Harnessing Extracellular Vesicles from Red Blood Cells for Targeted Delivery of Therapeutic Peptides and RNAs for Leukemia Treatment

Extracellular vesicles (EVs) are emerging as a new class of natural drug carriers with intrinsic ability to deliver bioactive cargo at high efficiency and low toxicity. However, clinical applications of EVs are limited by the production scale and the delivery specificity. We have recently established a new platform for purification and surface modification of EVs from red blood cells (RBCs) that are scalable and versatile for targeted delivery of small-molecule drugs and RNA therapeutics (Usman et al, Nat. Com. 2019 and Pham et al, J. Extracell. Vesicles 2021). Here, we describe a new development of the method for conjugation of RBC-EVs to obtain a higher targeting efficiency. We conjugate RBC-EVs, not only with peptides and nanobodies, but also with monoclonal antibodies, by coupling peptide conjugated EVs with streptavidin-bound biotinylated antibodies. Our data demonstrate that the conjugation is stable and does not affect the physicochemical characteristics of EVs. Conjugation of EVs with a cyclic peptide targeting CXCR4 or a monoclonal antibody targeting CD33 promotes specific binding and uptake of the conjugated EVs by leukemia cells expressing the corresponding receptors. We further use CXCR4-targeting RBC-EVs to specifically deliver the pro-apoptotic peptide KLA to CXCR4-expressing leukemia cells. Delivery of KLA using CXCR4-targeting EVs significantly suppresses leukemia burden and increases survival in a leukemia xenografted mouse model. Antibody-conjugated RBC-EVs are used to deliver RNA antisense oligonucleotides to knockdown FLT3 and miR-125b in cell lines and in patient-derived xenograft models of leukemia. Finally, we demonstrate that peptide/antibody conjugated RBC-EVs are biocompatible and nonimmunogenic. Our study provides a new platform for targeted delivery of therapeutic peptides and RNAs that is highly efficient, stable, versatile and biocompatible for potential clinical applications in leukemia treatment.

Migara Kavishka Jayasinghe, Marco Pirisinu, Huan Chen, and Yuqi Yang contributed equally to this work as first authors.