A clinical-scale manufacturing process can directly correct the mutation responsible for sickle cell disease in patients’ blood-forming stem cells, according to a new study.
The experimental therapy, CRISPR_SCD001, uses CRISPR-Cas9 and a short, single-stranded DNA template to repair the sickle mutation in the beta-globin gene, HBB. The edited autologous CD34-positive cells are then intended to be returned to the patient following conditioning.
The approach differs from the two gene therapies currently approved in the US for sickle cell disease. Lyfgenia uses a lentiviral vector to add an anti-sickling beta-globin gene, whereas Casgevy edits a regulatory region to restore fetal hemoglobin production. CRISPR_SCD001 instead attempts to repair the disease-causing mutation itself without using a viral donor template.
Moving the process toward the clinic required increasing production from hundreds of thousands of cells in laboratory experiments to hundreds of millions for a patient dose, UCLA explained in a press release.
The process begins with CD34-positive cells collected from mobilized peripheral blood. After two days in culture, the cells undergo electroporation, which temporarily opens pores in their membranes and allows the CRISPR-Cas9 complex and corrective DNA template to enter. The resulting drug product is washed, cryopreserved and tested before release.
The team conducted seven development runs, including four at full clinical scale, using cells from healthy donors and people with sickle cell disease. The large-scale lots had an average CD34-positive cell purity of 89 percent and viability of 84 percent. An average of 22 percent of HBB alleles were corrected, while insertions or deletions occurred in 51 percent.
When edited cells were differentiated into red blood cells in vitro, sickle hemoglobin fell from 89 percent to 22 percent, while adult and fetal hemoglobin reached 39 and 40 percent, respectively.
Editing was also detected at one previously identified off-target site in an intergenic region, at an average frequency of 4 percent. The researchers reported no HBB–off-target translocations above the assay’s 0.01 percent detection threshold, and cytogenetic testing found normal karyotypes in the two lots assessed.
In mice, edited human cells engrafted at lower levels than unedited controls but remained present in multiple hematopoietic tissues after 16 weeks. Gene-correction frequencies remained close to those measured before transplantation, and investigators found no noteworthy treatment-related toxicity or abnormal tissue changes.
The process has now been used to manufacture products for two participants in an ongoing Phase I/II trial. The study will assess safety, engraftment and whether corrected cells persist in patients over time.
Image Credit: National Center for Advancing Translational Sciences (NCATS), National Institutes of Health
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