CRISPR/Cas9在iPSC衍生肌肉预祖中恢复肌营养不良素表达的技术
Summary
在这里，我们提出了基于Cas9的外显23删除协议，从Dmdmdx小鼠衍生的皮肤成纤维细胞恢复iPSC中的营养不良素表达，并使用Tet-on MyoD激活系统将iPSC直接分化为肌源性祖细胞（MPC）。
Abstract
Duchenne 肌肉营养不良症 （DMD） 是一种由肌营养不良基因突变引起的严重渐进性肌肉疾病，最终导致肌肉祖细胞衰竭。聚类定期间隔短血循环重复/CRISPR相关9（CRISPR/Cas9）基因编辑有可能恢复肌营养不良素基因的表达。自体诱导多能干细胞（iPSCs）衍生的肌肉祖细胞（MPC）可以补充干细胞/祖细胞池，修复损伤，防止DMD的进一步并发症，而不会引起免疫反应。在这项研究中，我们介绍了CRISPR/Cas9和非集成iPSC技术的组合，以获得肌祖具有恢复的营养不良蛋白表达。简单地说，我们使用非集成仙台载体从Dmdmdx小鼠的皮成纤维细胞中建立iPSC线。然后，我们使用CRISPR/Cas9删除策略，通过重新框住的营养不良基因的非同源端联接来恢复肌营养不良症表达。在PCR验证了94个精选iPSC菌落的三个菌落的外显酶消耗后，通过多氧环素（Dox）诱导的肌D表达将iPSC分化为MPC，这是一种关键转录因子，在调节肌肉分化方面起着重要作用。我们的结果表明，使用CRISPR/Cas9删除策略在iPSC衍生的MPC中恢复营养不良素表达的可行性，这为开发未来治疗DMD的疗法具有巨大潜力。
Introduction
杜氏肌营养不良症 （DMD） 是最常见的肌肉营养不良症之一，其特征是缺乏营养不良症，影响全球约 5，000 名新生儿中的 1 个 。肌营养不良基因功能的丧失导致结构肌肉缺陷，导致渐进性肌纤维退化1，2。重组腺相关病毒（rAAV）介导的基因治疗系统已经过测试，以恢复肌营养不良症的表达和改善肌肉功能，例如使用微营养不良症（μ-Dys）替换基因。然而，rAAV方法需要反复注射来维持功能蛋白3，4的表达。因此，我们需要一种策略，能够有效和永久地恢复DMD患者的营养不良症基因表达。Dmdmdx小鼠是 DMD 的小鼠模型，在肌营养不良素基因的外显子 23 中具有点突变，该基因引入过早终止通香，导致缺乏 C-端营养不良症结合域的非功能性截断蛋白。最近的研究表明，使用CRISPR/Cas9技术，通过精确的基因校正或突变外源删除在大小动物5，6，7恢复营养不良蛋白基因表达。Long等人8报告了通过同源定向修复（HDR）基于CRISPR/Cas9基因组编辑来纠正Dmdmdx小鼠生殖系中营养不良蛋白基因突变的方法。El Refaey等人9日报告说，rAAV可以有效地切除营养不良小鼠体内的突变外生23。在这些研究中，gRNA被设计在20和23的内源中，导致双链DNA断裂，通过非同源端连接（NHEJ）修复DNA后部分恢复肌营养不良症表达。更令人兴奋的是，Amoasii等人10最近报告了rAAV介导CRISPR基因编辑在恢复犬型肌营养不良蛋白表达的疗效和可行性，这是未来临床应用的重要一步。
DMD也导致干细胞紊乱11。对于肌肉损伤，住宅肌肉干细胞补充肌肉分化后死亡的肌肉细胞。然而，连续的损伤和修复周期导致肌肉干细胞12端粒缩短，干细胞池过早耗尽13，14。因此，将自体干细胞治疗与基因组编辑相结合，以恢复营养不良素表达，是治疗DMD的实用方法。CRISPR/Cas9 技术提供了产生自体基因校正诱导多能干细胞 （iPSC） 的可能性，用于功能性肌肉再生，并在不引起免疫排斥的情况下防止 DMD 的进一步并发症。然而，iPSCs有肿瘤形成的风险，可以通过iPSC分化为肌原祖细胞来缓解。
在此协议中，我们描述了使用非集成仙台病毒将Dmdx小鼠的皮肤成纤维细胞重新编程到iPSC中，然后通过CRISPR/Cas9基因组删除恢复肌营养不良素表达。通过基因分型验证iPSC中的Exon23缺失后，通过MyoD诱导的肌基分化，将基因组校正的iPSC分化为肌原原体（MPC）。
Protocol
所有动物处理和外科手术均由奥古斯塔大学机构动物护理和使用委员会（IACUC）批准的协议进行。老鼠被喂食标准饮食和水。 1. 从成年Dmdmdx小鼠中分离原虫成纤维细胞 根据奥古斯塔大学佐治亚医学院批准的IACUC，通过CO2窒息和胸腔切除术，将成年Dmdx小鼠（雄性，2个月大）安乐死。在层罩下无菌条件下，用无菌手术刀切割尾部。用70%乙醇冲洗尾…
Representative Results
建立Dmdx皮肤成纤维细胞衍生iPSC。 我们使用无集成的重新编程载体，演示了从Dmdmdx小鼠衍生的皮肤成纤维细胞生成小鼠iPSC的效率。图1A表明，胚胎干细胞（ESC）样菌落在感染后三周出现。通过活碱性磷酸酶（AP）染色评估iPSC诱导效率;图1B显示，根据FACS分析，AP阳性细胞的百分比约为1.8%。SSEA1、Lin28、Nanog、OCT4和SOX2是?…
Discussion
杜琴肌肉萎缩症（DMD）是一种破坏性的，最终致命的遗传性疾病，其特征是缺乏营养不良症，导致渐进性肌肉萎缩1，2。我们的结果表明，通过CRISPR/Cas9介导的外显23缺失方法，Dmdmdx iPSC衍生的造血原细胞中恢复了营养不良蛋白基因表达。这种方法有三个优点。
首先，我们使用非集成RNA载体从Dmdmdx小鼠衍生的皮肤成纤…
Divulgations
The authors have nothing to disclose.
Acknowledgements
唐和温特劳布部分得到NIH-AR070029、NIH-HL086555、NIH-HL134354的支持。
Materials
| Surgical Instruments | |||
| 31-gauge needle | Various | ||
| Sharp Incision | Various | ||
| Sterile Scalpels | Various | ||
| Tweezers | Various | ||
| Fibroblast medium (for 100 mL of complete medium) | Company | Catalog Number | Volume |
