124 I‐labelled BMSC‐Derived Extracellular Vesicles Deliver CRISPR/Cas9 Ribonucleoproteins With a GFP‐Reporter System to Inhibit Osteosarcoma Proliferation and Metastasis

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ABSTRACT Metastasis constitutes the principal factor leading to the unfavourable prognosis of osteosarcoma patients. Hypoxia, as the inherent microenvironment of osteosarcoma, can upregulate HIF‐1α via multiple pathways, thereby facilitating osteosarcoma proliferation and metastasis. Our previous research indicated that the inwardly rectifying potassium channel subfamily J member 2 (KCNJ2) inhibits the degradation of HIF‐1α in osteosarcoma. Concurrently, HIF‐1α upregulates the expression of KCNJ2 through a positive feedback regulatory mechanism. This positive regulatory mechanism significantly promotes the proliferation and metastasis of osteosarcoma. Therefore, the development of a KCNJ2‐targeted therapeutic strategy capable of disrupting this reciprocal regulatory loop represents a crucial intervention for impeding osteosarcoma progression. The CRISPR/Cas9 targeted gene editing technology has garnered extensive attention in the field of tumour treatment due to its high efficiency and low off‐target rate. Nevertheless, the relative lag of the delivery systems has restricted its application. The extracellular vesicles (EVs) secreted by bone marrow mesenchymal stem cells (BMSCs) have a natural targeting specificity for osteosarcoma and possess superior biocompatibility, making them ideal carriers for in vivo delivery. However, it is essential to confirm whether the CRISPR/Cas9 system mediated by EVs can accurately function intracellularly. Hence, we developed a fluorescence‐based Cas9 editing efficiency reporter system. When CRISPR/Cas9 system induces double‐strand breaks at specific target sites and results in frameshift mutations, osteosarcoma cells will stably express GFP. This system enables the transformation of gene editing events into quantifiable fluorescence signals. Furthermore, we engineered radiolabelled EVs derived from BMSCs to deliver the CRISPR/Cas9 system targeting KCNJ2. Using this reporter system, we confirmed their efficient gene‐editing capabilities in vitro. Additionally, leveraging their radiolabelling properties, we validated their targeted distribution in vivo. Subsequent investigations revealed that our constructed 124 I@EVs‐Cas9 effectively suppresses the proliferation and metastasis of osteosarcoma by targeting the inhibition of KCNJ2 expression and promoting HIF‐1α ubiquitin‐dependent degradation (as depicted in Graphical Abstract).

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