Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg; Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, Freiburg.
Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, Freiburg; Institute for Immunodeficiency, Medical Center-University of Freiburg, Freiburg.
J Allergy Clin Immunol. 2024 Jan;153(1):243-255.e14. doi: 10.1016/j.jaci.2023.08.003. Epub 2023 Aug 16.
Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality.
We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV).
We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection.
Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH.
Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.
噬血细胞性淋巴组织细胞增生症（HLH）是一种以细胞因子风暴和免疫病理学为特征的过度炎症性疾病。家族性 HLH 型 3（FHL3）约占全球所有先天性 HLH 病例的 30%。它是由 UNC13D 基因突变引起的，导致细胞毒性囊泡脱颗粒受损，从而损害 T 细胞和自然杀伤细胞介导的杀伤。目前的治疗方案，包括异体造血干细胞（HSC）移植，仍然显示出高死亡率。
我们试图在临床前 FHL3 Jinx 小鼠模型中开发和评估一种有治愈潜力的基因组编辑策略。Jinx 小鼠在 26 号内含子中存在一个隐秘的剪接供体位点，感染淋巴细胞性脉络丛脑膜炎病毒（LCMV）后会发展出人类 FHL3 的临床症状。
我们采用成簇规律间隔短回文重复（CRISPR）-Cas 技术在 HSCs 中删除致病突变，并将编辑后的 Unc13d 干细胞移植到白消安预处理的 Jinx 受体小鼠中。安全性研究包括通过基于单靶链接介导 PCR 测序（CAST-Seq）的脱靶分析进行广泛的基因分型和染色体异常分析。通过 LCMV 感染评估对 HLH 易感性的治疗效果。
从移植小鼠中分离的造血细胞显示出高效的基因编辑（>95%）、T 细胞受体库的多克隆性，既没有脱靶效应的迹象，也没有白血病发生。编辑和野生型细胞的 Unc13d 转录水平相当。虽然 LCMV 挑战导致 mock 编辑 HSCs 移植的 Jinx 小鼠发生急性 HLH，但 Unc13d 编辑细胞移植的 Jinx 小鼠迅速清除病毒并免受 HLH 影响。
我们的研究表明，CRISPR-Cas 编辑 HSCs 的移植支持在没有遗传毒性相关克隆性生长的情况下，发育出功能性多克隆 T 细胞反应。
