Identification of a 144-amino-acid Scaffold Protein for Extracellular Vesicle Engineering

ABSTRACT: Extracellular vesicles (EVs) have emerged as promising drug-delivery vehicles. Genetic engineering of a membrane-bound, EV-sorting scaffold protein empowers these cell-borne nanosized vesicles by installing targeting moieties on the surface and enriching therapeutic cargo in the lumen. However, the choice of scaffold proteins with a simple structure and short sequence is scarce. Here, we conduct mass-spectrometry-based proteomic studies, identify 15 candidate proteins with EV-sorting properties, and examine their capability as the scaffold protein. ENPP1 stands out from our screening, outperforming the widely used EV-sorting proteins PTGFRN and Lamp2b with high abundance, homogeneity, stability, and engineerability. In particular, a 144-amino acid truncated version of ENPP1, called EN144 here, demonstrates high-density loading of cargos, including peptides, proteins, and mRNAs. Based on this finding, we engineer decoy EVs expressing IL-6 signal transducer (IL6ST, a.k.a. gp130) fused with EN-144, EN144-EVhgp130, to inhibit the IL-6 trans-signaling pathway. The resulting decoy EVs elicit potent anti-inflammatory effects and improve the survival rate of septic mice. Chondrocyte-targeting EN144-EVhgp130 ameliorates inflammation and remodels the synovial tissue microenvironment of cartilage in a rat model of osteoarthritis (OA), surpassing tocilizumab (TCZ), the clinically approved humanized anti-interleukin-6 receptor (IL-6R) antibody. Altogether, we report the identification and verification of EN144 as one of the smallest and simplest scaffold proteins for EV engineering and showcase systematic and local administrations of EN144-engineered decoy EVs for treating inflammatory diseases.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Wenjing Yan  

LAB HEAD: Wang shi zhi

PROVIDER: PXD057965 | Pride | 2026-04-27

REPOSITORIES: Pride