Mechanism of human hematopoietic stem cell loss during ex vivo manipulation and gene transfer

Hematopoietic stem cells (HSC) are excellent targets for gene therapy (GT) to cure inherited disorders. The HSC-GT success depends on the ability of the gene-modified (gm) HSC to differentiate into hematopoietic progenitor cells (HPC), while simultaneously self-renewing and regenerating themselves. For GT, CD34+ hematopoietic stem and progenitor cells (HSPC) containing the rare HSC are cultured for 2–4 days in cytokine-rich medium which enforces HSC division, making them more amenable to genetic manipulation, and then transplanted following pre-transplant chemotherapy conditioning. However, genetic manipulation causes significant loss of their long-term repopulating potential (LTRP), which has resulted in failure of numerous GT trials, or necessitated very high doses of HSCs and pre-transplant conditioning for success. Using primary and secondary transplants of human adult CD34+ HSPC and a GFP-encoding lentivirus (LV) or g-retrovirus (RV) vectors, we determined the LTRP of gmHSC and non-gmHSC in NSG mice following primary (24 wk) and secondary (6–12 wk) transplants. We found that non-cycling HSCs are more tolerant of genetic manipulation. Cycling HSCs largely lose LTRP, when genetically manipulated whether with RV or LV vectors, or with a gene editing (GE) nuclease (Cas9/gRNA). This loss is not from preferential gm-HPC versus HSC, nor from apoptosis in vitro. This loss is specific to gmHSC, not unmanipulated HSC within the same culture. Mechanistically, we show that cytokine-rich in vitro culture exits HSCs from dormancy, increasing p38MAPK stress signaling and activating the DNA damage response (DDR) pathway, which results in loss of LTRP of cultured HSC. Furthermore, gm-cycling-HSCs show further increase in DDR, accumulation of gm-HSCs in the G2M phase of cell cycle, activation of Chk1, depletion of HIF1α, and loss of LTRP. Inhibition of these pathways reduces DDR, stabilizes HIF1α, and reverses the G2M accumulation, to improve the LTRP of genetically manipulated HSC. Herein, we have identified fundamental mechanisms of loss of LTRP with genetic manipulation that can be targeted, and have important implications for the success of GT and GE.