Introduction: Ex vivo gene therapy targeting blood cells is a promising approach for the treatment of hematological malignancies and inherited genetic disorders. In particular, hematopoietic stem cells (HSCs) are considered an optimal source for ex vivo gene therapy in lysosomal storage diseases (LSDs), which are inherited monogenic disorders. We have previously demonstrated that HSC gene therapy (HSC-GT) can ameliorate systemic pathological phenotypes in murine models of several LSDs. To advance the clinical application of HSC-GT for LSDs, we focused on the automated manufacturing of gene-modified CD34+ cells and developed the production process of the HSC using a closed system.

Methods: We utilized the CliniMACS Prodigy system, which enables automated cell isolation, culture, and gene transduction within a closed environment. The KG1a and K562 leukemic cell lines, which are CD34+ and CD34- respectively, were used to assess the cell manufacturing process. Lentiviral vectors encoding green fluorescent protein were employed to analyze gene transduction efficiency. Additionally, peripheral blood mononuclear cells (PBMC) were used to evaluate the impact of manufacturing duration on the yield of primitive HSCs using the above automated cell manufacturing system.

Results: To assess the cell manufacturing process, a mixture of KG1a and K562 cells was processed for CD34+ cell isolation and subsequent gene transduction. The proportion of isolated cells closely reflected the initial ratio of KG1a cells in the input mixture. The isolated population exhibited high CD34 expression and viability rate. Flow cytometric analysis revealed that the majority of isolated cells were green fluorescence-positive. Quantitative PCR analysis confirmed that the vector copy number remained below five copies per cell in the isolated population. Furthermore, PBMC-based trials demonstrated that a higher proportion of primitive HSCs was retained during short-term manufacturing compared to extended production periods.

Conclusion: We developed a manufacturing process for gene-modified HSCs using a closed system. This automated cell manufacturing approach has the potential to provide a consistent quality product for the clinical development of HSC-GT in LSDs.