Cryopreserved human CD34+ HSC were thawed at 37°C, washed in phosphate-buffered saline (Life Technologies) and prestimulated in complete culture medium (Stemspan supplemented with stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt3-L), and thrombopoietic (TPO), each at 100ng/mL (Peprotech, NJ). For electroporation, 3–6 ×106 cells were spun at 100–300 × g for 10 minutes, followed by resuspension in 100 µL of MaxCyte electroporation buffer. The cell suspension was mixed with ZFN mRNA and pulsed with HSC-CL1 program (MaxCyte Systems, Gaithersburg, MD). Following electroporation, the cells were transduced with AAV6-donor (MOI 1–3 × 106 vector genomes/cell) and washed after 3 hours. Treated cells were kept in culture and either transplanted into NSG mice the following day (day 3 of culture), or maintained in vitro for analyses.
For transplant studies, following overnight recovery, treated cells (1–2 × 106 per mouse) were transplanted into 6- to 8-week old NSG mice (Jackson Laboratory) pre-conditioned with intraperitoneal busulfan (20mg/kg) 24 hours prior. The number of mice transplanted depended on amount of material available, particularly with patient CD34+ HSC. Following 6–8 weeks, the mice were euthanized for analysis.
For transplant studies, following overnight recovery, treated cells (1–2 × 106 per mouse) were transplanted into 6- to 8-week old NSG mice (Jackson Laboratory) pre-conditioned with intraperitoneal busulfan (20mg/kg) 24 hours prior. The number of mice transplanted depended on amount of material available, particularly with patient CD34+ HSC. Following 6–8 weeks, the mice were euthanized for analysis.