197

Center for Gene & Cell Therapy

Division of Molecular and Medical Genetics
分子遺伝医学分野
Professor
Project Senior Assistant Professor
Assistant Professor
Project Assistant Professor
Project Assistant Professor
Project Assistant Professor

Takashi Okada, M.D., Ph.D.
Yasunari Matsuzaka, Ph.D.
Yuji Tsunekawa, Ph.D.
Yuko Nitahara-Kasahara, Ph.D.
Hiromi Hayashita-Kinoh, Ph.D.
Ken Sugo, Ph.D.

教 授
特任講師
助 教
特任助教
特任助教
特任助教

博士（医学）
博士（医学）
博士（医学）
博士（医学）
博士（医学）
博士（工学）

岡松恒笠喜菅田坂川原納生

尚恭雄優裕巳成二子美健

In the Division of Molecular and Medical Genetics (DMMG), we focus on thedevelopment of virus vector-related technology, development of treatments for rare
intractable diseases, development of next-generation AAV vaccines, andidentification of biomarkers for Duchenne muscular dystrophy (DMD). A large-scale
short-term purification method for functional full-genome AAV particles wasestablished using two-step cesium chloride density-gradient ultracentrifugation witha zonal rotor. In addition, amnion-derived messenchymal stromal cells weresuggested to be promising tools for the treatment of DMD due to theirimmunomodulatory properties. Furthermore, the biomarkers we found weresuggested to be valuable for evaluating disease progression and monitoring
treatment response.

Virus vector-related technology development
With a final goal of commercializing improved adeno-associated virus (AAV) vectors, we are developing functionally enhanced host cells for vector production, investigating expression culture methods
involving bioreactors, and analyzing the efficient secretion of virus particles into the cell culture supernatant. While AAV vectors have attracted attention for
their clinical utility, issues remain regarding theirsafety, particularly their immunogenicity. Therefore,
it is desirable to establish a method for manufacturinghigh-quality and safe AAVs in large quantities, quickly, and at low cost. To resolve these issues, a largescale short-term purification method for functional
full-genome AAV particles was established using
two-step cesium chloride density-gradient ultracentrifugation with a zonal rotor. Another difficulty isthat viruses are generally stored at freezing temperatures reaching -80°C after production, which poseschallenges such as difficulty in quality control andpreserving their biological activity due to repeatedfreezing and thawing. To address this, we succeededin maintaining the activity of AAV by spraying theliquid condition purified AAV in a vacuum and immediately freeze-drying it into a powder, which does
not revert even after returning to room temperature,making quality control easier. In addition, tangential
flow filtration (TFF) is a widely used method for the
purification of adeno-associated virus (AAV) vectors.
The use of TFF for AAV vector purification has beenshown to be rapid, scalable, and cost-effective, making it a valuable technique in gene therapy and vaccine manufacturing.
Development of treatment methods for rare intractable diseases
The unique characteristics of amnion-derived
mesenchymal stromal cells (AMSCs) make them a

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clinically viable cell source for various applications.
These features include non-invasive cell isolation, mitotic stability, ethical acceptability, low immunogenicity, and low risk of tumorigenesis. AMSCs havebeen studied for their potential in the treatment ofconditions such as Duchenne muscular dystrophy(DMD) due to their immunomodulatory properties
and therapeutic benefits. Additionally, the immunosuppressive effects of human AMSCs have been already demonstrated, further supporting their potential for therapeutic applications.
Development of next-generation AAV vaccine
The development of a vaccine can be a lengthy
process, typically taking several years to complete.
However, due to the urgent need for COVID-19 vaccines, unprecedented financial investments and scientific collaborations are changing how vaccines are developed, for example with some clinical trialsevaluating multiple vaccines simultaneously. The establishment of the Strategic Center of Biomedical Advanced Vaccine Research and Development for Preparedness and Response (SCARDA) is part of Japan's
efforts to modernize and improve the government's
pandemic preparedness and response capabilities.
Research has shown that AAV-vectored vaccines havedemonstrated advantages such as thermostability,
high efficiency, safety, and the ability to induce robust immune responses. These vaccines have been
designed to avoid biased immune responses and mutational immune escape, making them a promisingavenue for future vaccine development. Extracellularvesicle (EV)-based vaccines have also recently received attention in vaccine development. Accordingly, we are also conducting research on extracellular
vesicle-based AAV-vectored vaccines against viruses,
including SARS-CoV-2. In addition, the “Japan Initia-

tive for World-leading Vaccine Research and Development Center” has been launched, and the University of Tokyo Pandemic preparedness, infection andAdvanced research center (UTOPIA) has been selected as the flagship center. At UTOPIA, we are working
to develop the use of empty particles as viral vectors.
Search for biomarkers in Duchenne muscular
dystrophy
Duchenne muscular dystrophy (DMD) is a genetic
disorder characterized by progressive muscle degeneration and weakness due to the alterations of a protein called dystrophin. Unfortunately, there is currently no known treatment that halts the progressionof the disease and available treatments are only palliative. Patients usually die in their twenties due to respiratory muscle weakness or cardiomyopathy. Biomarkers play a crucial role in the clinical managementof Duchenne muscular dystrophy (DMD). We havebeen working to identify proteomic and metabolomic
biomarkers for DMD, to monitor the disease progression and the effects of novel treatment strategies.
These biomarkers are valuable for evaluating diseaseprogression, monitoring treatment response, and improving the success of clinical trials in DMD. In addition, we have identified several microRNAs (miRNAs) as potential biomarkers for Duchenne muscular
dystrophy (DMD). These small non-coding RNAs
have shown promise as diagnostic and prognostic
markers for DMD. Dysregulation of serum circulating miRNAs has been observed in DMD, suggesting
their potential utility in monitoring the disease progression and response to therapy. While further research is needed to validate the clinical utility of thesemiRNAs as biomarkers, current evidence supports
their potential use in the management of Duchenne
muscular dystrophy.

