Rhscf

Frozen human mobilized peripheral blood (mPB) CD34⁺ cells were purchased from AllCells. Upon receipt, mPB CD34⁺ cells were thawed and counted prior to assessment of viability via trypan blue dye exclusion. For transduction, mPB CD34⁺ cells were re-plated at a cell density of 1 × 10⁶ cells/mL in RN-coated plates in stem cell growth media (SCGM) containing 300 ng/mL recombinant human Flt-3 Ligand (rhFlt-3L), 300 ng/mL recombinant human stem cell factor (rhSCF), 100 ng/mL recombinant human thrombopoietin (rhTPO), and 60 ng/mL recombinant human IL-3 (rhIL-3) and stimulated overnight at 37°C/5% CO₂. SCGM, rhFlt-3L, rhSCF, rhTPO, and rhIL-3 were all purchased from CellGenix.

For engineering runs, large-scale vector preparations were produced by transient transfection of HEK293T cells with LVV construct and packaging plasmids, followed by microfiltration, ion exchange chromatography, concentration, and diafiltration by tangential flow filtration and fill-finish. All vector preparations were titrated on HOS cells. Four large-scale validations were performed at Connell and O’Reilly Families Cell Manipulation Core Facility at Dana-Farber Cancer Institute (Boston, USA). Plerixafor mobilized CD34⁺ cells⁴⁴ (link) enriched by CliniMACS device (Miltenyi Biotec, Auburn, CA, USA) were pre-stimulated in CellGenix SCGM media supplemented with recombinant human (rh)TPO, rhSCF, and rhFLT3-L (all 100 ng/mL from CellGenix, Portsmouth, NH, USA) at a density of 1 × 10⁶ cells/mL for 44 ± 4 h at 37°C and 5% CO₂. Cells were then transduced at MOIs indicated in Table 1 at 5 × 10⁶ cells/mL with BCH-BB694 LVV for 22 ± 4 h as previously described.82 (link), 83 (link), 84 The final product was cryopreserved in Cryostor5 (BioLife Solutions, Bothell, WA, USA) prior to downstream analyses.