Clinical-Scale WAS Editing Meets GMP and Safety Benchmarks

Inserting a full therapeutic coding sequence by homology-directed repair (HDR) is attractive for monogenic disorders, but the process is hard to standardise and tends to exhaust long-term repopulating stem cells. To advance clinical readiness and use GMP-grade reagents, the team electroporated Cas9 ribonucleoproteins into healthy-donor CD34-positive cells to cut at the start codon of WAS, while AAV6 supplied a codon-optimised WAS cassette for homology-directed integration.

Clinical-scale runs editing approximately 115 million cells achieved 61% targeted integration, stable through two weeks of culture and cryopreservation, with viability, stemness and multilineage output preserved. After transplantation into immunodeficient mice, the edited cells engrafted almost as well as untouched cells, reaching a median of around 30% human chimerism in the mouse marrow. However, only 10% of the human cells recovered from the marrow after 14 weeks still carried the cassette – a sign that the correction reaches committed progenitors more readily than the stem cells that repopulate.

Transient inhibition of 53BP1 using i53 mRNA combined with a tenfold lower AAV6 dose reduced this post-transplant decline and produced up to fivefold more engrafted CD34-positive cells carrying the cassette. No treatment-related toxicity, disrupted haematopoiesis or tumour formation was detected in 55 transplanted mice over 14 weeks, although the authors note that confirming this will require secondary transplantation and extended surveillance in patients.

Layered safety testing found low-frequency indels at a single predicted off-target site in COL23A1 in one donor, no clonal selection, normal karyotypes, polyclonal integration, and a rare translocation to SLC47A2 detected only in engrafted cells post-transplant, underlining the need for longitudinal patient monitoring.

The study was led by Asma Naseem and Alessia Cavazza at University College London, with collaborators in Modena and Milan. It was published in Molecular Therapy: Advances on 10 September 2026.