Date: 11th September 2019
Unlocking a broad range of clinical therapeutic applications using an efficient, biodegradable CRISPR-Cas-9 delivery system.
CRISPR-Cas9 is the current most favoured gene editing tool. Its therapeutic potential is gathering momentum and, with the route to FDA approval under way, we reported back in February the first human to be treated with a CRISPR gene-edited therapy.
One key success factor in bringing this revolutionary tool to the clinical market will be the design and optimisation of an appropriate delivery system. Currently viral vectors can potentially be used but they do pose safety concerns. Alternatives systems such as liposomes or nanorobots have hit the news more recently however, limitations such as reliability of cargo loading, toxicity and poor stability still remain an issue.
Now scientists from the University of Wisconsin–Madison, US, have designed customisable synthetic nanoparticles for the delivery of Cas9 nuclease and a single-guide RNA (sgRNA). A system that enables a controlled stoichiometry of CRISPR components and so limits some of the safety concerns in vivo.
A ribonucleoprotein (RNP) is a complex of ribonucleic acid and RNA-binding protein. To achieve accurate, precise gene editing the authors preassembled a Cas9 nuclease and a single-guide RNA (sgRNA), RNP complex, which displayed a heterogenous surface charge. Then a ‘coat’ or nanocapsule (NC) was created to encase the RNP cargo by utilising the surface charge of the complex and a mixture of cationic and anionic monomers. A water-soluble shell containing specific ligands – with differing affinities for specific cell types – could then be complexed to the nanocapsules allowing them to hone-in on specific cell types in vitro and in vivo.
Part of the design of these nanocapsules included extra-cellular stability to enable them to reach their target cell types, however, once internalized, the biodegradable covalent links within the coating were broken down and the RNP complex entered the nucleus and was then available for gene editing.
Data showed that the NC efficiently generated targeted gene edits in vitro with little evidence of cytotoxicity.
When compared with other commercially available delivery vehicles, NCs functionally out-performed rival technologies. Further experiments in vivo, led to successful gene editing in murine retinal pigment epithelium tissue and skeletal muscle.
The strength of NCs lies in their small size and the ability to modulate their coat components – such as ligand incorporation and specialised monomers – so that they are highly modular and customisable.
Showing low cytotoxity, and a relatively short life-span inside the cell, it is hoped that off-target effects will be reduced. To add to the attraction of NCs they retain their biological functions after freeze-drying, allowing for long-term storage and transport.
The nanocapsule is certainly one technology we will be keeping an eye out for in the months that follow. Further tailoring could see it efficiently deliver gene editing machinery in a wide range of applications, and perhaps we will be looking at its use in the clinic in the not so distant future. The question of whether NCs can be used a delivery system for other cargo will also be an interesting development, one which may rely of the surface charge of the payload being compatible.
Chen, G., A. A. Abdeen, Y. Wang, P. K. Shahi, S. Robertson, R. Xie, M. Suzuki, B. R. Pattnaik, K. Saha and S. Gong (2019). “A biodegradable nanocapsule delivers a Cas9 ribonucleoprotein complex for in vivo genome editing.” Nature Nanotechnology.