Retroviral-mediated expression of recombinant Fancc enhances the repopulating ability of Fancc −/− hematopoietic stem cells and decreases the risk of clonal evolution by Laura S. Haneline, Xiaxin Li, Samantha L. M. Ciccone, Ping Hong, Yanzhu Yang, Hal E. Broxmeyer, Suk-Hee Lee, Attilio Orazi, Edward F. Srour, and D. Wade Clapp Blood Volume 101(4):1299-1307 February 15, 2003 ©2003 by American Society of Hematology

Fancc expression and mitomycin C sensitivity of BM cells from long-term reconstituted mice.(A) Fancc Western. Fancc expression and mitomycin C sensitivity of BM cells from long-term reconstituted mice.(A) Fancc Western. Whole cell lysates were extracted from the BM of long-term reconstituted mice transplanted withFancc−/− cells transduced with either MSCV-Fancc (lane 2) or MSCV-EGFP (lane 3).Fancc−/− murine embryonic fibroblasts transduced with MSCV-Fancc were used as a positive (+) control (lane 1). One of 4 representative blots is shown. (B) MMC progenitor assay. Six months following transplantation, BM cells were harvested from mice transplanted with either Fancc−/− cells transduced with MSCV-Fancc (▪), Fancc−/− cells transduced with a reporter gene only (●), or wild-type cells transduced with a reporter gene only (▴). BM cells were cultured in methylcellulose progenitor assays at 5 × 104 cells/mL with increasing concentrations of MMC. Each condition was plated in triplicate and scored on day 7 of culture. The percent maximal colony formation was determined by dividing the number of colonies scored at each MMC concentration by untreated control cultures. Error bars represent standard error of the means (SEM), Student t test *P ≤ .001. Laura S. Haneline et al. Blood 2003;101:1299-1307 ©2003 by American Society of Hematology

Schematic of gene therapy competitive repopulation experiments Schematic of gene therapy competitive repopulation experiments.Donor BM cells from Fancc−/− mice were transduced with either a retrovirus containing a reporter gene (MSCV-EGFP or MSCVpac) or a retrovirus encoding rFancc (MSCV-Fancc or MFG-FAC). Schematic of gene therapy competitive repopulation experiments.Donor BM cells from Fancc−/− mice were transduced with either a retrovirus containing a reporter gene (MSCV-EGFP or MSCVpac) or a retrovirus encoding rFancc (MSCV-Fancc or MFG-FAC). WT donor cells were transduced with a retrovirus containing a reporter gene only (MSCV-EGFP or MSCVpac). Each respective donor cell population was cotransplanted into 5-8 lethally irradiated recipient mice with competitor cells from B6.BoyJ mice that were genetically identical to C57Bl/6J mice with the exception of distinct CD45 isoantigen expression. Laura S. Haneline et al. Blood 2003;101:1299-1307 ©2003 by American Society of Hematology

Donor chimerism of individual mice transplanted with transduced test cells.Peripheral blood donor chimerism was determined by fluorescence cytometry 6 months following transplantation. Donor chimerism of individual mice transplanted with transduced test cells.Peripheral blood donor chimerism was determined by fluorescence cytometry 6 months following transplantation. Individual data points represent donor chimerism of single mice transplanted with eitherFancc−/− cells transduced with a reporter gene only (−/− control), Fancc−/− cells transduced with a retrovirus encoding rFancc (−/− rFancc), or WT cells transduced with a reporter only (+/+ control). All data from these 3 transduction groups in experiments 1-3 are shown. Mean donor chimerism of mice transplanted with −/− control cells was significantly lower than +/+ control (*P < .002). Transduction of Fancc−/− cells with rFancc (−/− rFancc) resulted in a significant increase in repopulating ability compared to −/− control test cells (**P ≤ .04). The lower bracket outlines expected chimerism of mice transplanted with −/− control cells, and the upper bracket outlines mice (mice 1-5, 7, and 8) with a marked increase in donor chimerism. Mouse 6 in the −/− rFancc transduction group exhibited low chimerism at this timepoint but had a marked increase in chimerism 16 months after transplant. A Fisher exact test confirmed that the incidence of aberrant chimerism in the −/− control (7 of 22) transduction group was significantly greater than in the −/− rFancc (1 of 22) transduction group (P < .05). Laura S. Haneline et al. Blood 2003;101:1299-1307 ©2003 by American Society of Hematology

