Summary:

The CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) gene-editing tool allows for sequence-specific cuts to genomic DNA, which can result in permanent silencing of a gene, or replacement of gene sequences. This has powerful implications for the treatment of diseases with a genetic origin. In this paper, the Siegwart Lab has identified several challenges to delivering CRISPR/Cas gene editing components to cells, which is crucial to its successful therapeutic development. To overcome these delivery challenges, the Siegwart Lab has developed a nanocarrier formulation based on a synthetic zwitterionic amino lipid (ZAL) compound to deliver the RNA-based tools needed for CRISPR/Cas activity. The Siegwart Lab used the NanoAssemblr™ Benchtop instrument to formulate their carrier material and RNA into nanoparticles for in vivo experiments. This constitutes the first demonstration of non-viral co-delivery of CRISPR/Cas components both in vitro and in vivo.

For CRISPR/Cas gene editing to work, both a Cas nuclease (ex: Cas9) and a short guide RNA (sgRNA) need to associate and be present in the nucleus where the guide strand can hybridize with the target sequence and allow the Cas nuclease to make a cut in the genome.  In vivo, this is typically achieved by transduction using an adeno-associated virus (AAV) that results in expression of Cas9 and a guide strand RNAs from viral DNA. Drawbacks of AAV transduction include possible immunogenicity that hinders repeat treatment, possible genome integration that would result in permanent expression of Cas9 and increase the risk of unintended genome edits, and limitations to the length of DNA that can be delivered to about 4500 – 4900 bases. The latter issue is important because it is desirable to co-deliver both a sequence encoding the Cas nuclease (~4500 bases) and one or more guide strands at once to ensure cells receive all the components needed for the desired edits.  This leaves little room for other regulatory elements. Furthermore, packaging custom DNA into AAVs is much more labourious and time consuming than formulating gene-delivery nanoparticles on the NanoAssemblr platform. Custom AAV preparation takes several weeks, while NanoAssemblr formulation can be accomplished in less than a day.

To demonstrate the effectiveness of their approach, the Siegwart Lab co-delivered mRNA encoding Cas9 and a guide strand to activate a reporter molecule in a transgenic mouse model.  The transgenic mouse model contains a floxed Stop cassette upstream of a tdTomato reporter homozygously in every cell. This is a challenging proof of concept because two cuts are needed to delete the Stop cassette and allow the reporter to be expressed. The ZAL nanoparticles formulated on the NanoAssemblr Benchtop containing both Cas9 mRNA and guide RNA – either LoxP or control – were injected intravenously at a 5mg/kg dose. Reporter activity was assayed by whole organ ex vivo imaging one week and two months after treatment. The reporter was detected in liver, kidneys and lungs at both time points demonstrating the long-term effects. No reporter activity was detected in the control group and no significant change in body weight was observed in either group. These findings represent proof-of-concept for co-delivering Cas9 mRNA and guide RNA in a single synthetic carrier that can be applied in vitro and in vivo. This has the potential to simplify the process of developing CRISPR/Cas treatments and cures for many genetic disorders.

Abstract:

CRISPR/Cas is a revolutionary gene editing technology with wide‐ranging utility. The safe, non‐viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95% decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro(<600 pM) and in vivo (1 mg kg⁻¹). Intravenous co‐delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.