Serum extracellular vesicles for delivery of CRISPR-CAS9 ribonucleoproteins to modify the dystrophin gene.

Abstract

Extracellular vesicles (EVs) mediate intercellular biomolecule exchanges in the body, making them promising delivery vehicles for therapeutic cargo. Genetic engineering by the CRISPR system is an interesting therapeutic avenue for genetic diseases such as Duchenne muscular dystrophy (DMD). We developed a simple method for loading EVs with CRISPR ribonucleoproteins (RNPs) consisting of SpCas9 proteins and guide RNAs (gRNAs). EVs were first purified from human or mouse serum using ultrafiltration and size-exclusion chromatography. Using protein transfectant to load RNPs into serum EVs, we showed that EVs are good carriers of RNPs in vitro and restored the expression of the tdTomato fluorescent protein in muscle fibers of Ai9 mice. EVs carrying RNPs targeting introns 22 and 24 of the DMD gene were also injected into muscles of mdx mice having a non-sense mutation in exon 23. Up to 19% of the cDNA extracted from treated mdx mice had the intended deletion of exons 23 and 24, allowing dystrophin expression in muscle fibers. RNPs alone, without EVs, were inefficient in generating detectable deletions in mouse muscles. This method opens new opportunities for rapid and safe delivery of CRISPR components to treat DMD.