DataSheet1_Genome Editing With TALEN, CRISPR-Cas9 and CRISPR-Cas12a in Combination With AAV6 Homology Donor Restores T Cell Function for XLP.PDF

X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the SH2D1A 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 SAP cDNA at the first exon of the SH2D1A 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 SAP gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients.

X-连锁淋巴增殖性疾病是一种罕见的遗传性免疫失调，由编码细胞内适配蛋白SAP（Slam相关蛋白）的SH2D1A基因的突变或缺失引起。SAP对于介导多种关键的免疫过程至关重要，特别是在其缺失的情况下，免疫系统中的T细胞将出现失调。患者表现出一系列的临床症状，包括噬血细胞性淋巴组织细胞增多症（HLH）、异常球蛋白血症、淋巴瘤和自身免疫性疾病。治疗选择有限，且患者很少在没有同种异体造血干细胞移植（HSCT）的情况下生存至成年。然而，在这种不匹配供体环境或活动性HLH存在的情况下，该手术可能产生不良后果，从而留下未满足的临床需求。自体造血干细胞或T细胞疗法可能提供替代治疗选择，消除了寻找HSCT合适供体的需要以及任何移植物抗宿主反应的风险。SAP的表达谱受到严格的调控，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表达的固有调控元件，从而可能提供更优化的治疗方案。在本研究中，我们评估了TALEN、CRISPR-Cas9和CRISPR-Cas12a核酸酶在利用包含供体模板的腺相关病毒血清型6（AAV6）载体驱动SAP cDNA在SH2D1A基因座第一个外显子上进行靶向插入的能力。所有核酸酶平台均能够实现高效的基因编辑，并使用无血清AAV6转染方案进行了优化。我们展示了经过基因编辑工具校正的XLP患者的T细胞恢复了SAP基因表达的生理水平，并恢复了SAP依赖的免疫功能，这表明为XLP患者提供了一种新的治疗机会。