Adenoviral vectors encoding CRISPR/Cas9 multiplexes rescue dystrophin synthesis in unselected populations of DMD muscle cells.
Adenoviral vectors encoding CRISPR/Cas9 multiplexes rescue dystrophin synthesis in unselected populations of DMD muscle cells.
复制标题
编码CRISPR/CAS9的腺病毒载体在未选择的DMD肌肉细胞中挽救肌营养不良蛋白的合成。
DOI：
10.1038/srep37051
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发表时间：
2016-11-15
影响因子：
4.6
通讯作者：
Gonçalves MA
中科院分区：
文献类型：
--
作者：
Maggio I;Liu J;Janssen JM;Chen X;Gonçalves MA
破坏约 2.4 Mb 抗肌营养不良蛋白编码 DMD 基因阅读框的突变会导致致命的 X 连锁肌肉萎缩症，称为杜氏肌营养不良症 (DMD)。基于 CRISPR/Cas9 系统的成对 RNA 引导核酸酶 (RGN) 的基因组编辑已被提议用于永久修复有缺陷的 DMD 基因座。然而，这种多重策略需要开发和测试能够将各种基因编辑工具引入靶细胞的递送系统。在这里，我们通过包装在编码化脓性链球菌 Cas9 核酸酶和序列特异性 gRNA 对的单个载体颗粒表达单元中，研究了腺病毒载体 (AdV) 用于多重 DMD 编辑的适用性。这些 RGN 组件经过定制，可触发短程和长程基因内 DMD 切除，包括患者来源的肌肉祖细胞中的阅读框破坏外显子。通过允许各种 RGN 成分的同步和化学计量表达，我们证明双重 RGN 编码 AdV 可以纠正超过 10% 的目标 DMD 等位基因，从而轻松检测未选择的肌肉细胞群中的 Becker 样肌营养不良蛋白。此外，我们报告基于 AdV 的基因编辑可以定制用于消除位于超过 500 kb 主要 DMD 突变热点内的突变。因此，这种单一的 DMD 编辑策略原则上可以解决 60% 以上 DMD 患者中存在的广泛突变。
Mutations disrupting the reading frame of the ~2.4 Mb dystrophin-encoding DMD gene cause a fatal X-linked muscle-wasting disorder called Duchenne muscular dystrophy (DMD). Genome editing based on paired RNA-guided nucleases (RGNs) from CRISPR/Cas9 systems has been proposed for permanently repairing faulty DMD loci. However, such multiplexing strategies require the development and testing of delivery systems capable of introducing the various gene editing tools into target cells. Here, we investigated the suitability of adenoviral vectors (AdVs) for multiplexed DMD editing by packaging in single vector particles expression units encoding the Streptococcus pyogenes Cas9 nuclease and sequence-specific gRNA pairs. These RGN components were customized to trigger short- and long-range intragenic DMD excisions encompassing reading frame-disrupting exons in patient-derived muscle progenitor cells. By allowing synchronous and stoichiometric expression of the various RGN components, we demonstrate that dual RGN-encoding AdVs can correct over 10% of target DMD alleles, readily leading to the detection of Becker-like dystrophin proteins in unselected muscle cell populations. Moreover, we report that AdV-based gene editing can be tailored for removing mutations located within the over 500-kb major DMD mutational hotspot. Hence, this single DMD editing strategy can in principle tackle a broad spectrum of mutations present in more than 60% of patients with DMD.
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DOI：
10.1038/mt.2015.164
发表时间：
2016-03
期刊：
Molecular therapy : the journal of the American Society of Gene Therapy
影响因子：
--
作者：
Chen X;Gonçalves MA
通讯作者：
Gonçalves MA
影响因子：
17.3
作者：
Gaj, Thomas;Gersbach, Charles A.;Barbas, Carlos F., III
通讯作者：
Barbas, Carlos F., III
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B
TREAT-NMD DMD 全球数据库：分析 7,000 多个杜氏肌营养不良症突变。
影响因子：
3.9
作者：
Bladen, Catherine L.;Salgado, David;Monges, Soledad;Foncuberta, Maria E.;Kekou, Kyriaki;Kosma, Konstantina;Dawkins, Hugh;Lamont, Leanne;Roy, Anna J.;Chamova, Teodora;Guergueltcheva, Velina;Chan, Sophelia;Korngut, Lawrence;Campbell, Craig;Dai, Yi;Wang, Jen;Barisic, Nina;Brabec, Petr;Lahdetie, Jaana;Walter, Maggie C.;Schreiber-Katz, Olivia;Karcagi, Veronika;Garami, Marta;Viswanathan, Venkatarman;Bayat, Farhad;Buccella, Filippo;Kimura, En;Koeks, Zaida;van den Bergen, Janneke C.;Rodrigues, Miriam;Roxburgh, Richard;Lusakowska, Anna;Kostera-Pruszczyk, Anna;Zimowski, Janusz;Santos, Rosario;Neagu, Elena;Artemieva, Svetlana;Rasic, Vedrana Milic;Vojinovic, Dina;Posada, Manuel;Bloetzer, Clemens;Jeannet, Pierre-Yves;Joncourt, Franziska;Diaz-Manera, Jordi;Gallardo, Eduard;Karaduman, A. Ayse;Topaloglu, Haluk;El Sherif, Rasha;Stringer, Angela;Shatillo, Andriy V.;Martin, Ann S.;Peay, Holly L.;Bellgard, Matthew I.;Kirschner, Jan;Flanigan, Kevin M.;Straub, Volker;Bushby, Kate;Verschuuren, Jan;Aartsma-Rus, Annemieke;Beroud, Christophe;Lochmueller, Hanns
通讯作者：
Lochmueller, Hanns