Identification of novel antisense-mediated exon skipping targets in DYSF for therapeutic treatment of dysferlinopathy

Dysferlinopathy is a progressive myopathy caused by mutations in the dysferlin (DYSF) gene. Dysferlin protein plays a major role in plasma-membrane resealing. Some patients with DYSF deletion mutations exhibit mild symptoms, suggesting some regions of DYSF can be removed without significantly impacting protein function. Antisense-mediated exon-skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Previous studies have focused on DYSF exon 32 skipping as a potential therapeutic approach, based on the association of a mild phenotype with the in-frame deletion of exon 32. To date, no other DYSF exon-skipping targets have been identified, and the relationship between DYSF exon deletion pattern and protein function remains largely uncharacterized. In this study, we utilized a membrane-wounding assay to evaluate the ability of plasmid constructs carrying mutant DYSF, as well as antisense oligonucleotides, to rescue membrane resealing in patient cells. We report that multi-exon skipping of DYSF exons 26–27 and 28–29 rescues plasma-membrane resealing. Successful translation of these findings into the development of clinical antisense drugs would establish new therapeutic approaches that would be applicable to ∼5%–7% (exons 26–27 skipping) and ∼8% (exons 28–29 skipping) of dysferlinopathy patients worldwide. Keywords: exon skipping, antisense, morpholino, dysferlin, dysferlinopathy, limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, distal myopathy, plasma membrane, membrane woundin

Clinical Guides for aHUS

Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. In 2013, we developed diagnostic criteria to enable early diagnosis and timely initiation of appropriate treatment for aHUS. Recent clinical and molecular findings have resulted in several proposed classifications and definitions of thrombotic microangiopathy and aHUS. Based on recent advances in this field and the emerging international consensus to exclude secondary TMAs from the definition of aHUS, we have redefined aHUS and proposed diagnostic algorithms, differential diagnosis, and therapeutic strategies for aHUS

Quantitative antisense screening and optimization for exon 51 skipping in Duchenne muscular dystrophy

International audienceDuchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping

Genome-Wide Analysis Reveals a Major Role in Cell Fate Maintenance and an Unexpected Role in Endoreduplication for the Drosophila FoxA Gene Fork Head

Transcription factors drive organogenesis, from the initiation of cell fate decisions to the maintenance and implementation of these decisions. The Drosophila embryonic salivary gland provides an excellent platform for unraveling the underlying transcriptional networks of organ development because Drosophila is relatively unencumbered by significant genetic redundancy. The highly conserved FoxA family transcription factors are essential for various aspects of organogenesis in all animals that have been studied. Here, we explore the role of the single Drosophila FoxA protein Fork head (Fkh) in salivary gland organogenesis using two genome-wide strategies. A large-scale in situ hybridization analysis reveals a major role for Fkh in maintaining the salivary gland fate decision and controlling salivary gland physiological activity, in addition to its previously known roles in morphogenesis and survival. The majority of salivary gland genes (59%) are affected by fkh loss, mainly at later stages of salivary gland development. We show that global expression of Fkh cannot drive ectopic salivary gland formation. Thus, unlike the worm FoxA protein PHA-4, Fkh does not function to specify cell fate. In addition, Fkh only indirectly regulates many salivary gland genes, which is also distinct from the role of PHA-4 in organogenesis. Our microarray analyses reveal unexpected roles for Fkh in blocking terminal differentiation and in endoreduplication in the salivary gland and in other Fkh-expressing embryonic tissues. Overall, this study demonstrates an important role for Fkh in determining how an organ preserves its identity throughout development and provides an alternative paradigm for how FoxA proteins function in organogenesis

