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bioRxiv · 10.1101/2023.09.14.557853

CRISPR-Cas9 Extracellular Vesicles for Treating Hearing Loss

Pan, X.·Huang, P.·Ali, S.·Hutchinson, T. E.·Erwin, N.·Greenberg, Z. F.·Ding, Z.·Li, Y.·Fernadez, N. E.·Staecker, H.·He, M.
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Abstract

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 (DES), 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 Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR). One sentence summaryThe 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.

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Pan, X., Huang, P., Ali, S., Hutchinson, T. E., Erwin, N., Greenberg, Z. F., Ding, Z., Li, Y., Fernadez, N. E., Staecker, H., He, M.. 2023-09-15. CRISPR-Cas9 Extracellular Vesicles for Treating Hearing Loss. https://doi.org/10.1101/2023.09.14.557853

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