A split-intein dual-mRNA cardiac-SORT-LNP strategy reconstitutes full-length MitoBE within the mitochondrial matrix o...

IF a dual-mRNA cardiac-SORT-LNP formulation — encoding two complementary split-intein fragments of MitoBE [Fragment A: N-terminal TALE-nickase–Npu DnaE-N intein, ~2.1 kb mRNA; Fragment B: Npu DnaE-C intein–TadA8e-V106W fused to a tandem mitochondrial targeting sequence (MTS), ~2.0 kb mRNA], co-encapsulated at 1:1 molar ratio with 20 mol% DOTAP substitution SORT-LNP at a total mRNA dose of 2.0 mg/kg — is administered as a single intravenous bolus to aged (18–20 month) C57BL/6 m.5024C>T heteroplasmic surrogate mice (≥70% cardiac mutant heteroplasmy, representing the best available murine proxy for the human m.3243A>G tRNA^Leu phenotype), with parallel validation in human m.3243A>G cybrid cardiomyocyte lines,

THEN cardiac mutant heteroplasmy will decrease from ≥70% to <50% within 28 days (measured by allele-specific NGS at ≥10,000× coverage at days 7, 14, and 28), accompanied by restoration of complex I activity to ≥70% of age-matched wild-type controls (spectrophotometric assay), improvement in ATP production rate (Seahorse XFe96), and ≥10 percentage-point improvement in echocardiographic ejection fraction, with hepatic heteroplasmy unaffected (demonstrating cardiac specificity conferred by the SORT formulation), and with off-target mtDNA mutation rate below 0.1% per site across the whole mitogenome,

BECAUSE the following mechanistic chain operates:

1. Hepatic tropism of standard LNPs is redirected to cardiac tissue by SORT lipid reformulation: permanent cationic lipid additives (DOTAP at ≥15 mol%) alter the LNP protein corona composition upon IV injection, favoring uptake by cardiac endothelium and cardiomyocytes over hepatocytes, as established in the SORT-LNP biodistribution framework described in the Evidence Set. [SPECULATIVE: the exact mol% required for ≥60% cardiac vs. <10% hepatic biodistribution has not been precisely titrated for this mRNA cargo size; pilot biodistribution studies are required.]

2. Splitting the 4.5 kb TALED mRNA into two ~2.0–2.1 kb halves solves the LNP cargo size-stability problem: the Evidence Set identifies that full-length TALE-deaminase mRNAs exceed 4.5 kb, approaching the translational efficiency and encapsulation stability limits of standard LNP formulations. Splitting into two individually small mRNAs, each well within the 2–3 kb optimal LNP encapsulation range, circumvents this constraint while preserving total coding information. This split-intein strategy is directly analogous to the split-AAV approach used in nuclear gene therapy, but has never been applied to mitochondrial base editors delivered via LNP-mRNA. [SPECULATIVE cross-discipline translation.]

3. Both mRNA halves are translated in the cytoplasm; tandem MTS sequences on both fragments direct independent import into the mitochondrial matrix via the TOM/TIM23 translocase machinery: MTS-fused proteins are canonical substrates for mitochondrial import. The N-terminal MTS on Fragment A (TALE-nickase half) and the ...

SENS category: LysoSENS