{"title":"基于CRISPR/Cas9的疾病建模和白细胞介素7受体α重度联合免疫缺陷症在T淋巴细胞和造血干细胞中的功能校正。","authors":"Rajeev Rai, Zohar Steinberg, Marianna Romito, Federica Zinghirino, Yi-Ting Hu, Nathan White, Asma Naseem, Adrian J Thrasher, Giandomenico Turchiano, Alessia Cavazza","doi":"10.1089/hum.2023.100","DOIUrl":null,"url":null,"abstract":"<p><p>Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the <i>IL7RA</i> gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the <i>IL7RA</i> gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets <i>IL7RA</i> exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":null,"pages":null},"PeriodicalIF":3.9000,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.\",\"authors\":\"Rajeev Rai, Zohar Steinberg, Marianna Romito, Federica Zinghirino, Yi-Ting Hu, Nathan White, Asma Naseem, Adrian J Thrasher, Giandomenico Turchiano, Alessia Cavazza\",\"doi\":\"10.1089/hum.2023.100\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the <i>IL7RA</i> gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the <i>IL7RA</i> gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets <i>IL7RA</i> exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.</p>\",\"PeriodicalId\":13007,\"journal\":{\"name\":\"Human gene therapy\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.9000,\"publicationDate\":\"2024-04-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Human gene therapy\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1089/hum.2023.100\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/2/29 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Human gene therapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1089/hum.2023.100","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/2/29 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
白细胞介素 7 受体 严重联合免疫缺陷症(IL7R-SCID)是一种由 IL7RA 基因同源突变引起的危及生命的疾病。人类体内 IL7R 的表达缺陷阻碍了淋巴细胞生成过程中 T 细胞前体的增殖和分化,导致新生儿体内缺乏 T 细胞,并在出生后早期死于严重感染。之前通过病毒基因疗法解决 IL7R-SCID 的尝试表明,IL7R 表达不正常易导致白血病,这表明应用靶向基因编辑技术在其基因组位点插入正确的 IL7RA 基因拷贝并介导其生理表达是一种更可行的治疗方法。为此,我们首先开发了基于CRISPR/Cas9的IL7R-SCID疾病模型系统,该系统能在原代人类T细胞和造血干细胞及祖细胞(HSPCs)中重现疾病表型。然后,我们设计了一种基因敲入策略,以 IL7RA 第 1 外显子为靶点,通过同源定向修复引入一个纠正性的、无启动子的 IL7RA cDNA,然后通过 AAV6 转导引入一个报告基因盒。在原代 T 细胞中靶向整合校正盒后,IL7R 的表达得到恢复,下游 IL7R 信号的功能也得到恢复。当应用于在人工胸腺器官系统中进一步诱导分化成 T 细胞的 HSPCs 时,我们的基因编辑策略克服了在 IL7R-SCID 患者中观察到的 T 细胞发育障碍,同时促进了具有生理和发育调控 IL7R 表达的 T 细胞的完全成熟。最后,利用偏倚和非偏倚技术对CRISPR/Cas9平台在HSPCs中的遗传毒性进行了评估,证实了该策略的安全性,为IL7R-SCID患者提供一种高效、安全的新治疗方案铺平了道路。
CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells.
Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in the IL7RA gene. Defective IL7R expression in humans hampers T cell precursors' proliferation and differentiation during lymphopoiesis resulting in the absence of T cells in newborns, who succumb to severe infections and death early after birth. Previous attempts to tackle IL7R-SCID by viral gene therapy have shown that unregulated IL7R expression predisposes to leukemia, suggesting the application of targeted gene editing to insert a correct copy of the IL7RA gene in its genomic locus and mediate its physiological expression as a more feasible therapeutic approach. To this aim, we have first developed a CRISPR/Cas9-based IL7R-SCID disease modeling system that recapitulates the disease phenotype in primary human T cells and hematopoietic stem and progenitor cells (HSPCs). Then, we have designed a knockin strategy that targets IL7RA exon 1 and introduces through homology-directed repair a corrective, promoterless IL7RA cDNA followed by a reporter cassette through AAV6 transduction. Targeted integration of the corrective cassette in primary T cells restored IL7R expression and rescued functional downstream IL7R signaling. When applied to HSPCs further induced to differentiate into T cells in an Artificial Thymic Organoid system, our gene editing strategy overcame the T cell developmental block observed in IL7R-SCID patients, while promoting full maturation of T cells with physiological and developmentally regulated IL7R expression. Finally, genotoxicity assessment of the CRISPR/Cas9 platform in HSPCs using biased and unbiased technologies confirmed the safety of the strategy, paving the way for a new, efficient, and safe therapeutic option for IL7R-SCID patients.
期刊介绍:
Human Gene Therapy is the premier, multidisciplinary journal covering all aspects of gene therapy. The Journal publishes in-depth coverage of DNA, RNA, and cell therapies by delivering the latest breakthroughs in research and technologies. Human Gene Therapy provides a central forum for scientific and clinical information, including ethical, legal, regulatory, social, and commercial issues, which enables the advancement and progress of therapeutic procedures leading to improved patient outcomes, and ultimately, to curing diseases.