Gates Package — page 828

Our current attempts are focused to further improve the safety and efficacy of the approach, including new HDAd vector platforms (53), alternative mobilization regimens (54), and in vivo selection strategies. The latter includes an in vivo selection strategy that would not involve low-dose BCNU (55). We believe that our study is an additional step towards the vision to treat hemoglobinopathies by a simple intravenous injection of a non-integrating vector. Methods The following information can be found under Supplementary Methods: Reagents for in vivo transduction and selection, generation of HDAd vectors, in vitro erythroid differentiation of CD34+ cells with O6BG/BCNU selection, blood analyses, colony-forming unit (CFU) assay, measurement of base conversion by Sanger sequencing and Next-Generation Sequencing (NGS), flow cytometry, globin HPLC, analysis of Reactive Oxygen Species (ROS) levels, cytospin slide preparation, HbF immunofluorescence, real-time reverse transcription PCR, measurement of vector copy number, off-target analyses, RNA-seq analysis. CD34+ cell culture: CD34+ cells from G-CSF-mobilized healthy adult donors were provided by the Fred Hutch Cell Processing Facility. CD34+ cells from thalassemic and SCD patients were previously collected during mobilization clinical trials conducted at G. Papanikolaou Hospital, Thessaloniki, Greece (56). CD34+ cells were recovered from frozen stocks and incubated overnight in StemSpan H3000 medium (STEMCELL Technologies, Vancouver, Canada) supplemented with penicillin/streptomycin, Flt3 ligand (Flt3L, 25 ng/ml), interleukin 3 (10 ng/ml), thrombopoietin (TPO) (2 ng/ml), and stem cell factor (SCF) (25 ng/ml). Cytokines and growth factors were from Peprotech (Rocky Hill, NJ). CD34+ cells were transduced with HDAd5/35++ vectors in low- attachment 12-well plates. Animal studies: C57BL/6-based transgenic mice that contained the human CD46 genomic locus and provide CD46 expression at a level and in a pattern similar to humans (human CD46tg mice) were described earlier (57). Transgenic mice carrying the wild-type 248-kb -globin locus yeast artificial chromosome (-YAC) were used (22). -YAC mice were crossed with human CD46tg mice to obtain CD46/-YAC mice for ex vivo and in vivo studies. The following primers were used for genotyping of mice: CD46 forward, 5'-GCCAGTTCATCTTTTGACTCTATTAA-3', and reverse, 5'-AATCACAGCAATGACCCAAA-3'; -