PDF(Pubmed)
Abstract：
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic disorder caused by mutations in the FOXP3 gene, required for generation of regulatory T (Treg) cells. Loss of Treg cells leads to immune dysregulation characterized by multi-organ autoimmunity and early mortality. Hematopoietic stem cell (HSC) transplantation can be curative, but success is limited by autoimmune complications, donor availability and/or graft-vs.-host disease. Correction of FOXP3 in autologous HSC utilizing a homology-directed repair (HDR)-based platform may provide a safer alternative therapy. Here, we demonstrate efficient editing of FOXP3 utilizing co-delivery of Cas9 ribonucleoprotein complexes and adeno-associated viral vectors to achieve HDR rates of >40% in vitro using mobilized CD34+ cells from multiple donors. Using this approach to deliver either a GFP or a FOXP3 cDNA donor cassette, we demonstrate sustained bone marrow engraftment of approximately 10% of HDR-edited cells in immune-deficient recipient mice at 16 weeks post-transplant. Further, we show targeted integration of FOXP3 cDNA in CD34+ cells from an IPEX patient and expression of the introduced FOXP3 transcript in gene-edited primary T cells from both healthy individuals and IPEX patients. Our combined findings suggest that refinement of this approach is likely to provide future clinical benefit in IPEX.
摘要：
免疫失调,多内分泌病,肠病,X连锁（IPEX）综合征是由FOXP3基因突变引起的单基因疾病，需要产生调节性T(Treg)细胞。Treg细胞的丧失导致以多器官自身免疫和早期死亡为特征的免疫失调。造血干细胞（HSC）移植可以治愈，但是成功受到自身免疫并发症的限制，供体可用性和/或移植物vs.-宿主病.利用基于同源定向修复(HDR)的平台校正自体HSC中的FOXP3可以提供更安全的替代疗法。这里,我们证明,使用来自多个供体的动员的CD34+细胞,利用Cas9核糖核蛋白复合物和腺相关病毒载体的共递送,在体外实现>40%的HDR率,对FOXP3进行有效编辑.使用这种方法来传递GFP或FOXP3cDNA供体盒，我们证实,在移植后16周,免疫缺陷受体小鼠中,约10%的HDR编辑细胞持续进行骨髓移植.Further,我们显示了来自IPEX患者的CD34+细胞中FOXP3cDNA的靶向整合,以及来自健康个体和IPEX患者的基因编辑的原代T细胞中引入的FOXP3转录物的表达.我们的综合发现表明，这种方法的改进可能会在IPEX中提供未来的临床益处。