2017～2019年度 関西大学研究拠点形成支援経費研究成果報告書

目次・研究成果の概要・2-1　工藤 宏人・宮前 翼・上田 正人・村山 憲弘・林 順一 "ノーリア骨格をテンプレートとした空孔内に水酸基を有する架橋化合物の合成とそれらの金属イオン包接性能" ネットワークポリマー論文集 vol.41, No.2, 65 - 71 (2020).・2-2　Mitsuaki Matsuoka, Kaho Yokoyama, Kohei Okura, Norihiro Murayama, Masato Ueda, Makio Naito " Synthesis of Geopolymers from Mechanically Activated Coal Fly Ash and Improvement of Their Mechanical Properties" Minerals 9, 791- 801 (2019).・2-3　Daisuke Shimoyama, Ryo Sekiya, Hiroto Kudo, Takeharu Haino, "Feet-to-Feet Connected Trisresorcinarenes" Organic Letters 22, 352 - 356 (2019).・2-4　Masato Ueda, Masahiko Ikeda, Shigeo Mori, Kenji Doi, Hisashi Kitagaki, Shuntaro Terauchi "Mechanical Properties of Additively Manufactured Porous Titanium with Sub-Millimetre Structural Units" Materials Transactions Vol.60, No.9, 1792 - 1798 (2019).・2-5　五十井 浩平・白杉 文香・松岡 光昭・林 順一・村山 憲弘 "種々のMg-Fe系複合酸化物を用いた希薄水溶液中のホウ素およびヒ素の除去" 環境資源工学 66, 29 - 35 (2019).・2-6　Toru Maruyama, Mitsuyoshi Tamaki, Keisuke Nakamura, Gou Nakamura "EFFECT OF MOLTEN METAL TEMPERATURE ON MOLD FILLING IN EVAPORATIVE PATTERN CASTING" International Journal of Metalcasting 13, 611–617 (2019).・2-7　Ryuta Saito, Toru Maruyama, Toshiki Nakamura, Hitoshi Yanagitani, Takahiro Sakai, Kouji Nakamoto "Influence of Tellurium Addition to Spheroidal Graphite Cast Iron on the Number of Graphite Particles" International Journal of Metalcasting Vol.13, 3, 571-577 (2018).・2-8　Masato Ueda, Rika Yamaguchi, Chika Fujita, Masahiko Ikeda "Control of Cell Adhesion on Titanium Dioxide by Light Irradiation" Materials Science Forum Vol.941, 2507 - 2512 (2018).・2-9　Hiroto Kudo, Mari Fukunaga, Kohei Shiotsuki, Hiroya Takeda, Hiroki Yamamoto, Takahiro Kozawa, Takeo Watanabe "Synthesis of hyperbranched polyacetals containing C-(4-t-butylbenz)calix[4]resorcinarene: Resist properties for extreme ultraviolet (EUV) lithography" Reactive and Functional Polymers 131, 361 - 367 (2018).・2-10　大隈 修・前 一廣・林 順一 "直接液化による豪州ビクトリア褐炭の高度利用 : 改新BCLプロセスによる化学原料の生産" Journal of the Japan Institute of Energy 98, 17 - 26 (2019).・2-11　Issei Suzuki, Ayako Kakinuma, Masato Ueda, Takahisa Omata "Flux growth of β-NaGaO₂ single crystals" Journal of Crystal Growth 504, 26 -30 (2018).・2-12　上田 正人、坂本 貴則、池田 勝彦 "電気抵抗率の精密測定による純チタンの組織評価" 環境資源工学 65, 74 -76 (2018).・2-13　Satoshi Imasaka, Hiroyasu Ishii, Jun\u27ichi Hayashi, Sadao Araki, Hideki Yamamoto "Synthesis of CHA-type titanosilicate zeolites using titanium oxide as Ti source and evaluation of their physicochemical properties" Microporous and Mesoporous Materials 273, 243-248 (2019).・2-14　Hiroto Kudo, Shizuya Ohori, Hiroya Takeda, Hiroki Ogawa, Takeo Watanbe, Hiroki Yamamoto, Takahiro Kozawa "Synthesis and Property of Tannic Acid Derivatives and Their Application for Extreme Ultraviolet Laser Lithography System" Journal of Photopolymer Science and Technology Vol.31, 221 - 225 (2018).・2-15　Hiroto Kudo, Tsubasa Miyamae, Kouta Kitagawa, Kohei Isoi, Norihiro Murayama, Jun\u27ichi Hayashi " Synthesis and Metal-Complexation Ability of Cross-Linking Materials Containing Noria-Templated Cavities with Pendant Carboxylic Acid Groups" Chemistry Select 3, 2223 - 2228 (2018).・2-16　上田 正人、池田 勝彦、土井 研児、 森 重雄、北垣 壽、寺内 俊太郎、関 あずさ "骨部分置換用ポーラスチタン : ポリグリコール酸 : 炭酸カルシウム複合体の開発" 高分子論文集 Vol.75, No.1, 69 - 74 (2018).・2-17　Alexandru C Sonoc, Jacob Jeswiet, Norihiro Murayama, Junji Shibata "A study of the application of Donnan dialysis to the recycling of lithium ion batteries" Hydrometallurgy 175, 133 - 143 (2018).2-3は、著作権の関係により非公開としております。2-8は、著作権の関係により非公開としております。2-9は、著作権の関係により非公開としております。2-10は、著作権の関係により非公開としております。2-11は、著作権の関係により非公開としております。2-16は、著作権の関係により非公開としております