CRISPR-Cas9 Engineered Extracellular Vesicles for Treating Genetic Hearing Loss

The biological treatment of inner ear disorders remains a significant challenge due to lack of biocompatible and efficient delivery system. To address these limitations, we present an innovative extracellular vesicle (EV)-based delivery system that efficiently carries sgRNA:Cas9 ribonucleoprotein (RNP) complexes to inner hair cells. The novel Microfluidic Droplet-based Electroporation System (µDES) is developed to load cargos into EVs through a rapid, low-voltage electroporation process within millisecond-pulsed, flow-through droplets, providing a scalable, high-throughput solution. This system achieved a ten-fold higher loading efficiency of sgRNA:Cas9 RNPs into EVs (RNP-EVs) compared to traditional bulk electroporation methods, while also significantly increasing throughput. The low-voltage electroporation minimizes Joule heating, preserving the native properties of the EVs’ surface membranes. Both in vitro and in vivo testing in the Shaker-1 mouse model confirmed the high biocompatibility and effective biodistribution of RNP-EVs within the cochlea, allowing for successful penetration of inner hair cells. In contrast, sgRNA:Cas9 RNP lipid-like nanoparticles (RNP-LNPs) are predominantly confined to neuron bundles associated with the organ of Corti. In heterozygous mice, the RNP-EVs demonstrated significantly higher gene-editing efficiency at the mRNA level of shaker-1 allele and led to marked hearing recovery, as measured by auditory brainstem response (ABR). This work presents a promising new approach to advancing gene therapy for sensorineural hearing loss, offering an efficient and biocompatible method for gene delivery to inner ear cells.