IF a self-inactivating AAV6-packaged adenine base editor (ABE8e) cassette — engineered with a dual-logic circuit comprising (i) an HSC-restricted synthetic promoter (RUNX1-SCL composite element) driving ABE8e expression, (ii) a single guide RNA (sgRNA) targeting the DNMT3A R882H or TET2 loss-of-function hotspot codon, and (iii) a secondary embedded "self-destruct" protospacer within the ABE8e coding sequence itself that is corrected from a functional to a non-functional reading frame upon base editor activity — is delivered via direct intra-femoral or intravenous injection into aged (18–22 month) C57BL/6J mice with confirmed CHIP clones,

THEN targeted A•T-to-G•C correction of DNMT3A R882H (or analogous TET2 frameshift rescue) will be detected in ≥10% of bone marrow HSCs (Lin⁻Sca1⁺cKit⁺; LSK fraction) within 8 weeks post-injection, with subsequent clonal contraction of the corrected mutant clone measurable by variant allele frequency (VAF) tracking via deep amplicon sequencing, and simultaneous transcriptional silencing of the ABE8e transgene within 4–6 weeks of initial expression, eliminating persistent on-target and off-target editing risk,

BECAUSE the causal chain proceeds as follows:

1. AAV6 preferentially transduces murine HSCs in the bone marrow niche following systemic or intra-osseous delivery, as demonstrated by AAV6's established use with Cas9 RNP and HDR templates achieving efficient on-target integration in CD34⁺/HSC-like cells under the control of lineage-restricted promoters such as CD68S (Engineering monocyte/macrophage-specific GBA expression in HSCs)[https://doi.org/10.1038/s41467-020-17148-x]; this tropism provides the in vivo delivery foundation without requiring HSC mobilization or ex vivo manipulation.

2. The RUNX1-SCL composite synthetic promoter restricts ABE8e transcription to HSCs and early multipotent progenitors, [SPECULATIVE] preventing expression in downstream committed lineages (myeloid, lymphoid), thereby concentrating editing activity in the clonally expanding CHIP-mutant HSC population where DNMT3A/TET2 mutations reside and conferring a therapeutic selectivity window.

3. ABE8e catalyzes precise A-to-I (read as G) conversion at the DNMT3A R882H locus (c.2644C>T mutation on the antisense strand, targetable as A•T at the protospacer position 6), restoring wild-type arginine 882, which re-enables DNMT3A methyltransferase activity at polycomb-regulated loci; analogously for TET2, a targeted ABE correction at splice-site or nonsense mutations restores functional dioxygenase activity, reversing the epigenetic reprogramming that confers clonal fitness advantage [SPECULATIVE: specific codon correction efficiency without transplant selection pressure].

4. Restoration of DNMT3A or TET2 function eliminates the clonal fitness advantage of CHIP HSCs — which depends on hypomethylation-driven upregulation of self-renewal genes (Tet2-null HSCs show increased symmetric self-renewal) — causing the corrected clon...

SENS category: OncoSENS

Key references: • doi.org/10.1038/s41467-020-17148-x]; • doi.org/10.1038/s41467-020-17148-x]. • doi.org/10.1038/s41467-020-17148-x