Date: 16th March 2020
Transient delivery systems are often ideal for therapeutic genome editing applications as they can reduce off-target edits and immunogenicity. Now scientists have developed an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC to deliver CRISPR-Cas9 in hard-to-transfect cell lines and mouse models.
The team led by Akitsu Hotta from Kyoto University, Japan, have devised two distinct homing mechanisms for their system, one to package the CRISPR-Cas9 protein and the other for the sgRNA (single guide). In brief, chemical ligand-dependent dimerisation was first used to recruit a tagged Cas9 protein into extracellular nanovesicles. The second part of the system used a viral RNA packaging signal, this directed sgRNAs, flanked by two self-cleaving riboswitches, into the vesicles.
Initial experiments tested the NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo) system, and showed that it could efficiently induce genome editing in various human cell types, such as T cells, monocytes, iPSCs (induced pluripotent stem cells), iPSC-derived cortical neurons, and myogenic cells.
As a further proof of concept, NanoMEDIC was then used to target the dystrophin gene of Duchenne muscular dystrophy (DMD) patient iPSCs. The team wanted to induce exon skipping to restore dystrophin protein expression in these cells. NanoMEDIC was able to induce highly efficient genome editing with up to 92% exon skipping achieved. Furthermore, cell toxicity did not occur, and off-target effects were almost eliminated when compared to DNA plasmid delivery of the CRISPR system.
The next question was whether exon skipping could be induced in vivo? To assess the duration and tissue specificity of exon skipping potential of NanoMEDIC targeting human DMD sequences in this setting, a transgenic (luciferase) reporter mouse model was created containing the appropriate human sequences. The percentage of genomic deletions of the targeted reporter sequence was ~ 7%, and activity was observed up to 160 days.
The final test was to target endogenous dystrophin gene in a mouse model of DMD. Once again, exon skipping was detected albeit at a lower level than seen in the reporter gene locus. The team detected a 1.1% large deletion between two edit target sites and exon skipping efficiency of around 1.6%.
Conclusion and future applications:
The use of CRISPRs in the fight against DMD has attracted much attention recently, we reported back in January attempts to relieve DMD symptoms (in pigs) and with large pharma and biotech companies such as Vertex, CRISPR Therapeutics and Exonics Therapeutics having a vested interest in this disease it is hoped that these efforts will accelerate a route to clinic.
The work performed here is likely to enhance the delivery of gene editing systems to a multitude of cell types in vivo. Whilst several CRISPR-Cas9-transient delivery systems have been reported, each system has unique characteristics, pit-falls and strengths, and further work on NanoMEDIC will likely determine which applications it is best suited. However, its use will likely expand into other Cas nuclease candidates, targeted delivery, or use in the delivery of other cargo. Whatever is on the horizon, increasing delivery options should be beneficial in the drive towards translating these methods into the clinic.
Gee, P., M. S. Y. Lung, Y. Okuzaki, N. Sasakawa, T. Iguchi, Y. Makita, H. Hozumi, Y. Miura, L. F. Yang, M. Iwasaki, X. H. Wang, M. A. Waller, N. Shirai, Y. O. Abe, Y. Fujita, K. Watanabe, A. Kagita, K. A. Iwabuchi, M. Yasuda, H. Xu, T. Noda, J. Komano, H. Sakurai, N. Inukai and A. Hotta (2020). “Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping.” Nature Communications 11(1): 1334.