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.

Lentiviral vector (LV)-based hematopoietic stem and progenitor cell (HSPC) gene therapy is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases. Clinical trials are currently underway to treat sickle cell disease using LVs expressing designed anti-sickling globin genes. However, because of the large size and complexity of the human β-globin gene, LV products often have low titers and transduction efficiency, requiring large amounts to treat a single patient. Furthermore, transduction of patient HSPCs often fails to achieve a sufficiently high vector copy number (VCN) and transgene expression for clinical benefit. We therefore investigated the combination of two compounds (PGE2 and poloxamer synperonic F108) to enhance transduction of HSPCs with a clinical-scale preparation of Lenti/G-AS3-FB. Here, we found that transduction enhancers increased the in vitro VCN of bulk myeloid cultures ∼10-fold while using a 10-fold lower LV dose. This was accompanied by an increased percentage of transduced colony-forming units. Importantly, analysis of immune-deficient NSG xenografts revealed that the combination of PGE2/synperonic F108 increased LV gene transfer in a primitive HSC population, with no effects on lineage distribution or engraftment. The use of transduction enhancers may greatly improve efficacy for LV-based HSPC gene therapy.

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.