IF CD117 (c-Kit)-antibody-conjugated lipid nanoparticles (LNPs) encapsulating ABE8e mRNA and a splice-acceptor-targeting sgRNA directed at the Mpl 5′ UTR exon-2 boundary are administered intravenously at 1.5 mg/kg weekly for 4 consecutive weeks beginning 8 weeks post-transplant to male and female C57BL/6 competitive bone marrow chimeras reconstituted with a 1:1 ratio of CD45.2+ Tet2−/− : CD45.1+ wild-type HSCs (2×10⁶ total cells, lethally irradiated CD45.1+ recipients),

THEN Tet2−/− clone chimerism in peripheral blood myeloid cells will decrease by ≥40% relative to vehicle-treated chimeras by week 20 post-editing, on-target A•T→G•C base editing at the Mpl splice-acceptor site will reach ≥20% frequency in sorted CD45.2+ Lin−Sca-1+c-Kit+CD150+CD48− (LT-HSC) populations, Mpl surface protein expression will be measurably reduced (≥30% MFI decline) on edited CD45.2+ HSCs, and critically, editing efficiency and functional consequence will be preferentially enriched in Tet2−/− over wild-type HSCs at equivalent LNP exposure, creating a selective competitive disadvantage specifically within the clonally expanded mutant compartment,

BECAUSE the following mechanistic chain operates synergistically:

1. TET2 loss causes genome-wide CpG hypomethylation that increases chromatin accessibility at regulatory elements including the Mpl locus in Tet2−/− HSCs, making the splice-acceptor target site more exposed to ABE8e deamination machinery relative to WT HSCs where TET2-mediated active demethylation maintains normal nucleosomal compaction. This predicts an intrinsic editing efficiency bias favoring the mutant clone without requiring any clone-selective delivery mechanism. [SPECULATIVE — epigenetic accessibility at the specific Mpl splice-acceptor locus in Tet2−/− HSCs has not been directly measured; indirect support from the established role of TET2 in maintaining chromatin architecture as reviewed in the Evidence Set].

2. CD117-conjugated LNPs achieve preferential HSC targeting following IV delivery, bypassing the hepatic tropism of conventional ionizable lipid formulations (DLin-MC3-DMA, ALC-0315) and concentrating ABE8e mRNA cargo in Lin−Sca-1+c-Kit+ bone marrow populations. Breda et al. (Science, 2023) demonstrated >90% editing efficiency in LT-HSCs using this platform to disrupt Mpl exon 2 splice sites in myeloproliferative neoplasm models, directly validating both the delivery vehicle and the exact genomic target relevant to this strategy. (CD117-LNP HSC targeting and Mpl splice-site disruption precedent)[Evidence Set narrative: Breda et al., Science, 2023, as cited in the Summary of Findings section].

3. ABE8e mediates efficient A•T→G•C transitions at splice-acceptor AG dinucleotides with transient mRNA expression, destroying consensus splice motifs to induce exon skipping or intron retention that generates non-functional Mpl protein. The hyperactive deamination kinetics of ABE8e relative to ABE7.10 are essential ...

SENS category: OncoSENS

Key references: • doi.org/10.1101/2023.11.16.567426].