PXD014527

PXD014527 is an original dataset announced via ProteomeXchange.

Title	Proteome analysis of NanoMEDIC particles
Description	CRISPR-Cas9 has tremendous potential as a therapeutic tool for treating human diseases. However, prolonged expression of the nuclease and gRNA from viral vectors in an in vivo context may cause off-target activity and immunogenicity. While extracellular vesicles have been recently demonstrated to be a promising option to transiently deliver the CRISPR-system, sufficient packaging of both Cas9 protein and gRNA is critical to achieve efficient genome editing in hard-to-transfect cells and tissues, such as skeletal muscle. Here, we developed a novel ribonucleoprotein delivery system utilizing two distinct homing mechanisms. The first is by chemical induced dimerization to recruit Cas9 protein into extracellular nanovesicles. The second utilizes a viral RNA packaging signal and two self-cleaving riboswitches to tether and release sgRNA into nanovesicles. We term our fully engineered delivery system NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo) and demonstrate efficient genome editing in various hard-to-transfect cell types, including human iPS cells and myoblasts. Furthermore, NanoMEDIC production is scalable for industrial production as a xeno-free suspension culture system. As a disease model, therapeutic exon skipping in the dystrophin gene locus was targeted and resulted in over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy patient iPS cells. Finally, we generated novel luciferase-based reporter mice to demonstrate that NanoMEDIC could induce exon skipping and sustain skipping activity for over 160 days, even though NanoMEDIC itself was rapidly degraded within 3 days, indicating its utility for transient in vivo genome editing therapy of DMD and beyond.
HostingRepository	jPOST
AnnounceDate	2020-01-30
AnnouncementXML	Submission_2022-09-18_03:28:27.342.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Iwasaki Mio
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; L-methionine sulfoxide; deamidated L-asparagine; deamidated L-glutamine; 2-pyrrolidone-5-carboxylic acid (Glu); 2-pyrrolidone-5-carboxylic acid (Gln); alpha-amino acetylated residue; Applied Biosystems iTRAQ(TM) multiplexed quantitation chemistry
Instrument	TripleTOF 5600

Revision	Datetime	Status	ChangeLog Entry
0	2019-07-09 02:43:15	ID requested
1	2020-01-29 20:34:01	announced
2	2020-03-22 22:32:27	announced	2020-03-23: Updated PubMed.
3	2021-02-04 17:09:44	announced	2021-02-04: Updated FTP location.
⏵ 4	2022-09-18 03:28:28	announced	2022-09-18: Updated FTP location.

Gee P, Lung MSY, Okuzaki Y, Sasakawa N, Iguchi T, Makita Y, Hozumi H, Miura Y, Yang LF, Iwasaki M, Wang XH, Waller MA, Shirai N, Abe YO, Fujita Y, Watanabe K, Kagita A, Iwabuchi KA, Yasuda M, Xu H, Noda T, Komano J, Sakurai H, Inukai N, Hotta A, Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping. Nat Commun, 11(1):1334(2020) [pubmed]

submitter keyword: exosomes

Mio Iwasaki
lab head
Iwasaki Mio
contact affiliation	Kyoto University
dataset submitter

jPOST dataset URI

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.jpostdb.org/JPST000623/