Selective RNA‐targeting of mTORC1 and 2 to ameliorate Alzheimer’s Disease pathogenesis

Abstract

AbstractBackgroundIt has been well described that inhibition of the kinase, mechanistic target of rapamycin (mTOR), improves age‐related pathologies including Alzheimer’s Disease (AD). However, mTOR exists as a complex and exerts differential functions depending on its binding partners. Suppression of mTORC1 has yielded positive outcomes for AD‐related measures (decreased amyloid beta/tau pathologies and improved cognition)1. In contrast, increased mTORC2 activity also resulted in positive outcomes in AD models (improved brain insulin sensitivity and neuronal survival)2, suggesting that a therapeutic which inhibits mTORC1 while upregulating mTORC2 may be desirable for AD. However, no such therapeutic exists.MethodTo inhibit mTORC1, we used a raptor siRNA which has been chemically modified to be stable for in vivo purposes. To upregulate mTORC2, we bioinformatically identified natural antisense transcripts (NATs) of rictor and designed custom siRNAs to target the various transcripts. Following an in vitro screen of the custom siRNAs, we determined which NATs yield upregulation of rictor when targeted and designed more siRNAs targeting the same transcript to optimize our mTORC2‐upregulating siRNA. Cellular RNA and protein were quantified using RT‐qPCR and western blot, respectively.ResultWe demonstrate efficient knockdown of raptor in all transfected cell lines, as well as in mouse brain. Raptor knockdown has been confirmed at both the RNA and protein level. Furthermore, we successfully upregulated expression of rictor using rictor NAT‐targeting siRNAs (AntagoNATs) in multiple cell lines. Downstream analyses suggest a therapeutic effect for both raptor knockdown and rictor upregulation in the context of AD.ConclusionNucleic acid‐based therapeutics which target RNA have been outcompeting and replacing small molecule protein‐targeting drugs in recent years. However, RNA‐targeted therapeutics have been largely unexplored for AD. Chemical linkage of raptor siRNA to rictor AntagoNAT siRNA yields the first therapeutic to selectively inhibit mTORC1 and upregulate mTORC2, providing a modern RNA‐targeting therapeutic for AD.