| 2-Mercaptoethanol (55 mM) | Gibco | 21-985-023 | 0.1 mL |
| Antibiotic Antimycotic Slution 100x | CORNING | MT30004CI | 1 mL |
| Dulbecco's Modified Eagle's Medium – high glucose | SIGMA | D6429 | 87 mL |
| Fetal Bovine Serum Characterized | HyClone | SH30396.03 | 10 mL |
| L-Glutamine solution | SIGMA | G7513 | 1 mL |
| MEM Non-Essential Amino Acids Solution (100x) | Gibco | 11140076 | 1 mL |
| TVP solution (for 500 mL of complete solution) | Company | Catalog Number | Volume |
| Chicken Serum | Gibco | 16110-082 | 5 mL |
| EDTA | Sigma-Aldrich | E6758 | 186 mg |
| Phosphat-buffered saline | to 500 mL | ||
| Trypsin (2.5%) | Thermo | 15090046 | 5 mL |
| mES growth medium(for 500 mL of complete solution) | Company | Catalog Number | Volume |
| 2-Mercaptoethanol (55 mM) | Gibco | 21-985-023 | 0.5 mL |
| Antibiotic Antimycotic Slution 100x | CORNING | MT30004CI | 5 mL |
| Dulbecco's Modified Eagle's Medium – high glucose | SIGMA | D6429 | 408.5 mL |
| Fetal Bovine Serum Characterized | HyClone | SH30396.03 | 75 mL |
| L-Glutamine solution | SIGMA | G7513 | 5 mL |
| Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106 U/mL | EMD Millipore Corp | CS210511 | 500 μL |
| MEK/GS3 Inhibitor Supplement | EMD Millipore Corp | CS210510-500UL | 500 μL |
| MEM Non-Essential Amino Acids Solution (100x) | Gibco | 11140076 | 5 mL |
| The ES cell media should not be stored for more than 4 weeks and with inhibitors not more than 2 weeks. | |||
| mES frozen medium(for 50 mL of complete solution) | Company | Catalog Number | Volume |
| Dimethyl sulfoxide (DMSO) | SIGMA | D2650 | 5 mL |
| Dulbecco's Modified Eagle's Medium – high glucose | SIGMA | D6429 | 24.9 mL |
| Fetal Bovine Serum Characterized | HyClone | SH30396.03 | 25 mL |
| Mouse recombinant Leukemia Inhibitory Factor (LIF), 0.5 x 106 U/mL | EMD Millipore Corp | CS210511 | 50 μL |
| Name of Material/ Equipment | Company | Catalog Number | RRID |
| 0.05% Trypsin/0.53 mM EDTA | CORNING | 25-052-CI | |
| 4% Paraformaldehyde | Thermo scientific | J19943-k2 | |
| Accutase solution | SIGMA | A6964 | Cell detachment solution |
| AgeI-HF | NEB | R3552L | |
| Alexa488-conjugated goat-anti-mouse antibody | Invitrogen | A32723 | AB_2633275 |
| Alexa488-conjugated goat-anti-rabbit antibody | Invitrogen | A32731 | AB_2633280 |
| Alexa555-conjugated goat-anti-rabbit antibody | Invitrogen | A32732 | AB_2633281 |
| anti-AFP | Thermo scientific | RB-365-A1 | AB_59574 |
| anti-α-Smooth Muscle Actin (D4K9N) XP | CST | 19245S | AB_2734735 |
| anti-Dystrophin | Thermo | PA5-32388 | AB_2549858 |
| anti-LIN28A (D1A1A) XP | CST | 8641S | AB_10997528 |
| anti-MYH2 | DSHB | mAb2F7 | AB_1157865 |
| anti-Nanog-XP | CST | 8822S | AB_11217637 |
