2017 Fiscal Year Research-status Report
CRISPR Cas9 protein delivery via virus-like particles for in vivo treatment of Duchenne muscular dystrophy
| Project/Area Number |
17K15048
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| Research Institution | Kyoto University |
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Principal Investigator |
Gee PeterDavid 京都大学, iPS細胞研究所, 特定研究員 (00754227)
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| Project Period (FY) |
2017-04-01 – 2019-03-31
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| Keywords | CRISPR Cas9 / Delivery / Virus-like particles |
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| Outline of Annual Research Achievements |
Research is progressing as planned. We have resolved the sgRNA packaging and have now obtained all-in-one Virus-like-particles containing the full Ribonucleotide protein complex of Cas9 protein and sgRNA. We have shown that these VLPs can be inoculated onto mouse C2C12 and Duchenne muscular dystrophy patient iPS cells. VLPs can be multiplexed to remove a genomic DNA region. The delivery of our VLPs versus DNA plasmid delivery of CRISPR Cas9 has over 7 times lower off-target cleavage which shows that they may be safer than other DNA vector delivering systems such as AAV. We compared our system to other delivery systems that have been published or are commercially available and our system dramatically outperforms all current RNP VLP delivery systems. Our next step is to characterize the VLPs, scale up the production, and look to purify them for further use in vivo.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The VLP delivery system is being assessed for toxicity and we are currently working on ways to increase the scale up of the VLPs using a suspension HEK293T system for producing higher yields. We are delivering the VLPs on various cell types including mouse myoblast progenitor cells. Also, we are quantifying the Cas9 amount in the VLPs. The development of alternative orthologue CRISPR nuclease incorporation is ongoing and we are cloning the plasmids now. Moreover, our system has lower off-target effects compared with DNA delivery of CRISPR Cas9.
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| Strategy for Future Research Activity |
Currently, we are planning in vivo studies in humanized DMD mice to deliver our VLPs and restore dystrophin expression. Genomic DNA, mRNA, and protein will be examined for dystrophin restoration. Moreover, muscle strength in the mice will be evaluated to assess whether the disease phenotype can be improved. Initially, we will look at the delivery of Luciferase by VLPs first to see if they can be delivered and visualized in vivo. Next we will deliver the VLPs containing CRIPSR Cas9 and sgRNA for inducing the exon skipping for the treatment of DMD.
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| Causes of Carryover |
I was planning on using money on travel but I did not attend an overseas conference. The money was used to hire an assistant for pushing my research forward. More costs were used to buy special software and equipment needed for research needs.
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Research Products
(2 results)
