CD47-Targeted Extracellular Vesicles Enable Enhanced Endolysosomal Escape for Oligonucleotide Therapy Delivery

Abstract

Despite significant advances in oligonucleotide therapeutics, translating small interfering RNA (siRNA) and antisense oligonucleotides (ASO) into clinical use remains challenging due to inadequate drug delivery systems (DDS). While these therapeutics hold promise for targeting "undruggable" pathogenic genes such as mutant KRAS in cancer and NLRP3 in inflammatory diseases, their efficacy is often hindered by rapid degradation, poor cellular uptake, and insufficient endosomal escape. The current lipid nanoparticle (LNP) delivery system has notable limitations, including high immunogenicity, predominant accumulation in the liver, and endosomal entrapment. To address these issues, we developed a CD47-mediated oligonucleotide delivery platform using engineered extracellular vesicles (EVs) expressing SIRPα membrane protein (SIRPα-EVs). By developing optimized lentiviral transduction and isolation protocols, we generated SIRPα-EVs and incorporated therapeutic oligonucleotides through cholesterol-based conjugation. Mechanistic studies revealed that SIRPα-EVs utilize clathrin-independent endocytosis for cellular uptake, allowing for efficient endolysosomal escape and enhanced therapeutic delivery compared to conventional endocytic pathways. In vivo imaging and flow cytometry confirmed selective accumulation of SIRPα-EVs in CD47-overexpressing pathological tissues. Therapeutic evaluation in two disease models highlighted the versatility as a DDS platform: KRAS siRNA-loaded SIRPα-EVs achieved 60% complete remission in combination with PD-1 blockade in a KRAS-mutant tumor model, while NLRP3 ASO-loaded SIRPα-EVs demonstrated superior anti-inflammatory effects in an LPS-induced inflammation model. Both applications exhibited significantly improved oligonucleotide delivery compared to naive EVs. This translational study establishes CD47-targeted EVs as a versatile platform for oligonucleotide delivery, demonstrating efficient endolysosomal escape and superior therapeutic outcomes across multiple disease models. Our findings provide compelling evidence for clinical applications in both cancer and inflammatory conditions characterized by CD47 overexpression.