Mouse il 3 mil 3

Lin⁻ mouse BM cells were isolated by flushing femurs, tibias, and iliac crests of 6- to 8-week-old CD45.2 C57BL/6 or CD45.2 Berkeley SCD mice (BERK-SCD, JAX stock #003342) followed by lineage depletion using the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Lin− cells were pre-stimulated at 1 × 10⁶ cells/mL in Stem Cell Growth Medium (CellGenix) supplemented with mouse SCF (100 ng/mL), hTPO (100 ng/mL), mouse IL-3 (mIL-3) (20 ng/mL), and hFlt3-L (100 ng/mL), all from Peprotech. Following a 36- 40-h pre-stimulation, cells were transduced at a density of 1 × 10⁶ cells/mL in the presence of LentiBOOST enhancer, and transduced cells (without sorting) were transplanted by retro-orbital injection into lethally irradiated (7 + 4 Gy, split dose) CD45.1 recipients (B6.SJL-Ptprca Pepcb/BoyJ, Jax Strain #002014) 24 h after transduction. PB samples were collected at weeks 4, 8, 12, and 16 to measure engraftment by flow cytometry (CD45.2/CD45.1), determine RBC indices, and quantitate sickled cells. At week 16, mice were euthanized, and BM cells were used to measure engraftment by flow cytometry (CD45.2/CD45.1), VCN, and mRNA expression, spleens were collected to weigh. All animal experiments were approved by the Boston Children's Hospital's Institutional Animal Care and Use Committee.