Nikoletta Y Papaioannou, Petros Patsali, Basma Naiisseh, Panayiota L Papasavva, Lola Koniali, Ryo Kurita, Yukio Nakamura, Soteroula Christou, Maria Sitarou, Claudio Mussolino, Toni Cathomen, Marina Kleanthous, Carsten W Lederer
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For many hematopoietic research applications, primary CD34<sup>+</sup> cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for <i>in vitro</i> manipulation. Moreover, <i>ex vivo</i> editing of CD34<sup>+</sup> cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. <b>Methods:</b> Here, we detail an <i>in vitro</i> transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34<sup>+</sup> cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal <i>in vitro</i> delivery of genome editing tools. <b>Results and Discussion:</b> Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. 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引用次数: 1
摘要
基因组编辑工具,如CRISPR/Cas, TALE核酸酶和最近的双链断裂独立编辑器,已成功用于基因治疗和反向遗传学。在该领域面临的各种挑战中,最紧迫的是如何容忍和有效地将编辑器运送到目标细胞和位点,以及如何独立于商业上可用的工具,以实现灵活性和快速采用新的编辑技术。对于许多造血研究应用,原代CD34+细胞和人脐带衍生祖红细胞2 (HUDEP-2)细胞系是信息丰富的底物,易于体外操作。此外,体外编辑CD34+细胞具有直接的治疗意义。这两种细胞类型都对标准的转染程序和试剂敏感,例如质粒DNA的脂质转染,这需要更合适的方法,以便通过选择编辑器实现高效率和可耐受的编辑。这些挑战可以通过RNA递送来解决,无论是作为CRISRP/ cas系统的向导RNA和mRNA的混合物,还是作为TALENs的mRNA混合物。与核糖核蛋白或蛋白质相比,RNA作为载体通过消除对新编辑蛋白的商业可用性或费力的内部制备的依赖而具有灵活性。与DNA相比,RNA毒性更小,并且通过避免核转录和mRNA的输出提供更快的动力学和更高的编辑效率。方法:在这里,我们详细介绍了一种基于质粒DNA模板的体外转录方案,使用T7 RNA聚合酶添加Anti-Reverse Cap Analog (ARCA),使用poly (A)聚合酶添加poly (A) tailtail,并结合HUDEP-2和患者来源的CD34+细胞的核感染。我们的基于rna的递送方案采用了广泛可用的试剂和设备,可以很容易地用于基因组编辑工具的普遍体外递送。结果和讨论:根据一个常见的用例,我们采用该方案来靶向β-珠蛋白突变和重新激活γ-珠蛋白表达,作为β-血红蛋白病的两种潜在治疗方法,随后进行红细胞分化和功能分析。我们的协议允许高编辑效率和未受损的细胞活力和分化,具有可扩展性,适合编辑结果的功能评估和应用于不同编辑器的高度灵活性。
High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis.
Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34+ cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34+ cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34+ cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools. Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.