Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells

Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10⁸ to 1 × 10⁹ CD34⁺ cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34⁺ cells uses LV inefficiently, because the majority of CD34⁺ cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34⁺CD38^- cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34⁺CD38^- cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34⁺ purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34⁺CD38^- cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38⁺ cells. Importantly, LV modification and transplantation of IB-purified CD34⁺CD38^- cells/non-modified CD38⁺ cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34⁺ cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.