Ex vivo culture of Fancc−/− cells compromises repopulating ability Ex vivo culture of Fancc−/− cells compromises repopulating ability.Peripheral blood donor chimerism was determined by fluorescence cytometry 3, 6, and 9 months following transplantation. Ex vivo culture of Fancc−/− cells compromises repopulating ability.Peripheral blood donor chimerism was determined by fluorescence cytometry 3, 6, and 9 months following transplantation. Individual data points represent donor chimerism of single mice transplanted with either WT or Fancc−/− donor cells that were freshly isolated or cultured for 4 days in hIL-6 and mSCF, identical to the culture conditions used to maintain donor cells in the gene transfer competitive repopulation experiments. Repopulating ability ofFancc−/− cells after ex vivo culture was significantly lower than the repopulating ability of freshly isolatedFancc−/− cells at all timepoints evaluated (Student t test *P < .0006). Donor chimerism of mice transplanted with ex vivo culturedFancc−/− cells increased significantly from 6 to 9 months after transplantation (Student t test **P < .001). Laura S. Haneline et al. Blood 2003;101:1299-1307 ©2003 by American Society of Hematology

Mice transplanted with either −/− control cells or a low frequency of −/− rFancc cells had progenitors that were resistant to multiple inhibitory cytokines.(A) Semiquantitative PCR. HEL cells with a single provirus copy were used as the standard DNA for dil... Mice transplanted with either −/− control cells or a low frequency of −/− rFancc cells had progenitors that were resistant to multiple inhibitory cytokines.(A) Semiquantitative PCR. HEL cells with a single provirus copy were used as the standard DNA for dilution, and untransduced HEL cells were used as the negative control. DNA extracted from the peripheral blood of mouse 6 was amplified with primers specific for proviral DNA. Approximately 1%-2% of peripheral blood cells from mouse 6 contained proviral DNA. (B) Inhibitory cytokine progenitor assay. Sorted CD45.2+ cells from mice 3, 5, and 6 were cultured in methylcellulose progenitor assays at 5 × 104 cells/mL with either IFN-γ (10 ng/mL), TNF-α (10 ng/mL), or MIP-1α (50 ng/mL). Each condition was plated in triplicate and scored on day 7 of culture. The percent maximal colony formation was determined by dividing the number of colonies scored with each inhibitory cytokine by untreated control cultures. Low-density cells fromFancc−/− and WT mice were used as controls. Baseline progenitor numbers scored in the untreated groups were 84 forFancc−/− , 82 forFancc+/+ , 195 for mouse 3, 106 for mouse 5, and 89 for mouse 6. Error bars represent SEM. Laura S. Haneline et al. Blood 2003;101:1299-1307 ©2003 by American Society of Hematology

Histologic evidence of BM hypoplasia and myelofibrosis Histologic evidence of BM hypoplasia and myelofibrosis.(A) H&E staining of the BM from a mouse transplanted withFancc+/+ cells (magnification × 100). Histologic evidence of BM hypoplasia and myelofibrosis.(A) H&E staining of the BM from a mouse transplanted withFancc+/+ cells (magnification × 100). (B) H&E staining of the spleen from a mouse transplanted withFancc+/+ cells (magnification × 100). (C) H&E staining of the BM from mouse 3 (magnification × 100). (D) Magnification × 200 revealed a hypoplastic marrow. (E) H&E staining of the spleen from mouse 3 (magnification × 200). (F) Magnification ×  400 revealed abnormal splenic architecture with an increased number of immature myeloid cells. (G) H&E staining of the BM from mouse 6 (magnification × 200). (H-I) Magnification ×  400 revealed a hypercellular BM with a marked degree of myeloid hyperplasia and significant myelofibrosis. (J) Reticulin staining of the BM from mouse 6 (magnification × 400) to identify collagen deposition, which confirms myelofibrosis. Laura S. Haneline et al. Blood 2003;101:1299-1307 ©2003 by American Society of Hematology

Donor chimerism of secondary recipients transplanted with BM cells from one of the 4 primary recipient experimental groups.Mean chimerism of primary recipients used as donor cells for secondary transplants are shown below each bar. Donor chimerism of secondary recipients transplanted with BM cells from one of the 4 primary recipient experimental groups.Mean chimerism of primary recipients used as donor cells for secondary transplants are shown below each bar. Secondary recipient donor chimerism was measured 8 months after transplantation. Error bars represent SEM. Laura S. Haneline et al. Blood 2003;101:1299-1307 ©2003 by American Society of Hematology