Houghton Benjamin C, Panchal Neelam, Haas Simone A, Chmielewski Kay O, Hildenbeutel Markus, Whittaker Thomas, Mussolino Claudio, Cathomen Toni, Thrasher Adrian J, Booth Claire
Molecular and Cellular Immunology, UCL Great Ormond Street Institute of Child Health, London, United Kingdom.
Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, Freiburg, Germany.
Front Genome Ed. 2022 May 23;4:828489. doi: 10.3389/fgeed.2022.828489. eCollection 2022.
X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of cDNA at the first exon of the locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients.
X连锁淋巴增生性疾病是一种罕见的遗传性免疫疾病，由编码细胞内衔接蛋白SAP（信号淋巴细胞激活分子相关蛋白）的基因突变或缺失引起。SAP对于介导多个关键免疫过程至关重要，缺乏它时免疫系统，尤其是T细胞，会出现失调。患者表现出一系列临床表现，包括噬血细胞性淋巴组织细胞增生症（HLH）、免疫球蛋白异常血症、淋巴瘤和自身免疫。治疗选择有限，若无同种异体造血干细胞移植（HSCT），患者很少能存活至成年。然而，在供体不匹配或存在活动性HLH的情况下，该手术可能效果不佳，仍存在未满足的临床需求。自体造血干细胞或T细胞疗法可能提供替代治疗选择，无需为HSCT寻找合适的供体，也不存在任何同种异体反应的风险。SAP具有严格控制的表达谱，传统的慢病毒基因递送平台可能无法完全复制。基因编辑方法可以保留更多控制SAP表达的内源性调控元件，有可能提供更优化的治疗方法。在此，我们评估了转录激活样效应因子核酸酶（TALEN）、CRISPR-Cas9和CRISPR-Cas12a核酸酶使用含有供体模板的腺相关病毒6型（AAV6）载体在该基因座的第一个外显子处驱动cDNA靶向插入的能力。所有核酸酶平台都能够进行高效基因编辑，通过无血清AAV6转导方案进行了优化。我们表明，经基因编辑工具校正的XLP患者的T细胞恢复了该基因表达的生理水平，并恢复了SAP依赖的免疫功能，这为XLP患者带来了新的治疗机会。
