Hi Jonathan, thank you so much! We think that - since the clonal advantage of the V617F mutation is mild - this re-growth would be very slow. It can take decades from acquisition of the JAK2 mutation to manifestation of clinical disease. But this is something we are keeping an eye on!

Huge thanks to my supervisors @jojdavies.bsky.social Adam Mead + all colleagues @imm.ox.ac.uk who contributed: @yuqi-shen.bsky.social, Zoë Wong, Ramy Slama, Maria Greco, Lai Cheng, @ellouka.bsky.social, Weijiao Zhang, Yavor Bozhilov, @jhamley98.bsky.social, Adam Wilkinson, @bethpsaila.bsky.social

We are excited about this data, since the decade-long progression of V617F MPNs means that – unlike for aggressive malignancy – a single genome editing intervention may provide meaningful long-term suppression of the mutant clone. Watch this space, especially as in vivo editing techniques mature!

Collectively, our data provides evidence that JAK2 V617F does not cause oncogene addiction, and that targeting the mutant copy reverts heterozygous cells to a more normal phenotype.

Intriguingly, cells with highly edited V617F copies can be detected at the experimental endpoint in the treatment group. This shows that editing V617F drastically reduces aggressiveness but preserves engraftment potential (presumably supported by the wildtype copy of JAK2 in SET-2 cells).

Finally, we undertook xenograft experiments. Targeting the V617F mutation in SET-2 cells prior to transplantation results in dramatically improved survival and fully reverted splenomegaly and bone marrow fibrosis phenotypes.

Transcriptionally, inactivation of V617F reverts inflammatory gene expression signatures caused by the mutation (more details in the preprint!).

Since editing creates many genotypes (edited/unedited for heterozygous/homozygous cells each), we used combined single-cell RNAseq and genotyping (TARGET-Seq) to resolve effects by genotype. Again, we see remarkable efficiency: 97% of mutant cells have all mutant copies inactivated by CRISPR.

To further validate that edited CD34+ cells retain normal haematopoiesis, we engrafted these cells into a bone marrow organoid system. We saw normal multilineage myeloid differentiation and deep and persistent drops in VAF – as much as 99% down from the baseline burden!

Editing preserves colony-forming ability while reducing erythroid output. Intriguingly, by genotyping individual colonies, we find that heterozygous mutant cells tolerate loss of their mutant alleles (MUTx).

After testing several strategies, we find that a Cas12 Ultra approach - which exploits a de novo PAM created by the V617F mutation - yields the best results. We achieve >97% V617F inactivation in primary patient CD34+ cells via electroporation, with minimal targeting of wildtype alleles!

Since MPNs originate from stem cells, it should be possible to genetically delete V617F alleles from CD34+ cells. If we can do this allele-selectively (AS) for the V617F alleles, we should selectively suppress mutant effects while preserving haematopoiesis from wildtype alleles.

JAK2 V617F is a common haematological driver mutation, but JAK2 inhibitors fail to clear mutant cells in most patients with myeloproliferative neoplasms (MPNs).