Robust loading and delivery of functional small RNAs <i>via</i> protein N-myristoylation-induced small extracellular vesicles.

Abstract

Small extracellular vesicles (sEVs) are naturally secreted nanovesicles that mediate intercellular communication by transporting biomolecules such as proteins and nucleic acids. Their inherent biocompatibility makes them promising platforms for RNA therapeutics; however, efficient encapsulation of small RNAs remains challenging. To address this, we developed the Protein N-Myristoylation-induced sEVs Loading (PMEVL) system. PMEVL employs a genetic construct encoding an N-Myristoylation peptide and, optionally, a small-RNA expression cassette in its 3'-untranslated region, enabling N-myristoylation-dependent, efficient, and specific RNA loading into sEVs. Mechanistically, PMEVL enhances sEVs biogenesis by activating ERK1/2 and inhibiting AMPK, while promoting RNA loading through recruitment of ANXA2 and key ESCRT components ALIX and TSG101. This system achieved highly efficient encapsulation of diverse functional RNAs, including exogenous/endogenous small RNAs (miRNAs, siRNAs) and messenger RNAs (e.g., GFP, mCherry), as well as co-loading of multiple siRNAs with proteins of interest. To demonstrate therapeutic potential, PMEVL-mediated delivery of Pcsk9 siRNA suppressed hepatic Pcsk9 expression <i>in vitro</i> and <i>in vivo</i>. In C57BL/6 mice, this treatment restored hepatic low-density lipoprotein receptor (LDLR) expression and significantly reduced serum levels of low-density lipoprotein cholesterol (LDL-C) and total cholesterol, without systemic toxicity. Furthermore, systematic screening of 181 peptides representing the N-terminal 15-18 residues of human N-myristoylated proteins identified candidates that substantially enhanced PMEVL loading efficiency. Collectively, PMEVL represents a versatile, efficient, and modular platform for loading RNA therapeutics into sEVs, with demonstrated co-loading capability for proteins <i>in vitro</i>.