CRISPR-Cas9 Extracellular Vesicles for Treating Hearing Loss
Authors: Xiaoshu Pan, Peixin Huang, Samantha Ali, Tarun E Hutchinson, Nina Erwin, Zachary Greenberg, Zuo Ding, Yanjun Li, Natalia Fernadez, Hinrich Staecker, and Mei He
The treatment of inner ear disorders remains challenging due to the intrinsic anatomical barriers. The majority treatments and delivery approaches for accessing inner hair cells are still engaged with surgical intervention, which is highly invasive and inconsistent in terms of efficacy and safety. In order to address this challenge for crossing anatomical barriers, we report an extracellular vesicle (EVs) -based delivery approach to inner hair cells, which enables carrying CRISPR/Cas9 ribonucleoprotein (RNP)-sgRNA complex in highthroughput and high efficiency. The novel Microfluidic Droplet-based Electroporation System (µDES) is developed to efficiently load cargos into EVs via millisecond pulsed, low-voltage electroporation within flowthrough droplets as enormous bioreactors in a continuous-flow and scalable manner. The observed loading efficiency of CRISPR/Cas9 RNA complex into EVs (RNP-EVs) is 10-fold higher than current bulk cuvette electroporation with hundred-fold increase of processing throughput. The low-voltage electroporation minimized the Joule heating influence on nanosized EVs, which retained the native surface membrane properties of cargoloaded EVs. Both ex vivo and in vivo testing in Shaker-1 mice model demonstrated the high biocompatibility and biodistribution of produced RNP-EVs in the mouse cochlea penetrating inner hair cells. In contrast, the CRISPR/Cas9 RNP lipid-like nanoparticles (RNP-LNPs) control group was unable to penetrate anatomical barriers to access inner hair cells. In the Shaker-1 mouse model, µDES produced RNP-EVs demonstrated much higher editing efficiency at Myo7ash1 mRNA level and showed significant hearing recovery in the Myo7aWT/Sh1 mice via Auditory Brainstem Response (ABR) testing. The report work will present a new solution to advance gene therapy in treating sensorineural hearing loss.
Fig. (A) Image of µDES device with illustration of continuous-flow droplet generation, droplet-based electroporation, and cargo-loaded EV harvesting and purification.
Keywords: CRISPR Cas9; droplet electroporation; pressure controller; droplet generation