CRISPR-Cas9 Engineered Extracellular Vesicles for the Treatment of Dominant Progressive Hearing Loss

Clinical translation of gene therapy has been challenging, due to limitations in current delivery vehicles such as traditional viral vectors. Herein, we report the use of gRNA:Cas9 ribonucleoprotein (RNP) complexes engineered extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a novel high-throughput microfluidic droplet-based electroporation system (μDES), we achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading RNP complexes into EVs (RNP-EVs), compared with conventional bulk electroporation. The flow-through droplets serve as enormous bioreactors for offering millisecond pulsed, low-voltage electroporation in a continuous-flow and scalable manner, which minimizes the Joule heating influence and surface alteration to retain natural EV stability and integrity. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7a sh1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR). One sentence summary The scalable microfluidic electroporation system enables the loading of gRNA:Cas9 ribonucleoprotein (RNP) complexes into extracellular vesicles, which leads to clinical translation potential employed in hearing disease gene therapy.