Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride

Takizawa, Hotake; Hara, Yuko; Mizobe, Yoshitaka; Ohno, Taisuke; Suzuki, Sadafumi; Inoue, Ken; Takeshita, Eri; Shimizu‐Motohashi, Yuko; Ishiyama, Akihiko; Hoshino, Mikio; Komaki, Hirofumi; Takeda, Shin’ichi; Aoki, Yosuke

doi:10.1038/s41598-019-40421-z

Cited by 21 publications

(29 citation statements)

References 31 publications

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“…The primers should generate products that can easily be distinguished by size on a MCE system or normal agarose gel electrophoresis if MCE is not available. 16 . Thus, only urine collected from the patient is required to generate the myoblasts, and no invasive procedure is necessary.…”

Section: Representative Resultsmentioning

confidence: 99%

Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides

Nordin

Mizobe

Nakamura

et al. 2020

JoVE

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Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations often cause a distorted reading frame and premature stop codons, resulting in an almost total lack of dystrophin protein. The reading frame can be corrected using antisense oligonucleotides (AONs) that induce exon skipping. The morpholino AON viltolarsen (code name: NS-065/NCNP-01) has been shown to induce exon 53 skipping, restoring the reading frame for patients with exon 52 deletions. We recently administered NS-065/NCNP-01 intravenously to DMD patients in an exploratory investigatorinitiated, first-inhuman trial of NS-065/NCNP-01. In this methods article, we present the molecular characterization of dystrophin expression using Sanger sequencing, RT-PCR, and western blotting in the clinical trial. The characterization of dystrophin expression was fundamental in the study for showing the efficacy since no functional outcome tests were performed.

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Section: Representative Resultsmentioning

confidence: 99%

Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides

Nordin

Mizobe

Nakamura

et al. 2020

JoVE

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“…According to our previous report, we performed direct reprogramming of UDCs into myotubes [21,22]. In short, we transduced MYOD1 into patient-derived UDCs by a retroviral vector containing a doxycycline-inducible MYOD1 expression system, and differentiated MYOD1-transduced UDCs (MYOD1-UDCs) into myotubes by changing the growth medium to differentiation medium containing doxycycline and 5 μM 3-deazaneplanocin A hydrochloride.…”

Section: Myogenic Differentiation Of Myod1-transduced Udcsmentioning

confidence: 99%

“…We performed immunoblotting as described in our previous report [21]. Briefly, total protein was extracted from cultured cells using radio immunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche, Indianapolis, IN, USA).…”

Section: Immunoblottingmentioning

confidence: 99%

“…The most significant limitation of previous reports is the insufficient direct-reprogramming efficiency of UDCs into myotubes as an ideal muscle model of DMD. To overcome some of the challenges mentioned above, we recently reported efficient cellular skeletal muscle modeling of DMD by using MYOD1-UDCs obtained from patients with DMD [21,22]. Since the DMD skeletal muscle model could be induced efficiently in a short period of time and the protocol is straightforward, we expected this to be a new screening system for therapeutic agents instead of using rhabdomyosarcoma cells.…”

Section: Introductionmentioning

confidence: 99%

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Highly sensitive screening of antisense sequences for different types of DMD mutations in patients' urine-derived cells

Takizawa

Takeshita

Sato

et al. 2021

Journal of the Neurological Sciences

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Exon skipping using short antisense oligonucleotides (AONs) is a promising treatment for Duchenne muscular dystrophy (DMD). Several exon-skipping drugs, including viltolarsen (NS-065/NCNP-01), have been approved worldwide. Immortalized human skeletal muscle cell lines, such as rhabdomyosarcoma cells, are frequently used to screen efficient oligonucleotide sequences. However, rhabdomyosarcoma cells do not recapitulate DMD pathophysiology as they express endogenous dystrophin. To overcome this limitation, we recently established a direct human somatic cell reprogramming technology and successfully developed a cellular skeletal muscle DMD model by using myogenic differentiation 1 (MYOD1)-transduced urine-derived cells (MYOD1-UDCs). Here, we compared in vitro drug screening systems in MYOD1-UDCs and rhabdomyosarcoma cells. We collected UDCs from patients with DMD amenable to exon 51 skipping, and obtained MYOD1-UDCs. We then compared the efficiency of exon 51 skipping induced by various morpholino-based AONs, including eteplirsen in differentiated MYOD1-UDCs (UDC-myotubes) and rhabdomyosarcoma cells. Exon skipping was induced more efficiently in UDCmyotubes than in rhabdomyosarcoma cells even at a low AON concentration (1 μM). Furthermore, exon 51 skipping efficiency was higher in UDC-myotubes with a deletion of exons 49-50 than in those with a deletion of exons 48-50, suggesting that the skipping efficiency may vary depending on the DMD mutation pattern. An essential finding of this study is that the sequence of eteplirsen consistently leads to much lower efficiency than other sequences. These findings underscore the importance of AON sequence optimization by our cellular system, which enables highly sensitive screening of exon skipping drugs that target different types of DMD mutations.

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“…Lastly, the authors assessed if the model was suitable for exon-skipping studies in USCs derived from DMD patients. They showed that AONs targeting DMD exons, including 44, 50, 51 and 55, induced the skipping of the specific targeted exon [31].…”

Section: Application Of Urine-derived Stem Cells In Genetic Diseasesmentioning

confidence: 99%

Urinary Stem Cells as Tools to Study Genetic Disease: Overview of the Literature

Falzarano

Ferlini

2019

JCM

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Urine specimens represent a novel and non-invasive approach to isolate patient-specific stem cells by easy and low-cost procedures, replacing the traditional sources (muscle/skin biopsy/adipose tissue) obtained with invasive and time-consuming methods. Urine-derived stem cells (USCs) can be used in a broad field of applications, such as regenerative medicine, cell therapy, diagnostic testing, disease modelling and drug screening. USCs are a good source of cells for generating induced pluripotent stem cells (iPSCs) and importantly, they can also be directly converted into specific cell lines. In this review, we show the features of USCs and their use as a promising in vitro model to study genetic diseases.

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Modelling Duchenne muscular dystrophy in MYOD1-converted urine-derived cells treated with 3-deazaneplanocin A hydrochloride

Cited by 21 publications

References 31 publications

Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides

Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides

Highly sensitive screening of antisense sequences for different types of DMD mutations in patients' urine-derived cells

Urinary Stem Cells as Tools to Study Genetic Disease: Overview of the Literature

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