{"doi":"10.1101/2023.09.14.557853","title":"CRISPR-Cas9 Engineered Extracellular Vesicles for the Treatment of Dominant Progressive Hearing Loss","abstract":"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 (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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":390590,"datarank":0.3596842909197557,"base_score":2.3978952727983707,"endowment":2.3978952727983707,"self_citation_contribution":0.3596842909197557,"citation_network_contribution":0.0,"self_endowment_contribution":0.3596842909197557,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":10,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.947,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1162382,"name":"Peixin Huang","orcid":"0000-0002-1883-1975","position":1,"is_corresponding":false},{"id":1162383,"name":"Samantha Ali","orcid":"0009-0004-4582-1678","position":2,"is_corresponding":false},{"id":1110032,"name":"Bryan Renslo","orcid":"0000-0001-6093-9421","position":3,"is_corresponding":false},{"id":846336,"name":"Tarun E. Hutchinson","orcid":"0000-0002-9175-7222","position":4,"is_corresponding":false},{"id":651306,"name":"Nina Erwin","orcid":"0000-0002-2692-3714","position":5,"is_corresponding":false},{"id":651304,"name":"Zachary Greenberg","orcid":"0000-0001-9626-2694","position":6,"is_corresponding":false},{"id":1162384,"name":"Zuo Ding","orcid":"0000-0002-5901-4621","position":7,"is_corresponding":false},{"id":88599,"name":"Yanjun Li","orcid":"0000-0002-6277-4189","position":8,"is_corresponding":false},{"id":734724,"name":"Athanasia Warnecke","orcid":"0000-0001-9652-1035","position":9,"is_corresponding":false},{"id":1162858,"name":"Natalia E. Fernandez","orcid":null,"position":10,"is_corresponding":false},{"id":734729,"name":"Hinrich Staecker","orcid":"0000-0002-0348-3015","position":11,"is_corresponding":false},{"id":620697,"name":"Mei He","orcid":"0000-0001-5316-9326","position":12,"is_corresponding":false},{"id":268375,"name":"Xiaoshu Pan","orcid":"0000-0003-1896-2198","position":0,"is_corresponding":true}],"reference_count":70,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T01:18:37.743462Z","pmid":"38168224","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}