Figure 2. Efficient selection of methylguanine methyltransferase (MGMT) P140K expressing hematopoietic stem cells leads to high-level and sustained hFIX expression in primary and secondary mice.

hFIX plasma levels monitored over 27 weeks in (a) Busulfan treatment group (n = 4) and (b) irradiation control group (n = 5). Three BG/BCNU selections (30 and 5 mg/kg, respectively) were performed in Busulfan-conditioned mice only (a, c, d) (indicated by black arrows). The 5 and 10% levels in a and b corresponding to human physiological levels of plasma FIX are indicated. These levels are considered therapeutic and curative, respectively, in human therapy. (c) Peripheral blood vector copy number (VCN) was determined by Taqman quantitative PCR analysis and was monitored over the same period. (d) hFIX expression in Busulfan-conditioned mice normalized by peripheral blood VCN. (e) The hFIX production and corresponding proportion of Ter119/hFIX double positive cells in total Ter119 positive cells analyzed by flow cytometry in three long-term chimeras (S1.1, S1.2, T2.2). (f) High levels of hFIX expression was sustained in secondary chimeras over 30 weeks (n = 4). Statistical analyses were done for a-d, and f. The probability of a statistically significant difference between the mean values of two data sets was determined by unpaired t-test with Welch's correction using GraphPad Prism version 4.0 (GraphPad software). In a and c, data sets marked with asterisk symbol indicates that there was a statistical significance when compared the present data set with the data set from the previous time point. No statistical significance in data sets between each time point was found in b, d and f. BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; BG, O⁶-benzylguanine.