Humanized Mouse Model Generation

Recombinant AAV9 (rAAV9) vectors encoding human IL-3, IL-15, GM-CSF, and HLA-A*0201 were constructed as previously described (Huang et al., 2014 (link)). Four-week-old NSG mice were transduced with rAAV9 encoding HLA-A*0201 by perithoracic injection and with rAAV9-encoding HLA-A*0201 and AAV9-encoding human IL-3, IL-15, and GM-CSF, by IV injection, as previously described (Huang et al., 2014 (link)). Two weeks later, mice were subjected to 150-Gy total body sub-lethal irradiation for myeloablation, and several hours later, each transduced, irradiated mouse was engrafted IV with 1 × 10⁵ HLA-A*0201⁺-matched, CD34⁺ human HSCs. CD34⁺ HSCs among lymphocytes derived from HLA-A*0201⁺ fetal liver samples were isolated using a Human CD34 Positive Selection Kit (STEMCELL Technologies, Vancouver, BC, United States) (Lepus et al., 2009 (link)). At 14 weeks post-HSC engraftment, the reconstitution status of human CD45⁺ cells in the blood of HIS-CD8 mice was determined by flow cytometry analysis, as previously described (Huang et al., 2014 (link)).