Figure 1.
Integrating HDAd5/35++ Vector
(A) Vector structure. In HDAd-γ-globin/mgtm, the 11.8-kb transposon is flanked by inverted transposon repeats (IRs) and FRT sites for integration through a hyperactive Sleeping Beauty transposase (SB100x) provided from an HDAd-SB vector.10 The γ-globin expression cassette contains a 4.3-kb version of the β-globin LCR consisting of four DNase hypersensitivity (HS) regions and the β-globin promoter.51 The full-length γ-globin cDNA, including that 3′ UTR (for mRNA stabilization in erythrocytes) was used. The mgmtP140K gene is under the control of the ubiquitously active EF1α promoter. The bidirectional SV40 poly-adenylation signal is used to terminate transcription. To avoid interference between the LCR/β-promoter and EF1α promoter, a 1.2-kb chicken HS4 chromatin insulator25 was inserted between the cassettes. The second vector (HDAd-SB) provides both Flpe recombinase and the SB100x transposase in trans. Both vectors are helper-dependent adenovirus (HDAd) vectors containing the CD46 affinity-enhanced Ad35++ fiber knob and Ad35 fiber shaft.16 Upon co-infection of both vectors, Flpe mediates the circularization of the transposon through FRT sites. SB100x then randomly integrates the transposon into the host genome through interaction with the IRs. (B) Schematic of the experiment. Bone marrow was harvested from hCD46tg mice and lineage-negative cells (Lin–) were isolated by magnetic-activated cell sorting (MACS). Lin− cells were transduced with HDAd vectors at a total MOI of 1,000 vp/cell at a ratio of 1:1. After 1 day in culture, 1 × 106 transduced cells/mouse were transplanted into lethally irradiated C57BL/6 mice. At week 4, O6-BG/BCNU treatment was started and repeated every 2 weeks for a total of 3 times. With each cycle, 30 mg/kg O6-BG was applied, whereas the BCNU dose was increased from 5 mg/kg to 7.5 mg/kg and 10 mg/kg. Animals were sacrificed at week 12, and bone marrow Lin− cells were transplanted into secondary recipients, which were then followed for 16 weeks.