How a CRISPR sickle cell therapy moved from lab to clinical trial

Findings

A UCLA-led team has described how they advanced an experimental CRISPR approach for sickle cell disease into a blood stem cell therapy that can be manufactured at the scale and quality required for a clinical trial.

The therapy uses CRISPR-Cas9 gene editing to correct the single-point mutation that causes sickle cell disease in a patient’s own blood-forming stem cells. In preclinical studies, the researchers established a reproducible manufacturing process and showed that red blood cells in vitro differentiated from edited stem cells produced less sickle hemoglobin than those grown from unedited cells. They also found that the proportion of corrected gene copies did not significantly decrease after the edited human cells were transplanted into mice and allowed to engraft for 16 weeks.

Background

Sickle cell disease is a genetic blood disorder caused by a mutation in the beta-globin gene, which is responsible for producing one of the two types of protein chains that make up hemoglobin — the protein in red blood cells that carries oxygen. The mutation causes red blood cells to become rigid and sickle-shaped, leading to blocked blood flow, anemia, severe pain, organ failure and a shortened lifespan. In the United States, sickle cell disease disproportionately affects African Americans, occurring in approximately 1 in every 365 Black or African American births.

The U.S. Food and Drug Administration has approved two gene therapies for sickle cell disease: One uses a viral vector to add a modified, anti-sickling version of the beta-globin gene to a patient’s stem cells; the other uses CRISPR to boost production of fetal hemoglobin, which helps prevent red blood cells from sickling. The experimental therapy instead uses CRISPR-Cas9 to directly correct the sickle cell mutation in a patient’s own blood-forming stem cells, using a nonviral DNA template to guide the correction.

Method

Moving the therapy from the research laboratory to the clinic required dramatically increasing the scale of the process. Laboratory experiments can involve a few hundred thousand cells, but manufacturing a therapy for a single patient requires processing and editing hundreds of millions of cells. The researchers developed a manufacturing process in which blood-forming stem cells collected from blood are maintained in culture for two days, then exposed to electrical pulses that opened temporary pores in the cells, allowing CRISPR-Cas9 and a short strand of corrective DNA to enter.

The team completed seven manufacturing runs, four at full clinical scale, using cells from people with sickle cell disease and healthy donors. All large-scale manufacturing runs met the established quality requirements, producing cell products with high stem cell purity and viability and successful correction of the sickle cell mutation. When the edited cells were grown into red blood cells in the laboratory, they produced substantially less sickle hemoglobin and increased levels of healthy adult and fetal hemoglobin. Additional studies in mice showed that the edited human cells successfully engrafted and that the proportion of corrected gene copies remained stable over 16 weeks. They also found no noteworthy treatment-related toxicity or evidence of abnormal tissue changes, supporting the preclinical safety of the cell product and its advancement toward clinical testing.

Impact

If enough corrected, long-lasting blood-forming stem cells successfully engraft, the therapy could offer a one-time treatment with the potential to cure sickle cell disease, allowing those corrected stem cells to continually produce red blood cells containing normal adult hemoglobin. The manufacturing process has been implemented at UCLA to produce cell products for two trial participants in the ongoing phase 1 clinical trial led by Dr. Mark Walters at UCSF Benioff Children’s Hospital Oakland. This trial will evaluate the treatment’s safety and efficacy, including whether corrected cells persist in patients over time. The approach could also provide a framework for moving other gene-editing therapies for inherited blood disorders from the laboratory to the clinic.     

Journal

The study was published in Molecular Therapy.

Authors

The study's corresponding author is Zulema Romero Garcia, assistant adjunct professor of microbiology, immunology and molecular genetics and a member of the UCLA Broad Stem Cell Research Center and the UCLA Health Jonsson Comprehensive Cancer Center. The co-first authors are Mark DeWitt and Beatriz Campo-Fernandez. 

UCLA authors include Sohini Roy, Alejandra Davila, Mallory Lennon, Suzanne Said, Chin-hong Tseng and Donald Kohn.

Other authors are Alissa L.W. Kleinhenz, Jonnathan T. Vu, Netravathi Krishnappa, So Hyun Park, Gang Bao, Wendy Magis, David I.K. Martin, Dario Boffelli, Stacia K. Wyman and Mark C. Walters.

Disclosures

DeWitt, Walters, Kohn, Martin, Magis and Romero Garcia are named inventors on a patent application related to this work, which has been assigned to the Regents of the University of California. Walters also serves as a scientific advisor to Ensoma Inc.