NanoMEDIC gene editing delivery system

Technical Summary

➤Particle construction and production

First, the researchers designed NanoMEDIC delivery system which relies on two homing mechanisms to package Cas9 protein and sgRNA separately. The first utilizes chemical ligand-dependent incorporation of SpCas9 to GagHIV. The second relies on Gag-mediated HIV Ψ packaging signal to direct sgRNA flanked by HH and HDV self-cleaving ribozymes into NanoMEDIC. In both cases, Gag is the beacon for recruitment and the dual-homing approach synergistically recruits active RNP complexes into NanoMEDIC particles (Gee et al. 2020). The particles are produced by transfecting producer cells with five plasmid DNAs expressing FRB-Cas9, Ribozyme-gRNA-Ribozyme, FKBP12-Gag, Tat, and VSV-G.The inventors also established a straightforward large-scale NanoMEDIC production (Watanabe et al. 2023, Fig.1).

Figure 1.Schematic of experimental procedures for NanoMEDIC production.

➤Particle application

The researchers then used NanoMEDIC system to target exon 45 in DMD gene to restore out-of-frame dystrophin protein by exon skipping. They delivered two sgRNAs independently without mutual inhibition achieving a high exon-skipping activity. They confirmed the system efficiency in skeletal muscle cells differentiated from DMD patient-derived iPSCs: dystrophin protein expression was restored.

Long-term (160 days) exon 45 skipping was also achieved in mice accompanied by a transient delivery, which was cleared within 3 days. Importantly, off-target cleavage activity was almost eliminated using NanoMEDIC compared with DNA plasmid delivery. Thus,NanoMEDIC system can be used to deliver CRISPR-Cas9 for genome editing into living skeletal muscle tissue. Moreover,NanoMEDIC efficiently induces genome editing in various human cell types, such as T cells, monocytes, iPSCs, and iPSC-derived cortical neurons (Gee et al. 2020).

Technology Readiness Level

3

Approach has been validatedin vitro (various cell types) and in vivo (mice)

Potential Applications

Gene therapy for DMD

Gene therapy for other diseases
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Possible Collaboration Mode(s)

R&D collaboration

Licensing

IP Acquisition

Other

Patent No

WO2020/013317

Publication(s)

Gee P, Lung MSY, Okuzaki Y, Sasakawa N, Iguchi T, Makita Yet al. Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.Nat Commun 2020; 11: 1334.

Watanabe K, Gee P, Hotta A. Preparation of NanoMEDIC Extracellular Vesicles to Deliver CRISPR-Cas9 Ribonucleoproteins for Genomic Exon Skipping.Methods Mol Biol 2023;2587: 427–453.