Publications
Hirohata K, Yamaguchi Y, Maruno T, Shibuya R, Torisu T, Onishi T, Chono H, Mineno J, Yuzhe Y, Higashiyama K, Masumi-Koizumi K, Uchida K,Yamamoto T, Uchida E, Okada T, Uchiyama S. Applications and Limitations of Equilibrium Density
Gradient Analytical Ultracentrifugation for the
Quantitative Characterization of Adeno-AssociatedVirus Vectors. Anal Chem. doi: 10.1021/acs.analchem.3c01955. Online ahead of print. 2024 Jan 2.
Yoshida K, Tsunekawa Y, Kurihara K, Watanabe K,
Makino-Manabe Y, Wada M, Tanaka T, Ide T, Okada T. Engineering a highly durable adeno-associated virus receptor for analytical applications. MolTher Methods Clin Dev. 2023 31: 101157 doi.
org/10.1016/j.omtm.2023.101157. Dec 14.
Arakawa M, Sakamoto Y, Nito C, Takahashi S, Nitahara-Kasahara Y, Suda S, Yamazaki Y, Sakai M, Ki-mura K, Okada T, Miyagawa Y. Induced pluripotent stem cell-derived mesenchymal stem cells
attenuate cerebral ischemia-reperfusion injury via
anti-inflammation and anti-oxidative stress in rats.
Mol Ther Methods Clin Dev. 2023 Jul 15:30:333-349.
doi: 10.1016/j.omtm.2023.07.005. eCollection 2023
Sep 14.
Miyaoka R, Tsunekawa Y, Kurosawa Y, Sasaki T, Onodera A, Sakamoto K, Kakiuchi Y, Wada M, Nitahara-Kasahara Y, Hayashita-Kinoh H, Okada T. Development of a novel purification method for AAV
vectors using tangential flow filtration. Biotechnol
Bioeng., 2023 Nov;120(11):3311-3321. doi: 10.1002/bit.28524. Epub 2023 Aug 16.
Maruyama M, Sakai A, Fukunaga T, Miyagawa Y,Okada T, Hamada M, Suzuki H. Neat1 lncRNA organizes the inflammatory gene expressions in the199
dorsal root ganglion in neuropathic pain caused bynerve injury. Front. Immunol. 14:1185322. doi.:
10.3389/fimmu.2023.1185322. 2023 Aug 8
Li D, Johmura Y, Morimoto S, Doi M, Nakanishi K,Ozawa M, Tsunekawa Y, Inoue-Yamauchi A,Naruse H, Matsukawa T, Takeshita Y, Suzuki N,Aoki M, Nishiyama A, Zeng X, Konishi C, Suzuki
N, Nishiyama A, Harris AS, Morita M, Yamaguchi
K, Furukawa Y, Nakai K, Tsuji S, Yamazaki S,Yamanashi Y, Shimada S, Okada T, Okano H, Toda
T, Nakanishi M. LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes
late-onset neurological deficits. Nat Aging doi:
10.1038/s43587-023-00464-4. Online ahead of print
2023 Jul 20
Nitahara-Kasahara Y, Nakayama S, Kimura K, Yamaguchi S, Kakiuchi Y, Nito C, Hayashi M, Nakaishi T,Ueda Y, Okada T. Immunomodulatory amnion
mesenchymal stem cells preserve muscle function
in a mouse model of Duchenne muscular dystrophy. Stem Cell Res Ther. 14(1):108. doi: 10.1186/
s13287-023-03337-0. 2023 Apr 27

Wada M, Uchida N, Posadas-Herrera G, Hayashita-Kinoh H, Tsunekawa Y, Hirai Y, Okada T. Largescale purification of functional AAV particles packaging the full genome using short-term
ultracentrifugation with a zonal rotor. Gene Ther.
doi: 10.1038/s41434-023-00398-x, Online ahead ofprint 2023 Mar 28
Pilaz LJ, Liu J, Joshi K, Tsunekawa Y, Musso CM,
D'Arcy BR, Suzuki IK, Alsina FC, Kc P, Sethi S, Vanderhaeghen P, Polleux F, Silver DL. SubcellularmRNA localization and local translation of Arhgap11a in radial glial progenitors regulates corticaldevelopment. Neuron 111(6):839-856.e5. doi:
10.1016/j.neuron.2023.02.023. 2023 Mar 15
Hashimoto A, Hirose T, Hashimoto K, Mizumoto S,
Nitahara-Kasahara Y, Saka S, Yoshizawa T, Okada
T, Yamada S, Kosho T, Watanabe T, Miyata S, Nomura Y. Collagen network formation in in vitro
models of musculocontractural Ehlers-Danlos syndrome. Genes 14(2):308. doi: 10.3390/genes14020308.
2023 Jan 24