Development of a CRISPR/Cas9 gene editing platform to correct Severe Combined Immunodeficiency caused by mutations in the IL7RA gene

严重联合免疫缺陷(SCID)是一种罕见的遗传性疾病，由大量基因突变引起，以免疫系统发育障碍为特征。IL7RA基因突变导致第三种最常见的SCID形式。IL7RA SCID患者不会发展T细胞，通常在生命早期受到严重的细菌、病毒或真菌感染。这些婴儿如果不接受治疗，通常会在一年内死于严重感染，除非他们成功接受了造血干细胞移植。最近，针对不同形式的SCID的基因治疗已被成功地尝试作为骨髓移植的替代方案。在基因治疗过程中，造血干细胞在实验室得到纠正，然后注入患者体内，在那里它们开始产生健康的免疫细胞。然而，基因疗法试图在造血干细胞中引入正确的IL7RA拷贝，表明该基因的结构性和非调控表达易患白血病。另一种方法是通过使用基因编辑将正常的基因拷贝直接引入其内源位置，从而保持其生理调节，从而纠正造血干细胞。我们的初步数据证明了这种应用于IL7RA SCID的可行性。我们确实已经开发了基因编辑试剂和方案，可以有效地将报告基因插入到人类造血干细胞中的IL7RA基因位点。我们现在的目标是向前推进并插入正确的IL7RA拷贝，以便在SCID患者IL7RA缺陷的造血干细胞中恢复IL7RA的表达。我们还旨在证明，矫正后的干细胞移植允许所有血细胞的发育，特别是T细胞的发育，而且这一过程造成的毒性最小，甚至没有。开发一种基于基因编辑的治疗方法来治疗IL7R蛋白缺乏症，将为患有这种疾病的儿童提供一种有价值的选择。由于我们在基因编辑和造血干细胞操作方面的专业知识，我们认为我们处于实现这一目标的最佳位置。

Severe combined immunodeficiency (SCID) is a rare genetic disorder caused by numerous genetic mutations and characterized by the disturbed development of the immune system. Mutations in the IL7RA gene cause the third most common form of SCID. IL7RA SCID patients do not develop T-cells and are usually affected by severe bacterial, viral, or fungal infections early in life. These babies, if untreated, usually die within one year due to severe infections unless they have undergone successful hematopoietic stem cell transplantation. More recently, gene therapy for different forms of SCID has been successfully attempted as an alternative to bone marrow transplant. For the gene therapy procedure, hematopoietic stem cells are corrected in a laboratory and then infused back to the patient where they start producing healthy immune cells. However, gene therapy attempts to introduce a correct copy of IL7RA in hematopoietic stem cells showed that constitutive and unregulated expression of the gene pre-disposes to leukemia. An alternative approach is to correct the hematopoietic stem cells by introducing a normal copy of the gene directly in its endogenous locus, thus maintaining its physiological regulation, through the use of gene editing. Our preliminary data demonstrates the feasibility of such application for IL7RA SCID. We have indeed developed gene editing reagents and protocols to efficiently knock-in a reporter gene into the IL7RA locus in human hematopoietic stem cells. We now aim to move forward and knock-in a correct copy of IL7RA, in order to restore IL7RA expression in IL7RA-deficient hematopoietic stem cells from SCID patients. We also aim to demonstrate that transplantation of corrected stem cells allows the development of all blood cells, and in particular of T-cells, and that the process causes minimal to no toxicity. The development of a gene editing-based therapeutic approach to treat IL7R protein deficiency will provide a valuable alternative for children with this disease. Thanks to our expertise in gene editing and manipulation of hematopoietic stem cells we feel we are in the best position to achieve this goal.