"All-in One" Engineered Exosomes For Tumor Killing Effect

BackgroundMalignant tumor is a disease of abnormal cell proliferation caused by gene mutation,with high spatial and temporal heterogeneity.All malignancies,including hematologic tumors,are thought to have a common pathogenesis,that is,genetic variation of individual cells acquired through continuous random mutation,thus enabling cells to acquire abnormal growth and metastasis capabilities.Evasion of cell death is one of the fundamental changes in cells undergoing malignant transformation,and reducing apoptosis or its resistance plays a crucial role during tumorigenesis.Tumor cells can reduce apoptosis or inhibit apoptosis through multiple pathways.It has been found that up-regulation of some genes or down-regulation of some genes can reduce the apoptosis of tumor cells and thus promote the occurrence and development of tumors,and tumor cells survive dependent on defects in apoptotic signaling pathways.Thus,drugs or therapeutic strategies that return apoptotic signaling pathways back to normal have the potential to eliminate tumor cells.In addition,eradicating tumor cells while preserving non-tumor cells remains a daunting task in cancer therapy.Non-targeted therapies can cause negative side effects when the cancer treatment impacts healthy tissues or organs.These side effects cause great pain to patients and hinder the progress of cancer treatment.Based on these reasons,it is very challenging to establish a strategy with a broad spectrum across tumor types while maintaining tumor killing specificity as well as limiting host toxicity.Currently,many new cancer treatment strategies are being developed,and gene therapy is one of them.Exosomes have received much attention as delivery vehicles for gene therapy in recent years.Exosomes are extracellular vesicles released by various cells and are approximately 30-150 nm in diameter.Compared with other delivery vectors,exosomes have the advantages of low immunogenicity,good cell and tissue penetration,and are advanced platforms for gene therapy.However,not all exosomes produced by host cells carry a large number of destination cargoes,and only a small proportion of exosomes have sufficient therapeutic levels.Therefore,purification of therapeutic exosomes from collected exosomes by engineering techniques would provide a multifunctional tool for nanomedicine.The aim of this study was to deliver tumor-specific killing molecules by exosomes as drug delivery carriers,and purify therapeutic exosomes by engineering technology to provide a new strategy for gene therapy of tumors by highly specific,highly purified,and efficient drug delivery.Methods and results1.Consturction of“all-in-one”exosome engineering strategyIn order to improve the antitumor efficacy of exosomes,we optimized their processing,isolation and delivery strategies.By constructing a fusion protein vector（Flag-TCS-PTGFRN-CTSL-MCP）and transfecting donor cells,the collected exosomes were named Exo^Sorter.The Flag and thrombin cleavage site（TCS）are displayed outside the membrane of the exosome,Cathepsin L（CTSL）and the RNA binding protein MCP is displayed in the lumen of the exosome.It was found that the expression of the exogenous marker protein Flag on the exosome membrane can bring a specific label to the engineered exosome,which is specifically recognized by the anti-flag magnetic beads and distinguished from the rest of the exosome.Further under the action of thrombin,the magnetic beads can be separated from the purified engineered exosomes.Previous studies have shown that RNA-binding proteins can specifically bind m RNA containing MS2 and sort m RNA into the lumen of exosomes,which provides us with ideas to improve exosome loading efficiency.We constructed a plasmid containing tumor-specific killing fragments CD95DD and MS2.After co-transfection（fusion protein vectors（Flag-TCS-PTGFRN-CTSL-MCP）and MS2-CD95DD）,we obtained therapeutic exosomes.It was found that exosomes loaded with MCP-MS2 system had higher loading efficiency.In addition,after acidifying exosomes,CTSL will separate MCP/MS2-CD95DD from the fusion protein by autohydrolysis and enhance the therapeutic effect.2.Evaluation of tumor killing efficacy based on“all-in-one”enginnered exosomesWe established a human lymphoma model by subcutaneous tumor bearing in nude mice.By tracking fluorescently labeled exosomes,it was clarified that engineered exosomes were able to be delivered into mice.The results showed that mice treated with engineered exosomes had smaller tumor tissue,increased apoptosis,and prolonged survival compared with PBS-treated mice.The purified exosome group achieved the same therapeutic effect at a lower dose compared to the unpurified exosome group.ConclusionsIn this study,we developed an exosome sorter,which can not only efficiently enrich therapeutic m RNA in exosome lumen,but also display exogenous tag protein Flag on exosome surface,facilitating the purification of the target exosome.Through proof-of-concept experiments,the"all-in-one"strategy was demonstrated to be effective both in the efficient encapsulation of therapeutic m RNA into exosomes and in exosome subsets for specific purification purposes.Compared with unpurified exosomes,purified exosomes loading CD95DD-MS2 m RNA achieved the same killing effect on tumor tissue at a smaller dose.