When it comes to gene therapy, blood diseases are prime candidates. Blood cells are constantly turning over, so you can quickly fix the blood supply if you replace the blood-producing stem cells in bone marrow; i.e., hematopoietic stem/progenitor cells (HSPCs). Transplanting marrow from a healthy donor is one approach, but a match can be difficult to find. In the age of CRISPR gene editing, a better option would be to remove, edit, and re-transplant the patient’s own HSPCs.
In a recent paper, Mark DeWitt and Jacob Corn at UC Berkeley demonstrate just such a gene-editing pipeline for HSPCs. While the pipeline is pluripotent, this paper differentiated into a correction for sickle cell disease (SCD). SCD is particularly promising for gene editing because healthy red blood cells survive much longer in the bloodstream than sickle cells. This means even a small starting percentage of healthy cells will outlast their diseased counterparts, significantly improving a patient’s blood.
The endothelium-lined pipeline includes the following steps:
- Design libraries of both Cas9 sgRNAs targeting the mutation and single-stranded DNA oligonucleotide donors (ssODNs) as templates for homology-directed repair (HDR).
- Test the sgRNAs and ssODNs in K562 cells (a human leukemia line) by electroporating them along with Cas9 as RNP complexes.
- Using the best sgRNA and ssODN, find the optimal dose in human HSPCs.
- Test how well corrected HSPCs can engraft into immunocompromised mice.
Interestingly, many of the optimizations the authors tested didn’t turn out to be very important for editing HSPCs:
- Protecting the ssODN with phosphorothioate did not improve HDR.
- Similarly, SCR, an inhibitor of nonhomologous end-joining, did little to improve HDR.
- Optimizing the ssODN to anneal to the non-target strand with a longer 5’ arm improved efficiency, but only slightly.
- Two improved versions of Cas9 – HF1 and eSpCas9-1.1 – reduced off-target editing in K562 cells, but they reduced on-target editing as well.
However, even without selecting for edited cells, the authors achieved some impressive results:
- The optimized protocol corrected 25% of sickle cell alleles in HSPCs.
- Treated pools of HSPCs produced about ⅓ correct hemoglobin.
- When edited HSPCs were transplanted into mice, they formed stable, self-renewing stem cell populations.
- Off-target editing in HSPCs was much lower than in K562 cells, suggesting that you should wait until moving on from cancer cell lines (like K562) before worrying too much about off-target effects.
You may have seen your fill of blood during Halloween, but if the season only served to whet your appetite, definitely check out this paper at Science Translational Medicine, 2016.