Erol Özgür Doğuş, Şenocak Şimal, Özçimen Burcu, Güney Esken Gülen, Kiliç Hasan Basri, Kocaefe Çetin, VAN Til Niek P, Aerts Kaya Fatima
Department of Stem Cell Sciences, Center for Stem Cell Research and Development, Graduate School of Health Sciences, Hacettepe University, Ankara, Turkiye.
Hacettepe University Advanced Technologies Application and Research Center, Hacettepe University, Ankara, Turkiye.
Turk J Biol. 2024 Jul 31;48(5):290-298. doi: 10.55730/1300-0152.2705. eCollection 2024.
BACKGROUND/AIM: Griscelli Syndrome Type 2 (GS-2) is a rare, inherited immune deficiency caused by a mutation in the gene. The current treatment consists of hematopoietic stem cell transplantation, but a lack of suitable donors warrants the development of alternative treatment strategies, including gene therapy. The development of mutation-specific clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 gene editing technology has opened the way for custom-designed gene correction of patient-derived stem cells. In this study, we aimed to custom design CRISPR/Cas9 constructs and test their efficiency on homology-directed repair (HDR) on the correction of exon 3 and exon 7 mutations in the gene of GS-2 patient-derived mesenchymal stem cells (MSCs) and induced pluripotent stem cells.
We assessed gene and protein expression using qRT-PCR, Western Blot, and immune fluorescence in GS-2 patient-derived MSCs and induced pluripotent stem cells (iPSCs). Guide RNAs (gRNAs) and donor DNAs were designed based on patient mutations in exon 3 and exon 7 using the CHOPCHOP online tool and transfected into GS-2 MSCs and iPSCs by electroporation. The cells were cultured for 2 days and then used for mutation analysis using DNA sequencing.
MSCs and iPSCs from the GS-2 patients lacked gene and protein expression. After gRNA and donor DNAs were designed and optimized, we found HDR efficiency with gRNA3.3 (10% efficiency) and gRNA7.3 (27% efficiency) for MSCs but lower efficiency in iPSCs (<5%). However, transfection of both MSCs and iPSCs resulted in massive cell death, loss of colony formation, and spontaneous differentiation.
The use of CRISPR/Cas9 to genetically correct MSCs and iPSCs from GS-2 patients with different mutations through HDR is feasible but requires optimization of the procedure to reduce cell death and improve stem cell function before clinical application.
背景/目的：2型格里塞利综合征（GS-2）是一种由该基因突变引起的罕见遗传性免疫缺陷病。目前的治疗方法是造血干细胞移植，但缺乏合适的供体促使人们开发替代治疗策略，包括基因治疗。突变特异性成簇规律间隔回文重复序列（CRISPR）/Cas9基因编辑技术的发展为对患者来源的干细胞进行定制化基因校正开辟了道路。在本研究中，我们旨在定制设计CRISPR/Cas9构建体，并测试其对GS-2患者来源的间充质干细胞（MSCs）和诱导多能干细胞中外显子3和外显子7突变进行同源定向修复（HDR）的效率。
我们使用qRT-PCR、蛋白质免疫印迹和免疫荧光评估了GS-2患者来源的MSCs和诱导多能干细胞（iPSCs）中的该基因和蛋白表达。使用CHOPCHOP在线工具根据外显子3和外显子7中的患者突变设计向导RNA（gRNAs）和供体DNA，并通过电穿孔转染到GS-2 MSCs和iPSCs中。将细胞培养2天，然后用于DNA测序进行突变分析。
GS-2患者的MSCs和iPSCs缺乏该基因和蛋白表达。在设计并优化gRNA和供体DNA后，我们发现MSCs使用gRNA3.3（效率为10%）和gRNA7.3（效率为27%）时HDR效率较高，但iPSCs中的效率较低（<5%）。然而，MSCs和iPSCs的转染均导致大量细胞死亡、集落形成丧失和自发分化。
通过HDR使用CRISPR/Cas9对患有不同突变的GS-2患者的MSCs和iPSCs进行基因校正是可行的，但在临床应用前需要优化该程序以减少细胞死亡并改善干细胞功能。
