造血干细胞和祖细胞的CRISPR/Cas9基因编辑用于基因治疗应用
Summary
本协议描述了一种优化的造血干细胞和祖细胞（HSPC）培养程序，用于 体内基因编辑细胞的稳健植入。
Abstract
CRISPR / Cas9是一种高度通用和高效的基因编辑工具，广泛用于纠正各种基因突变。体 外 对造血干细胞和祖细胞（HSPCs）进行基因操作的可行性使HSPCs成为基因治疗的理想靶细胞。然而，HSPC在 离体 培养中适度失去其植入和多谱系再种群潜力。在本研究中，描述了改善HSPC植入并在 体内产生更多基因修饰细胞的理想培养条件。当前报告显示了优化的 体外 培养条件，包括培养基类型、独特的小分子混合物补充剂、细胞因子浓度、细胞培养板和培养密度。除此之外，还提供了优化的HSPC基因编辑程序，以及基因编辑事件的验证。为了 体内 验证，显示小鼠受体的基因编辑HSPC输注和植入后分析。结果表明，培养系统增加了 体外 功能性HSC的频率，从而在 体内对基因编辑细胞进行了稳健的植入。
Introduction
在同种异体移植环境中无法获得人类白细胞抗原（HLA）匹配的供体，以及高度通用和安全的基因工程工具的快速发展，使自体造血干细胞移植（HSCT）成为遗传性血液疾病的治愈性治疗策略1，2。自体造血干细胞和祖细胞（HSPC）基因治疗涉及收集患者的HSPC，基因操作，纠正致病突变以及将基因校正的HSPC移植到患者体内3，4。然而，基因治疗的成功结果取决于可移植基因修饰移植物的质量。HSPC的基因操作步骤和离体培养通过降低长期造血干细胞（LT-HSCs）的频率来影响移植物的质量，因此需要输注大剂量的基因操纵HSPCs2，5，6。
目前正采用几种小分子，包括SR1和UM171，以稳健地扩增脐带血HSPCs7，8。对于成年HSPC，由于在动员中获得更高的细胞产量，因此不需要稳健的扩增。然而，在离体培养中保留分离的HSPC的干性对其基因治疗应用至关重要。因此，使用小分子组合开发了专注于造血干细胞（HSC）培养富集的方法：白藜芦醇，UM729和SR1（RUS）7。优化的HSPC培养条件促进了HSC的富集，导致体内基因修饰HSC的频率增加，并减少了基因操纵大剂量HSPC的需求，促进了具有成本效益的基因治疗方法8。
在这里，描述了HSPC培养的综合方案，以及 体内基因编辑细胞的输注和分析。
Protocol
免疫缺陷小鼠的体内实验由印度韦洛尔基督教医学院动物伦理委员会（IAEC）批准并按照其指南进行。在获得机构审查委员会（IRB）批准后，在知情同意的情况下，从健康人类供体收集粒细胞集落刺激因子（G-CSF）动员的外周血样本。 1. 外周血单核细胞（PBMNCs）的分离和CD34 + 细胞的纯化 按照以下步骤执行 PBMNC 隔离。注意：对于体外</em…
Representative Results
本研究确定了理想的HSPC培养条件，以促进CD34 + CD133 + CD90 + HSC在离体培养中的保留。为了证明HSC的培养富集以及基因修饰HSC的增强生成，提供了PBMNC分离，CD34 +细胞纯化，培养，基因编辑，移植，植入表征和体内基因修饰细胞的优化程序（图1）。纯化后，进行流式细胞术评估以检查HSPC标志物，并将HSPC培养72 h。培养72小时后，用C…
Discussion
HSPC基因治疗的成功结果主要取决于移植物中可移植HSC的质量和数量。然而，HSC的功能特性在基因治疗产品的准备阶段受到很大影响，包括体外培养和与基因操作程序相关的毒性。为了克服这些限制，我们已经确定了理想的HSPC培养条件，这些条件在离体培养中保留了CD34 + CD133 + CD90 + HSC的干性。许多研究小组使用SR1或UM171或其他分子作为独立分子在体外?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢CSCR流式细胞术设施和动物设施的工作人员。A.C.由ICMR-SRF研究金资助，K.V.K.由DST-INSPIRE研究金资助，P.B.由CSIR-JRF研究金资助。 这项工作由印度政府生物技术部资助（拨款号BT/PR26901/MED/31/377/2017和BT/PR31616/MED/31/408/2019）
Materials
| 4D-Nucleofector® X Unit | LONZA BIOSCIENCE | AAF-1003X | |
| 4D-Nucleofector™ X Kit ( 16-well Nucleocuvette™ Strips) | LONZA BIOSCIENCE | V4XP-3032 | |
| Antibiotic-Antimycotic (100X) | THERMO SCIENTIFIC | 15240096 | |
| Anti-human CD45 APC | BD BIOSCIENCE | 555485 | |
| Anti-human CD13 PE | BD BIOSCIENCE | 555394 | |
| Anti-human CD19 PerCP | BD BIOSCIENCE | 340421 | |
| Anti-human CD3 PE-Cy7 | BD BIOSCIENCE | 557749 | |
| Anti-human CD90 APC | BD BIOSCIENCE | 561971 | |
| Anti-human CD133/1 | Miltenyibiotec | 130-113-673 | |
| Anti-human CD34 PE | BD BIOSCIENCE | 348057 | |
| Anti-mouse CD45.1 PerCP-Cy5 | BD BIOSCIENCE | 560580 | |
| Blood Irradator-2000 | BRIT (Department of Biotechnology, India) | BI 2000 | |
| Cell culture dish (delta surface-treated 6-well plates) | NUNC (THERMO SCIENTIFIC) | 140675 | |
| CrysoStor CS10 | BioLife solutions | #07952 | |
| Busulfan | CELON LABS (60mg/10mL) | - | |
| Guide-it Recombinant Cas9 | TAKARA BIO | 632640 | |
| Cas9-eGFP | SIGMA | C120040 | |
| Centrifuge tube-15ml | CORNING | 430790 | |
