Press ReleaseNano-micelle Packaging CRISPR/Cas9 Components Enables
Brain Genome Editing
Summary:
The search of safe and efficient delivery methods of CRISPR/Cas9 is still ongoing. We
fabricated nano-micelle co-loading Cas9 mRNA and gRNA in the core, and proved for the
first time that RNA-based delivery of CRISPR/Cas9 induces genome editing in mousebrain parenchyma. This was possible because: first, co-loading gRNA with Cas9 mRNA
protected gRNA from degradation, and second, polyethylene glycol-covered nano-micelleallowed better diffusion in brain tissue.
March 10, 2021 – Kawasaki, Japan: The research group of Deputy Principal Research
Scientist Dr. Satoshi Uchida (Associate Professor, Kyoto Prefectural University of Medicine)at the Innovation Center of NanoMedicine (Director General: Prof. Kazunori Kataoka,
Location: Kawasaki-Japan, Abbreviation: iCONM) reported that optimized nano-micelles
can induce efficient genome editing in the mouse brain.
The 2020 Chemistry Nobel Prize-winning technology CRISPR/Cas9 holds great promisefor treating various diseases such as congenital disorders and viral infections, by correcting
the disease-specific genomic sequences. This technology attracted increasing attention in
the last decade as it allows easy targeting of genomic sequences with high precision andlow level of undesired off-target gene editing. However, safe and efficient delivery of theCas9 DNA cutting enzyme and guide RNA (gRNA), which is a short piece of RNA that guides
Cas9 to its genomic targets, remains an obstacle.
We fabricated nano-micelle for the delivery of Cas9-encoding messenger RNA (mRNA)and gRNA, which condenses these RNAs in the micelle core and forms a protective shell ofpolyethylene glycol. The efficiency of genome editing was evaluated in transgenic mice
which express a red fluorescent protein upon successful genome editing in the mouse brain.
Cas9 mRNA (4500 base) and gRNA (100 base) are largely different in size, and when eachof them was loaded separately in a nano-micelle, gRNA was rapidly released and degraded.

Interestingly, when both Cas9 mRNA and gRNA were loaded in same nano-micelle, thestability of gRNA was increased and this co-loaded nano-micelle allowed more efficientgenome editing in various brain cells, such as neurons, astrocytes and microglia (see figurebelow). To the best of our knowledge, this is the first report validating the utility of RNAbased introduction CRISPR/Cas9 in brain parenchyma. Additionally, the presence ofpolyethylene glycol on the surface of nano-micelle was found to play a pivotal role in diffusionin the brain tissue after injection, allowing to induce higher genome editing. This nanomicelle represents an efficient and safe tool that could be used for the treatment of genetic
brain diseases such as Huntington’s disease, and other neurodegenerative disorders suchas Alzheimer’s disease. Saed Abbasi et al published these results in “Journal of ControlledRelease” (Impact Factor: 7.727) on March 4th as follows:“Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome
editing in mouse brain”, Saed Abbasi, Satoshi Uchida et al., J. Controlled Release, 332,260-268 (2021). https://doi.org/10.1016/j.jconrel.2021.02.026

About Nanomicelle
Polymeric micelles were one of the first polymer self-assemblies reported as a nano-DDS, andare composed of distinct two domains, a drug-loading core and a hydrophilic shell. Amphiphilic
block copolymers, containing a hydrophilic block and a hydrophobic block, are firstly revealed
to construct those distinct domains in a micelle structure through spontaneous self-assemble asa result of hydrophobic interactions in aqueous.
H. Cabral, K. Miyata, K. Osada, K. Kataoka, “Block copolymer micelles in nanomedicineapplications” Chem. Rev. 2018, 118 6844-6892. (DOI: 10.1021/acs.chemrev.8b00199)

Public Interest Incorporated Foundation KAWASAKI INSTITUTE OF INDUSTRIAL PROMOTIONKAWASAKI INSTITUTE OF INDUSTRIAL PROMOTION was established in 1988 funded 100%from Kawasaki City for the purpose of coping with the hollowing out of industry and changes in thedemand structure.
In order to realize a higher level of market development, transforming R&D type companies, training
technological capabilities to support it, human resources development, understanding market needs,etc., by utilizing the functions of the Kawasaki, KAWASAKI INSTITUTE OF INDUSTRIALPROMOTION has been contributing to revitalize the local economy by promoting exchanges of local
industry information, advancing technology and corporate exchanges with establishment of a R&D
institutions, developing creative human resources through workshops and promoting businesses suchas expanding sales channels through exhibition business.
http://www.kawasaki-net.ne.jp/

Innovation Center of NanoMedicine (iCONM)
Innovation Center of NanoMedicine (iCONM) started its operation in April 2015 as a core research
center in life science field at King SkyFront on the request of Kawasaki city that KAWASAKIINSTITUTE OF INDUSTRIAL PROMOTION utilized national policies as a business operator andproposer.
It is a unique research center that the world has ever seen which is designed for the purpose ofpromoting open innovation through industry-academia-government/medical-engineeringcollaboration, prepared with state-of-the-art facilities and experimental equipment, that enables
comprehensive research and development from organic synthesis / microfabrication to preclinicaltesting.
https://iconm.kawasaki-net.ne.jp/en/

Center of Innovation Program (COI)The COI program is a research and development program under the Ministry of Education, Culture,Sports, Science and Technology and the Japan Science and Technology Agency. The program
employs the backcasting approach and set interdisciplinary and collaborative R&D themes thatshould be challenged at the present from the issues that are underlying in the future society. Eighteen
centers have been established nationwide to realize radical innovation through industry-academia
collaboration which cannot be accomplished by industry and academia alone.
The Kawasaki center is the only COI center managed by local governments, not universities, and theresearch projects carried out there are called COINS (Center of Open Innovation Network for Smart
Health).
COI: https://www.jst.go.jp/tt/EN/platform/coi.html
COINS: https://coins.kawasaki-net.ne.jp/en/
Kyoto Prefectural University of Medicine
In 2022, it will be 150 years since the Kyoto Prefectural University of Medicine, which was first
facilitated in Shoren-in Temple and has developed into the present organization, was founded.
Fundamental of our university is the education of professionals who have advanced specialized
knowledge. With the professional nurses and doctors, our research as a base, we provide world top
level of medicine to our region.
https://www.kpu-m.ac.jp/doc/english/

March 10, 2021