By Adrien Morin
Publication (December 2021)
Kenjo, E., Hozumi, H., Makita, Y. et al.
Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice.
Nat Commun 12, 7101 (2021). https://doi.org/10.1038/s41467-021-26714-w. PMID: 34880218; PMCID: PMC8654819
Nowadays, therapies for the fatal X-linked progressive disease, Duchenne muscular dystrophy, hold great promise, particularly exon skipping strategies aiming to restore the open reading frame, either at the genomic DNA level through CRISPR-Cas9, or at the RNA level with antisense oligonucleotides (ASO). The size of ASO molecules allows body wide targeting through intravenous injection, however, the transient effects of ASOs lead to the need for lifelong injections. On the contrary, CRISPR-Cas9 modify the genomic DNA with in theory long-lasting effect, however, the AAV vectors commonly used for delivery have some restrictions. Indeed, repetitive administrations are not possible due to acquired immunity while the quantity for one unique effective injection is in theory too high.
Recently, non-viral distribution systems such as lipids nanoparticles (LNPs) have been successfully used to transport siRNA and mRNA as a treatment for various conditions. LNPs are chemical compounds generally consisting of 4 lipids encapsulating RNA, the delivery is provided by the presence of an ionizable lipid: uncharged LNP can bind to the cell surface, after internalization the ionizable lipid is cationized, thus breaking the endosome membrane and releasing RNA molecules into the cytoplasm. The use of LNPs to transport Cas9 and sgRNA has been demonstrated to target liver, muscle and brain in mice, however, this technology had not yet been characterized for broad muscle and repetitive injections.
In this study, Kenjo et al. optimized the structure of LNP to successfully deliver Cas9 and sgRNA into mouse muscle. They also confirmed that the co-transfection of LNP-Cas9 mRNA and LNP-hEx45 sgRNA #1 (sgRNA targeting the splicing acceptor site of DMD exon 45) + sgRNA #23 (sg RNA targeting the donor site of exon 45 in humans) cause exon 45 skipping and restoration of dystrophin expression in myoblasts derived from DMD patients presenting a deletion of exon 44.
Moreover, in order to test exon 45 skipping in vivo, the authors generated a new mouse model of DMD, the hEx45KI-mdx44 mice. The latter were produced by replacing wild-type mouse Dmd exon 45 via the knock-in of human exon 45 in addition to deleting the mouse exon 44. With this mouse, they showed that LNP-Cas9 mRNA delivery in combination with the dual sgRNA allowed around 10% of exon skipping and 1.3 % of dystrophin protein restoration which was sustained up to 1 year after the initial intramuscular injection. As opposed to ASO injection which showed up to 15% of exon skipping efficiency and 7% of dystrophin protein restoration but presented a disappearance of exon skipping and protein restoration level after 6 months. In addition, they demonstrated the feasibility of intravenous limb perfusion as a means to treat multiple muscles compared to intramuscular injection.
Finally, using a reporter mouse model for a ubiquitously expressed luciferase gene disrupted by the insertion of hEx45, they were able to detect luciferase bioluminescence after a first and second, 28 days later, injection of LNPs as opposed to AAV vectors.
To conclude they show a cumulative effect of LNP injections, up to 6 consecutive injections, on exon skipping efficiency and dystrophin restoration level in the hEx45KI-mdx44 mice proving that the low immunogenicity of LNPs allows repeated administration.
In this study, the authors describe in detail the potential therapeutic values of LNPs as a CRISPR-Cas9 delivery system for dystrophin restoration in DMD. Indeed, the low immunogenicity of LNP mediated CRISPR-Cas9 is a hope for long-term and stable dystrophin restoration for patients. Further in-depth research of this new delivery system is however necessary to evaluate the possibility of body-wide dystrophin restoration as well as efficacy and safety of LNPs.
Eriya Kenjo is a principal scientist at Takeda Pharmaceutical Company Limited and a member of T-CiRA Discovery and Innovation which runs a joint research program by the Center for iPS Cell Research and Application (CiRA), Kyoto University and Takeda Pharmaceutical Company Limited.
He is currently working on genome editing therapy for Duchenne muscular dystrophy (DMD) and is responsible for lipid nanoparticle formulation to deliver genome editing tools.
His research interests focus on genome editing therapy, gene therapy and non-viral delivery technologies.
He holds a master’s degree from the University of Shizuoka Graduate School of Pharmaceutical Sciences where he studied nucleic acid delivery using liposomes modified with tumor-targeting peptides for cancer therapy.
Adrien Morin: After a master’s degree in Genome and Cells differentiation in Paris I started my Ph.D. under the supervision of Pr. Helge Amthor at Paris-Saclay University. My project aims to characterize how the sarcolemmal dystrophin distribution can impact the response to therapy in our Duchenne muscular dystrophy mouse model.
Published on 21 April 2022.
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This article is presented by the Publication Highlights Committee.