P-CB-1 - Click-Engineered Small Extracellular Vesicles Displaying CARs for Precision Therapy of Acute Liver Failure

Acetaminophen (APAP) overdose is a major cause of acute liver failure (ALF), often resulting in severe hepatic injury and life-threatening systemic complications. Although N-acetylcysteine (NAC) is the standard therapeutic agent for APAP detoxification, its efficacy is limited in cases of severe overdose, and it is associated with side effects such as nausea, vomiting, and hypersensitivity reactions. Mesenchymal stromal cell (MSC)-based therapies have shown promise for liver regeneration through paracrine signaling, with growing interest in MSC-derived small extracellular vesicles (sEVs) as cell-free therapeutic alternatives. sEVs carry bioactive molecules that contribute to tissue repair; however, their lack of intrinsic targeting specificity limits their therapeutic potential in vivo.

Study

Design/Methods:

To overcome this challenge, we developed a strategy to enhance the liver-targeting capacity of MSC-derived sEVs using bioorthogonal click chemistry. MSCs were metabolically labeled with the azide-functionalized sugar N-azidoacetylmannosamine (Ac4ManNAz), producing azide-modified sEVs (N3-sEVs). These were subsequently conjugated to dibenzocyclooctyne (DBCO)-tagged single-chain variable fragments (scFv) specific for the asialoglycoprotein receptor 1 (ASGR1), which is highly expressed on hepatocytes. The resulting chimeric antigen receptor-modified sEVs (CAR-sEVs) demonstrated efficient and selective targeting of liver tissue.

Results/Findings:

In an APAP-induced mouse model of ALF, CAR-sEVs targeting ASGR1 demonstrated robust therapeutic efficacy. Treatment with MSC-derived CAR-sEVs significantly reduced serum liver enzyme levels, alleviated hepatic injury, and promoted hepatocyte proliferation. These findings underscore the potential of CAR-sEVs as a targeted, cell-free therapeutic strategy for acute liver injury and support further development of engineered sEVs in regenerative medicine.

Conclusions:

In conclusion, this study presents a novel and effective approach to enhance the therapeutic potential of MSC-derived sEVs for acute liver failure by engineering them for targeted delivery to hepatocytes. Through metabolic labeling and click chemistry-based conjugation with ASGR1-specific scFvs, CAR-sEVs achieved precise liver targeting and conferred significant hepatoprotective effects in an APAP-induced ALF mouse model. These results highlight the promise of CAR-sEVs as a targeted, cell-free therapy for liver regeneration and pave the way for future clinical translation of engineered sEVs in treating acute and possibly chronic liver diseases.