Ex vivo expansion of hematopoietic stem and progenitor cells from human mobilized peripheral blood for gene therapy applications.

Ex vivo expansion of mobilized peripheral blood (mPB) hematopoietic stem cells (HSCs) represents a promising approach to advance cell and gene therapy strategies yet is hampered by loss of stem cell function when applying commonly used culture protocols. We performed in-depth characterization of mPB expansion cultures by single-cell RNA sequencing, which highlighted differentiation trajectories with preservation of lineage fidelity in committed progenitors. Defining a putative HSC cluster allowed an estimation of transduction efficiency in ex vivo cultures, which correlated with long-term gene marking in xenografts and patients enrolled in a gene-therapy study. We then developed a clinically translatable, good manufacturing practice-compliant process to expand lentiviral (LV)-transduced HSCs from mPB of pediatric patients and adult donors by biologically informed protocol improvements of cytokine supplementation, media choice, timing of LV transduction, and combinations of small molecules preventing the activation of differentiation programs. Our optimized process outperforms validated state-of-the-art cord-blood expansion protocols when applied to mPB. LV integration site analysis and genomic barcode-based clonal tracking provided definitive proof for symmetric HSC self-renewal divisions occurring during ex vivo culture. These results warrant clinical testing of this HSC transduction/expansion process in an upcoming clinical gene-therapy trial for autosomal recessive osteopetrosis (EU CT 2024-518972-30).