Autophagic plasmid expressing SIRT3 and SIRT4 to improve Alzheimer’s disease

Abstract Background: Improving mitochondria dysfunction using gene therapy effectively treats Alzheimer’s disease (AD). Meanwhile, the molecular chaperone-mediated autophagy (CMA) mechanism in mammalian cells helps achieve effective and safe gene therapy. Methods: We introduced an internal ribosome entry site (IRES) into a plasmid vector co-expressing Sirtuin (SIRT)3 and SIRT4. This plasmid was then combined with a nucleic acid autophagic peptide (NAP) that activates and expresses CMA under the tetracycline-on (Tet-on) regulation system to construct a two-gene autophagic plasmid pSIRT3/4-NAP. The plasmid was then transfected into 293T cells, and its effect on autophagy mechanisms and dysfunctional mitochondria were assessed. Additionally, the plasmids were encapsulated into nanoparticles with transferrin-coupled cationic liposomes and injected into AD mouse models for behavioral tests and mitochondrial function detection in brain cells.Results: NAP expression is induced by doxycycline (DOX). Thereafter NAP activates the CMA mechanism to remove plasmid DNA in 293T cells. Meanwhile, SIRT3 and SIRT4 are simultaneously expressed in mitochondrial dysfunction cells induced by high glucose concentration, and significantly improve mitochondrial structure and function. Moreover, the liposome-plasmid effectively targets the brain tissue of AD mice, significantly improving mitochondrial dysfunction in the cerebral cortex and hippocampus, and restoring the learning and memory functions. Importantly, following induction by DOX, the intracellular autophagic pathway activates and clears the plasmid.Conclusions: The two-gene autophagic plasmid realizes safe and controllable expression of target genes by activating the intracellular CMA mechanism, thus, providing an effective strategy for controllable gene therapy in AD.