In an independent publication  researchers at UCLA cited using leukopaks obtained from HemaCare in order to investigate a new gene editing method.
The group is researching strategies to treat genetic blood disorders such as sickle cell anemia. While there has been a great deal of research focused on using CRISPR/Cas9 gene therapy to treat these illnesses , current strategies for gene editing in hematopoietic stem cells are inefficient, and thus costly.Hematopoietic stem cells differ from other cells in the body because they have the ability to divide and differentiate; these properties are the reason the cells are so valuable. But gene therapy only works if “repaired” stem cells can be delivered effectively, and the process of editing stem cell DNA is not terribly efficient.
Hematopoietic stem cells are commonly isolated via identification of the CD34 cell surface maker, but only a small percentage of CD34+ cells are actually capable of long-term repopulation in a patient. Scientists looking for long-term cures can’t depend on stem cells that will only persist in a patient’s body for a few short weeks. Added to this complication is the fact that gene editing technologies, including the CRISPR/Cas9 system, are simply not that efficient. Gene editing systems can miss their target or have low incorporation rates, leading to poor transplant outcomes.
The UCLA researchers devised a strategy for increasing gene editing efficiency by taking advantage of the stem cell replication cycle. There are 2 main DNA repair pathways by which gene editing can take place, and both are cell-cycle dependent. The first pathway uses “homology-directed repair” (HDR), which is highly site specific, and takes place during the S/G2 phase of the cycle. The second pathway (NHEJ) is non-specific and more error prone, and it takes place throughout the cell cycle. The authors reasoned that if they could skew gene editing towards the more site-specific pathway, it would be more efficient.
To carry out their strategy, the group isolated hematopoietic stem cells from HemaCare sourced leukopaks. HemaCare runs the largest recallable donor network in the industry, making it easier for researchers to request specific donor or disease-state criteria. Leukapheresis collection methods, including mobilization techniques, can be customized to match individual studies. For this study, G-CSF mobilized peripheral blood leukopaks from normal, healthy donors were processed to enrich for CD34+ hematopoietic stem cells. These cells were cryopreserved until they were used for the study.
The UCLA scientists controlled the timing of the hematopoietic stem cell cycle by temporarily synchronizing the cells in the HDR-directed S/G2 phase. At the same time, the authors used a modified version of Cas9 that has reduced DNA cutting activity in the phases of the cell cycle when HDR cannot occur. The combined tactic was highly successful, demonstrated by the fact that the authors achieved a 4-fold improvement in the proportion of stem cells using the site-specific HDR pathway. This result was reaffirmed by a lower, but still significant improvement in the ratio of stem cells edited by the HDR pathway following their transplantation into immune-deficient mice.
Improving gene editing outcomes in hematopoietic stem cells is an important step forward for improving gene therapy outcomes. The UCLA scientists noted that this work functions as proof-of-principle, since this is the first time researchers have successfully shown an improvement in HDR directed gene editing in human primary stem cells as opposed to cell lines.
- Lomova, et al. Improving Gene Editing Outcomes in Human Hematopoietic Stem and Progenitor Cells by Temporal Control of DNA Repair. Stem Cells. 1-17. Oct 2018.
- Hoban MD, et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood.125(17):2597–2604.2015.