be retrieved at different time points, thus demonstrating that multipotent progenitor cells were genetically corrected and engrafted in this clinical trial. Note that the 2 additional tracking PCR amplicons found with tracking primers for clone 5522 (P1; fourth panel from top) 24 and 38 months after gene transfer have been sequenced and correspond to individual unique integration sites different from clone 5522, detected due to partial genome homology of the flanking primers used. Arrows with numbers denote position and size of specific PCR amplicons in base pairs. White asterisks on gels (C) indicate sequenced; (Pat. 4) 37° indicates after chemotherapy; and 41°° indicates after allotransplantation; months, time after gene therapy; CD3, T cells; CD19, B cells; PB, peripheral blood mononuclear cells; CD14, granulomonocytic cells; CD15, granulocytes; -C, 1.0 μg nontransduced human leukocyte DNA was used as a negative control; first lane, 100-bp ladder.