Hitting ‘undo’ to cure Sickle Cell Disease

From the Lieber and Kiem Labs, Hematologic Malignancies Program, Cancer Consortium.

For biologists, the advent of CRISPR/Cas9 meant an entirely new toolset for adding, removing, and modulating genes to accelerate the pace of biological discovery. But for many, the larger promise of CRISPR was an idea long fantasized by philosophers and science fiction writers alike: the capability to edit human DNA to cure genetic disease. To be clear, we’re not quite there yet. This grand challenge comes with many important considerations: how to best apply CRISPR systems to human tissues, how to avoid potentially disastrous off-target gene editing, and how to develop this technology within the bounds of societal ethics. But thanks to CRISPR, we’ve never been closer to a future where huge swaths of human disease become curable—not merely treatable.

One of the researchers on the forefront of this field is Dr. Chang Li, a research assistant professor in the Division of Medical Genetics at the University of Washington. Dr. Li works with colleagues Dr. André Lieber of the University of Washington and Dr. Hans-Peter Kiem at Fred Hutch to develop gene therapy treatments for hematological (blood) disorders. In a recent paper published in the journal Blood, the team report exciting proof-of-concept towards using gene editing to correct Sickle Cell Disease-causing mutations in a mouse model of the disease.

In many ways, Sickle Cell Disease (SCD) is a poster child for prospective gene therapies: it’s globally prevalent, but especially deadly in the developing world. The disease is caused by an inherited mutation to a single gene, the beta subunit of hemoglobin (that’s the protein which allows your red blood cells to carry oxygen). Remarkably, the vast majority of SCD cases are caused by a single nucleotide mutation to β-globin: an adenine-to-thymine substitution which changes the corresponding amino acid in the β-globin protein from a glutamic acid to valine. This single amino acid swap causes red blood cells to become misshapen (‘sickle-shaped’) in the absence of oxygen and aggregate into long filaments, a state which causes both acute and chronic health complications (and, in resource-poor settings, early mortality). SCD has no cure: symptoms can be managed, and experimental approaches ranging from bone marrow transplantation to genetic reactivation of non-mutated fetal globin genes are in development, but SCD continues to kill thousands across the globe each year.

Together with colleagues, Dr. Li sought to provide a proof-of-concept for a simple (sounding) cure for SCD: what if we could use gene editing to correct the pathogenic β-globin mutation in a patient’s blood cell precursors, known as hematopoietic stem cells (HSCs)? This approach was enabled by two crucial, state-of-the-art technologies. The first was a precision DNA editing system called PE5max, developed in the lab of Dr. David Liu (a coauthor on the study) at Harvard University. PE5max is one of a class of prime editors, a variation of the CRISPR/Cas9 system which ‘nicks’ DNA instead of cutting it and uses engineered enzymes to precisely mutate a DNA sequence of choice in the genome. The second key tool was a modified viral vector system which allowed the team to package their prime editor and inject it directly into the bloodstream, where the virus ‘infects’ HSCs, introducing the PE5max editor and allowing it to do its work where it matters.

A digital artwork depicting a hand holding a scalple up to a molecule of DNA
An artistic interpretation of precision DNA editing. Image created by the author using DALL-E 2.

After testing several iterations of prime editors and verifying that they are capable of correcting the SCD mutation in human and mouse cultured cell lines, Li and colleagues turned their attention to the mouse as a model organism. A crucial detail here was their use of so-called Townes mice—a strain of mice which have been genetically modified to express the human hemoglobin genes in place of their mouse counterparts. For their penultimate investigation, the team opted to test both ex vivo and in vivo approaches in tandem. For the first approach, they isolated bone marrow HSCs from mice, used PE5max to correct the SCD mutation in culture, then reintroduced these edited cells back into wild-type mice (which were irradiated to deplete their endogenous HSCs and “make room” for the edited HSCs). After following these recipient mice for only four weeks, Li observed a roughly 85% editing frequency in the peripheral blood monocytes (PBMCs) of the mice, which levelled out at 100% after 16 weeks. Reassuringly, taking HSCs from these mice and transplanting them into a cohort of irradiated wild-type mice for a second 16-week period resulted in near 100% editing efficiency in a variety of relevant organs, demonstrating that they had achieved mutation correction in long-lived HSCs which reestablished themselves in the primary host.

And now, the moment we’ve all been waiting for: this time, Li and colleagues used a drug to ‘mobilize’ HSCs into the bloodstream of Townes mice before injecting their viral PE5max construct. To increase the efficiency, the team also took advantage of a selection marker expressed along with their PE5max; this way, they could select for edited cells, which have been made resistant against a specific chemotherapeutic cocktail (O6BG and BCNU, for the aficionados). At 16 weeks, these mice showed averaged a 43.6% editing frequency in PBMCs—well above the level shown to confer therapeutic benefit (since SCD is caused by blood cells aggregating, you don’t need to fix all of them to make a difference!). Remarkably, transplanting HSCs into another cohort of mice resulted in ~40% correction frequency in a variety of tissues throughout the 16-week follow up period, proving that the team had successfully edited a stable population of long-lived HSCs. Importantly, Li and colleagues showed a <2% frequency of undesirable indels (insertions/deletions) in their target tissues, and they used a variety of in vitro techniques on isolated blood to show a significant improvement in sickle-cell phenotypes as a result of editing.  

All in all, Dr. Li’s study is a taste of the future—the team anticipates several details to troubleshoot as they move from a proof of concept into a potential therapy, and more studies are warranted to evaluate the long-term safety of in vivo gene editing. Nevertheless, the concept of using prime editing to cure SCD (and other diseases) is exciting from multiple angles. Especially prescient is the fact that the team can achieve therapeutic benefit by administering their editor in vivo, sidestepping the requirement to isolate, culture, and reintroduce HSCs and making their approach much more attractive for the resource-poor settings where SCD does the most damage—where an innovative cure can have the greatest impact.

The spotlighted research was funded by the National Institutes of Health, Ensoma Bio, the Bill and Melinda Gates Foundation, the Howard Hughes Medical Institute, the National Science Foundation, and a Helen Hay Whitney Postdoctoral Fellowship

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Dr. André Lieber and Dr. Hans-Peter Kiem contributed to this study.

Li, C., Georgakopoulou, A., Newby, G. A., Chen, P. J., Everette, K. A., Paschoudi, K., Vlachaki, E., Gil, S., Anderson, A. K., Koob, T., Huang, L., Wang, H., Kiem, H.-P., Liu, D. R., Yannaki, E., & Lieber, A. (2023). In vivo HSC prime editing rescues sickle cell disease in a mouse model. Blood, 141(17), 2085–2099.