Abstract Body: [Introduction]
Danon Disease is a life-threatening Lysosomal storage disorder (LSD) caused by mutations in the Lysosome-associated membrane protein 2 (LAMP2) gene, leading to autophagy dysfunction and severe cardiomyopathy. We used patient-derived Danon iPSC-Cardiomyocytes (iPSC-CMs) and delivered lipid nanoparticle (LNP)-encapsulated LAMP2 mRNA to restore LAMP2 protein expression and lysosomal function, leading to phenotypic recovery. This approach directly addresses LAMP2 deficiency, offering a novel therapeutic strategy for Danon disease and establishing the LNP-mRNA platform as a promising protein replacement therapy.

[Method]
LAMP2 mRNA was synthesized via in vitro transcription (IVT) and encapsulated into lipid nanoparticles (LNPs). LNP-mRNA complexes were characterized for particle size, encapsulation efficiency, and stability, with morphology analyzed using cryogenic transmission electron microscopy (Cryo-TEM). LAMP2 expression and functionality were evaluated in HEK293T cells and iPSC-CMs via Western blot and immunofluorescence. Subcellular localization of LAMP2 was confirmed by confocal microscopy with LysoTracker staining. Autophagy flux and autolysosomal function were assessed through LC3-I/II ratio, p62 levels, and pH-sensitive markers.

[Result]
LNP-mRNA delivery conditions in iPSC-CMs were optimized using LNP-FLuc mRNA, determining optimal uptake conditions including ApoE and FBS concentrations. LAMP2 protein expression was confirmed in iPSC-CMs via Western blot and immunocytochemistry. Confocal microscopy with LysoTracker co-staining verified lysosomal localization of newly synthesized LAMP2 from LNP-mRNA with Pearson correlation analysis. Autophagic function was evaluated through autolysosome formation and autophagosome differentiation using pH-responsive EGFP markers, assessing functional restoration in Danon iPSC-CMs.

[Conclusion]
Our study demonstrates that LNP-mRNA therapy restores LAMP2 protein levels in cardiac cells, presenting a novel therapeutic strategy for Danon disease. These findings suggest LNP-mediated mRNA delivery as a potential treatment for LSDs with cardiomyopathy. Further studies on long-term efficacy and safety may position this approach as a promising strategy for diverse genetic disorders requiring protein replacement therapy.