Editor s Summary:The clinical translation of gene therapy has been challenging, due to limitations from current delivery approaches. Herein, we report an efficient non-viral genome editor delivery approach using single guide RNA (sgRNA):CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes mediated by extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet-based electroporation system ( DES), we achieved a 10-fold enhancement in loading efficiency and more than a 1000-fold increase in processing throughput for loading RNP complexes into EVs compared to conventional high-voltage pulsed electroporation. DES generates uniform microdroplets containi... More
Editor s Summary:The clinical translation of gene therapy has been challenging, due to limitations from current delivery approaches. Herein, we report an efficient non-viral genome editor delivery approach using single guide RNA (sgRNA):CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes mediated by extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet-based electroporation system ( DES), we achieved a 10-fold enhancement in loading efficiency and more than a 1000-fold increase in processing throughput for loading RNP complexes into EVs compared to conventional high-voltage pulsed electroporation. DES generates uniform microdroplets containing EVs and RNPs, applying direct current (DC) controlled low voltage (up to 60V) to transiently permeabilize membranes and enable efficient cargo encapsulation while maintain EV integrity at both protein and morphological level.. In the Myo7a WT/Sh1 mouse model of autosomal dominant progressive hearing loss which potentially represent Myo7a-associated DFNA11 hearing loss in humans, we demonstrated the effective delivery of RNP mediated by EVs into cochlear hair cells by cross-sectional and whole-mount confocal imaging. The posterior semicircular canal injection of RNP-EVs in Myo7a WT/Sh1 (4-week old) resulted in a notable reduction of mRNA expression of Myo7a Sh1 allele and evidence of hearing recovery as measured by auditory brainstem responses (ABR), compared to untreated side of ears and EV only control groups. This study highlights the potential of DES-produced RNP-EVs for gene editing as a treatment for progressive non-syndromic hearing loss in patients.One Sentence Summary:High-throughput loading of sgRNA:Cas9 RNPs targeting Myo7a Sh1 into extracellular vesicles rescues progressive hearing loss in an adult mouse model.
