Extracellular vesicle-mediated gene editing for the treatment of nonsyndromic progressive hearing loss in adult mice.

Abstract

The clinical translation of gene therapy has been challenging in part because of the limitations of current delivery approaches. Herein, we report an efficient nonviral genome editor delivery approach using extracellular vesicles (EVs) carrying single-guide RNA (sgRNA): CRISPR-Cas9 ribonucleoprotein (RNP) complexes for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet-based electroporation system (μDES), we achieved a 10-fold enhancement in loading efficiency and more than 1000-fold increase in processing throughput for loading RNP complexes into EVs compared with conventional high-voltage pulsed electroporation. μDES generated uniform microdroplets containing EVs and RNPs by applying direct current-controlled low voltage (up to 60 V) to transiently permeabilize membranes and enable efficient cargo encapsulation while maintaining EV integrity at both the protein and morphological levels. In the <i>Myo7a^WT/Sh1</i> mouse model of autosomal dominant progressive hearing loss, which may model MYO7A-associated DFNA11 hearing loss in humans, we demonstrated the effective delivery of RNPs by EVs into cochlear hair cells by cross-sectional and whole-mount confocal imaging. The injection of RNP-EVs via the posterior semicircular canal in 4-week-old <i>Myo7a^WT/Sh1</i> mice resulted in a reduction in <i>Myo7a^Sh1</i> messenger RNA expression and evidence of hearing preservation, as measured by auditory brainstem responses, compared with untreated ears and EV only-injected mice. This study highlights the potential of μDES-produced RNP-EVs for gene editing as a treatment for progressive nonsyndromic hearing loss in patients.