IF an AAV-PHP.eB vector encoding a codon-optimized, intracellularly retained engineered subtilisin variant (optimized from the Bacillus subtilis subtilisin-family scaffold, analogous to KSM-N440 but with an appended fibril-binding domain derived from β-sheet affinity peptides) is delivered via stereotaxic injection into the substantia nigra pars compacta (SNpc) of aged (18-month-old) male C57BL/6J mice carrying established nigral α-synuclein pathology (AAV-A53T α-synuclein pre-seeding model), followed 8 weeks later by intrastriatal transplantation of iPSC-derived dopaminergic neurons (A9-subtype, matured in vitro),

THEN transplanted neuron survival at 6 months post-transplantation (measured by stereological TH+ cell counting in striatum and SNpc) will be increased ≥2-fold compared to transplantation without pre-clearance, and propagation of phospho-Ser129 α-synuclein (pSyn) immunoreactivity into grafted neurons will be reduced by ≥60% compared to transplant-only controls,

BECAUSE the causal chain operates as follows:

1. Intraneuronal α-synuclein fibrils and high-molecular-weight oligomers accumulate in the aged SNpc and propagate in a prion-like trans-cellular manner; this aggregate burden in the recipient microenvironment is the primary mechanism by which transplanted fetal and iPSC-derived dopaminergic neurons acquire Lewy body pathology and ultimately fail, as demonstrated in post-mortem analyses of long-term fetal transplant recipients — establishing that aggregate clearance before engraftment is necessary, not merely beneficial. (AAV leakage confounds PTBP1 reprogramming interpretation)[https://pmc.ncbi.nlm.nih.gov/articles/PMC11613593/]

2. Subtilisin-family serine proteases (including nattokinase and its engineered derivatives) possess broad amyloidolytic activity against β-sheet-rich aggregated substrates through hydrolysis of exposed amide bonds at aggregate surfaces, with evidence from in vitro work suggesting degradation of diverse amyloidogenic proteins; however, direct fibril-specific activity against α-synuclein by KSM-N440 or nattokinase is currently unvalidated — the engineered variant proposed here adds a fibril-targeting peptide (e.g., a D-amino acid peptide known to bind α-synuclein β-sheet surfaces) to direct proteolytic activity to the fibril substrate. [SPECULATIVE for the specific engineered fusion]

3. AAV-PHP.eB achieves robust retrograde and anterograde transduction of SNpc neurons at titers of ≥1×10¹³ vg/mL with minimal off-target astrocytic expression when using the synapsin-1 (SYN1) promoter — critically, this avoids the AAV leakage artifact that confounded PTBP1 reprogramming claims, where GFAP promoter-driven AAV mis-labeled endogenous neurons and generated false-positive conversion results. (Stringent lineage tracing with Aldh1l1-CreERT2 refuted PTBP1 conversion)[https://elifesciences.org/articles/75636] By restricting expression to neurons via SYN1, the intracellular protease acts within the pathologically lo...

SENS category: GlycoSENS

Key references: • PMID: 32581380 • PMID: 34582785