Objective:

Develop and evaluate a clinical-scale manufacturing process for CRISPR_SCD001, an autologous stem-cell therapy designed to directly correct the sickle mutation in HBB.

Approach:

• Cell processing: CD34-positive cells collected from mobilized peripheral blood were cultured for two days, electroporated with CRISPR-Cas9 and a corrective single-stranded DNA template, then washed, cryopreserved, and tested.
• Manufacturing evaluation: The team conducted seven development runs, including four at clinical scale, using cells from healthy donors and people with sickle cell disease.
• Functional and preclinical testing: Researchers assessed edited cells differentiated into red blood cells, off-target editing and chromosomal findings, and engraftment and toxicity in mice.

Key Findings:

• Clinical-scale lots averaged 89% CD34-positive cell purity and 84% viability.
• An average of 22% of HBB alleles were corrected; insertions or deletions occurred in 51%.
• In vitro, sickle hemoglobin decreased from 89% to 22%; adult and fetal hemoglobin reached 39% and 40%, respectively.
• Editing at one previously identified intergenic off-target site averaged 4%. No HBB–off-target translocations were detected above the 0.01% assay threshold; the two lots assessed had normal karyotypes.
• In mice, edited cells engrafted at lower levels than unedited controls but persisted in multiple hematopoietic tissues after 16 weeks. Correction frequencies remained close to pre-transplant levels, and investigators found no noteworthy treatment-related toxicity or abnormal tissue changes.
• The process has been used to manufacture products for two participants in an ongoing Phase I/II trial.

Interpretation:

The study reports a clinical-scale process for manufacturing CRISPR_SCD001 and preclinical findings on editing, cell function, and engraftment. The ongoing Phase I/II trial will assess safety, engraftment, and persistence of corrected cells in patients.

Limitations:

• The supplied context does not report clinical outcomes from the two trial participants.
• Normal karyotypes were reported for only two lots, and mouse engraftment was lower for edited cells than for unedited controls.

Sources:

• Article context provided in the prompt