CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells
CRISPR/Cas9-Based Disease Modeling and Functional Correction of Interleukin 7 Receptor Alpha Severe Combined Immunodeficiency in T-Lymphocytes and Hematopoietic Stem Cells
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DOI：
10.1089/hum.2023.100
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发表时间：
2024-02-29
影响因子：
4.2
通讯作者：
Cavazza，Alessia
中科院分区：
文献类型：
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作者：
Rai，Rajeev;Steinberg，Zohar;Cavazza，Alessia
白细胞介素7受体严重联合免疫缺陷（IL7R-SCID）是一种由il7基因纯合突变引起的危及生命的疾病。人类IL7R表达缺陷阻碍了T细胞前体在淋巴形成过程中的增殖和分化，导致新生儿缺乏T细胞，他们在出生后不久就死于严重感染和死亡。先前通过病毒基因治疗解决IL7R- scid的尝试表明，不受调节的IL7R表达易导致白血病，这表明应用靶向基因编辑将IL7R基因的正确拷贝插入其基因组位点并介导其生理表达是一种更可行的治疗方法。为此，我们首先开发了一种基于CRISPR/ cas9的IL7R-SCID疾病建模系统，该系统概括了原代人T细胞和造血干细胞和祖细胞（HSPCs）中的疾病表型。然后，我们设计了一种靶向sil7raexon 1的敲入策略，并通过同源性定向修复引入一个纠正的、无启动子的IL7RA cDNA，然后通过AAV6转导引入一个报告子盒。在原代T细胞中靶向整合纠正盒恢复了IL7R表达，并恢复了功能性下游IL7R信号传导。当应用于在人工胸腺类器官系统中进一步诱导分化为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细胞发育阻滞，同时促进了生理和发育调节IL7R表达的T细胞的完全成熟。最后，使用偏倚和无偏倚技术对CRISPR/Cas9平台在HSPCs中的遗传毒性评估证实了该策略的安全性，为IL7R-SCID患者提供一种新的、有效的、安全的治疗选择铺平了道路。
Interleukin 7 Receptor alpha Severe Combined Immunodeficiency (IL7R-SCID) is a life-threatening disorder caused by homozygous mutations in theIL7RAgene. 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 theIL7RAgene 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 targetsIL7RAexon 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.