IF a codon-optimized chimeric enzyme — designated MTS-iSelectPLB — consisting of (i) the N-terminal mitochondrial targeting sequence from human SOD2 (matrix-directing, cleavable), (ii) the substrate-recognition and lipid-sensing lid domain of human iPLA2γ (PNPLA8, residues responsible for its kinetic preference for oxidized acyl chains), and (iii) the catalytic serine-hydrolase domain of Pseudomonas aeruginosa PaPlaB (pa2927) — is encoded in a bicistronic AAV9-CMV vector and delivered at 1×10¹² vector genomes via tail vein injection to aged (18–20 month), male and female C57BL/6J mice,

THEN by 8 weeks post-injection, ≥30% reduction in 4-hydroxynonenal-modified tetralinoleoyl cardiolipin (4-HNE-CL) species in isolated left ventricular mitochondria (quantified by LC-MS/MS), ≥15% improvement in respiratory Complex I activity (NADH:ubiquinone oxidoreductase assay in cardiac homogenates), and maintenance of left ventricular ejection fraction (echocardiography) relative to baseline and AAV9-CMV-GFP controls will be observed,

BECAUSE the following causal chain is supported by converging evidence:

1. In aged C57BL/6J hearts (18–24 months), native tetralinoleoyl-CL is progressively depleted while oxidized CL species — particularly 4-HNE adducts of C18:2 acyl chains — accumulate in the inner mitochondrial membrane (IMM), directly impairing the lipid-protein contacts required to stabilize respiratory supercomplexes (Paradies et al., Free Radical Biology and Medicine, 2002; Petrosillo et al., Cell Death & Differentiation, 2013).
2. 4-HNE-modified CL is the principal driver of Complex I inhibition in aged interfibrillar mitochondria; its accumulation is not fully reversed by endogenous remodeling enzymes (tafazzin, ALCAT1) whose activity diminishes with age, making enzymatic degradation — not merely ROS scavenging — the only strategy capable of removing already-accumulated covalent adducts (Petrosillo et al., Cell Death & Differentiation, 2013). [REPAIR rationale, not prevention]
3. The "lipid whisker" model demonstrates that oxidized acyl chains, by virtue of their increased polarity and reduced hydrophobicity, protrude from the bilayer into the aqueous phase, adopting a conformationally distinct geometry inaccessible within the tightly packed native CL bilayer (Free Radic Biol Med. 2017; 111:245-261). This structural divergence is the molecular basis on which selectivity can be rationally engineered.
4. Endogenous iPLA2γ (PNPLA8) has evolved a substrate-recognition lid domain that exploits precisely this conformational difference, demonstrating kinetic preference for oxidized over native fatty acyl chains on cardiolipin (mBio 2013; 4:e00643-13). However, its endogenous catalytic throughput is insufficient to clear the oxidized CL burden in aged tissue.
5. Wild-type PaPlaB (pa2927) provides potent, broad-spectrum PLB (phospholipase B) hydrolytic activity at both sn-1 and sn-2 positions with a characterized serine-hydrolase catalyti...

SENS category: LysoSENS