| anti-Oct-4A (D6C8T) | CST | 83932S | AB_2721046 |
| anti-Sox2 | abcam | ab97959 | AB_2341193 |
| anti-SSEA1(MC480) | CST | 4744s | AB_1264258 |
| anti-TH (H-196) | SANTA CRUZ | sc-14007 | AB_671397 |
| Alkaline Phosphatase Live Stain (500x) | Thermo | A14353 | |
| Blasticidin S | Sigma-Aldrich | 203350 | |
| BsmBI/Esp3I | NEB | R0580L/R0734L | |
| Carbenicillin | Millipore | 205805-250MG | |
| Collagenase IV | Worthington Biochemical Corporation | LS004189 | |
| Competent Cells | TakaRa | 636763 | |
| CutSmart | NEB | B7204S | |
| CytoTune-iPS 2.0 Sendai Reprogramming Kit | Thermo | A16517 | |
| DirectPCR Lysis Reagent (cell) | VIAGEN BIOTECH | 302-C | |
| Dispase (1 U/mL) | STEMCELL Technologies | 7923 | |
| Doxycycline Hydrochloride | Fisher BioReagents | BP26535 | |
| EcoRI-HF | NEB | R3101L | |
| Fibronectin bovine plasma | SIGMA | F1141 | |
| QIAEX II Gel Extraction Kit (500) |
QIAGEN | 20051 | |
| Gelatin from porcine skin, type A | SIGMA | G1890 | |
| HardSet Antifade Mounting Medium with DAPI | Vector | H-1500 | |
| Hygromycin B (50 mg/mL) | Invitrogen | 10687010 | |
| Ketamine HCL Injection | HENRY SCHEIN ANIMAL HEALTH | 45822 | |
| KpnI-HF | NEB | R3142L | |
| lenti-CRISPRv2-blast | Addgene | 83480 | |
| lenti-Guide-Hygro-iRFP670 | Addgene | 99377 | |
| Lipofectamin 3000 Transfection Kit | Invitrogen | L3000015 | |
| LV-TRE-VP64-mouse MyoD-T2A-dsRedExpress2 | Addgene | 60625 | |
| LV-TRE-VP16 mouse MyoD-T2A-dsRedExpress2 | Addgene | 60626 | |
| Mouse on Mouse (M.O.M.) Basic Kit | Vector | BMK-2202 | |
| NotI-HF | NEB | R3189L | |
| Opti-MEM I Reduced Serum Media | ThermoFisher | 31985070 | |
| Polyethylene glycol 4,000 | Alfa Aesar | AAA161510B | |
| Polybrene | SIGMA | TR1003 | |
| Corning BioCoat Poly-D-Lysine/Laminin Culture Slide | CORNING | CB354688 | |
| PowerUp SYBR Green Master Mix | ThermoFisher | A25742 | |
| PrimeSTAR Max Premix | TakaRa | R045 | |
| Proteinase K | VIAGEN BIOTECH | 507-PKP | |
| Puromycin Dihydrochloride | MP Biomedicals | ICN19453980 | |
| qPCR Lentivirus Titration Kit | abm | LV900 | |
| Quick ligation kit | NEB | M2200S | |
| QIAprep Spin Miniprep Kit (250) | QIAGEN | 27106 | |
| QIAGEN Plasmid Plus Midi Kit (100) | QIAGEN | 12945 | |
| RevertAid RT Reverse Transcription Kit | Thermo scientific | K1691 | |
| RNAzol RT | Molecular Research Center, INC | RN 190 | |
| T4 DNA Ligase Reaction Buffer | NEB | B0202S | |
| T4 Polynucleotide Kinase | NEB | M0201S | |
| Terrific Broth Modified | Fisher BioReagents | BP9729-600 | |
| ViralBoost Reagent (500x) | ALSTEM | VB100 | |
References
- Mendell, J. R., et al. Evidence-based path to newborn screening for Duchenne muscular dystrophy. Annals of Neurology. 71 (3), 304-313 (2012).