| Centrifuge tube-50ml | NUNC (THERMO SCIENTIFIC) | 339652 | |
| DMSO | MPBIO | 219605590 | |
| DNAase | STEMCELL TECHNOLOGIES | 6469 | |
| Dulbecco′s Phosphate Buffered Saline- 1X | HYCLONE | SH30028.02 | |
| EasySep™ Human CD34 Positive Selection Kit II | STEMCELL TECHNOLOGIES | 17856 | |
| EasySep magnet | STEMCELL TECHNOLOGIES | 18000 | |
| Electrophoresis unit | ORANGE INDIA | HDS0036 | |
| FBS | THERMO SCIENTIFIC | 10270106 | |
| Flow cytometer – ARIA III | BD BIOSCIENCE | - | |
| FlowJo | BD BIOSCIENCE | - | |
| Flt3-L | PEPROTECH | 300-19-1000 | |
| Gel imaging system | CELL BIOSCIENCES | 11630453 | |
| HighPrep DTR reagent | MAGBIOGENOMICS | DT-70005 | |
| Human BD Fc Block | BD BIOSCIENCE | 553141 | |
| IL6 | PEPROTECH | 200-06-50 | |
| IMDM media | THERMO SCIENTIFIC | 12440053 | |
| Infrared lamp | MURPHY | – | |
| Insulin syringe 6mm 31G | BD BIOSCIENCE | 324903 | |
| Ketamine | KETMIN 50 | – | |
| Loading dye 6X | TAKARA BIO | 9156 | |
| Lymphoprep | STEMCELL TECHNOLOGIES | 7851 | |
| Mice Restrainer | AVANTOR | TV-150 | |
| Nano drop spectrophotometer | THERMO SCIENTIFIC | ND-2000C | |
| Neubauer cell counting chamber | ROHEM INSTRUMENTS | CC-3073 | |
| NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) | The Jackson Laboratory | RRID:IMSR_JAX:005557 | |
| NOD,B6.SCID Il2rγ−/−KitW41/W41 (NBSGW) | The Jackson Laboratory | RRID:IMSR_JAX:026622 | |
| Nunc delta 6-well plate | THERMO SCIENTIFIC | 140675 | |
| Polystyrene round-bottom tube | BD | 352008 | |
| P3 primary cell Nucleofection solution | LONZA BIOSCIENCE | PBP3-02250 | |
| Pasteur pipette | FISHER SCIENTIFIC | 13-678-20A | |
| PCR clean-up kit | TAKARA BIO | 740609.25 | |
| Mouse Pie Cage | FISCHER SCIENTIFIC | 50-195-5140 | |
| polystyrene round-bottom tube (12 x 75 mm) | STEMCELL TECHNOLOGIES | 38007 | |
| Primer3 | Whitehead Institute for Biomedical Research | https://primer3.ut.ee/ | |
| QuickExtract™ DNA Extraction Solution | Lucigen | QE09050 | |
| Reserveratrol | STEMCELL TECHNOLOGIES | 72862 | |
| SCF | PEPROTECH | 300-07-1000 | |
| SFEM-II | STEMCELL TECHNOLOGIES | 9655 | |
| sgRNA | SYNTHEGO | - | |
| SPINWIN | TARSON | 1020 | |
| StemReginin 1 | STEMCELL TECHNOLOGIES | 72342 | |
| ICE analysis tool | SYNTHEGO | https://ice.synthego.com/ | |
| Tris-EDTA buffer solution (TE) 1X | SYNTHEGO | Supplied with gRNA | |
| Thermocycler | APPLIED BIOSYSTEMS | 4375305 | |
| TPO | PEPROTECH | 300-18-1000 | |
| Trypan blue | HIMEDIA LABS | TCL046 | |
| UM171 | STEMCELL TECHNOLOGIES | 72914 | |
| UM729 | STEMCELL TECHNOLOGIES | 72332 | |
| Xylazine | XYLAXIN – INDIAN IMMUNOLOGICALS LIMITED | – |
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Cite This Article
Venkatesan, V., Christopher, A. C., Karuppusamy, K. V., Babu, P., Alagiri, M. K. K., Thangavel, S. CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications. J. Vis. Exp. (186), e64064, doi:10.3791/64064 (2022).