Engineering of extracellular vesicles for efficient intracellular delivery of multimodal therapeutics including genome editors

Nat Commun. 2025 Apr 29;16(1):4028. doi: 10.1038/s41467-025-59377-y.

Abstract

Intracellular delivery of protein and RNA therapeutics represents a major challenge. Here, we develop highly potent engineered extracellular vesicles (EVs) by incorporating bio-inspired attributes required for effective delivery. These comprise an engineered mini-intein protein with self-cleavage activity for active cargo loading and release, and fusogenic VSV-G protein for endosomal escape. Combining these components allows high efficiency recombination and genome editing in vitro following EV-mediated delivery of Cre recombinase and Cas9/sgRNA RNP cargoes, respectively. In vivo, infusion of a single dose Cre loaded EVs into the lateral ventricle in brain of Cre-LoxP R26-LSL-tdTomato reporter mice results in greater than 40% and 30% recombined cells in hippocampus and cortex respectively. In addition, we demonstrate therapeutic potential of this platform by showing inhibition of LPS-induced systemic inflammation via delivery of a super-repressor of NF-ĸB activity. Our data establish these engineered EVs as a platform for effective delivery of multimodal therapeutic cargoes, including for efficient genome editing.

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems
  • Drug Delivery Systems* / methods
  • Extracellular Vesicles* / genetics
  • Extracellular Vesicles* / metabolism
  • Gene Editing* / methods
  • HEK293 Cells
  • Humans
  • Inflammation / chemically induced
  • Inflammation / therapy
  • Integrases / genetics
  • Integrases / metabolism
  • Mice
  • NF-kappa B / metabolism

Substances

  • Cre recombinase
  • Integrases
  • CRISPR-Associated Protein 9
  • NF-kappa B