- Batchelor, C. L., Winder, S. J. Sparks, signals and shock absorbers: how dystrophin loss causes muscular dystrophy. Trends Cell Biology. 16 (4), 198-205 (2006).
- Gregorevic, P., et al. Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nature Medicine. 10 (8), 828-834 (2004).
- Bengtsson, N. E., Seto, J. T., Hall, J. K., Chamberlain, J. S., Odom, G. L. Progress and prospects of gene therapy clinical trials for the muscular dystrophies. Human Molecular Genetics. 25 (R1), R9-R17 (2016).
- Tabebordbar, M., et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 351 (6271), 407-411 (2016).
- Long, C., et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 351 (6271), 400-403 (2016).
- Nelson, C. E., et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 351 (6271), 403-407 (2016).
- Long, C., et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 345 (6201), 1184-1188 (2014).
- El Refaey, M., et al. In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice. Circulation Research. 121 (8), 923-929 (2017).
- Amoasii, L., et al. Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science. 362 (6410), 86-91 (2018).
- Eisen, B., et al. Electrophysiological abnormalities in induced pluripotent stem cell-derived cardiomyocytes generated from Duchenne muscular dystrophy patients. Journal of Cellular and Molecular Medicine. 23 (3), 2125-2135 (2019).
- Marion, R. M., et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell. 4 (2), 141-154 (2009).
- Dumont, N. A., et al. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Nature Medicine. 21 (12), 1455-1463 (2015).
- Sacco, A., et al. Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice. Cell. 143 (7), 1059-1071 (2010).
- Su, X., et al. Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice. Journal of Visualized Experiments. (146), (2019).
- Su, X., et al. Exosome-Derived Dystrophin from Allograft Myogenic Progenitors Improves Cardiac Function in Duchenne Muscular Dystrophic Mice. Journal of Cardiovascular Translational Research. 11 (5), 412-419 (2018).
- Ousterout, D. G., et al. Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nature Communications. 6, 6244 (2015).
- Barde, I., et al. Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector. Molecular Therapy. 13 (2), 382-390 (2006).
- Glass, K. A., et al. Tissue-engineered cartilage with inducible and tunable immunomodulatory properties. Biomaterials. 35 (22), 5921-5931 (2014).
Citer Cet Article
Jin, Y., Shen, Y., Su, X., Weintraub, N., Tang, Y. CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors. J. Vis. Exp. (151), e59432, doi:10.3791/59432 (2019).