Herreno-Pachón Angélica María, Leal Andrés Felipe, Khan Shaukat, Alméciga-Díaz Carlos Javier, Tomatsu Shunji
Nemours Children's Health, Wilmington, DE 19803, USA.
Faculty of Arts and Sciences, University of Delaware, Newark, DE 19716, USA.
Int J Mol Sci. 2025 May 2;26(9):4334. doi: 10.3390/ijms26094334.
Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously, we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice, respectively. In this study, we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system, targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion, and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally, MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture, MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction, pro-oxidant profile amelioration, mitochondrial mass recovery, and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction.
黏多糖贮积症IVA型（MPS IVA）是一种影响骨骼的溶酶体贮积病（LSD），由于N - 乙酰半乳糖胺 - 6 - 硫酸酯酶（GALNS）酶活性不足，导致硫酸角质素（KS）和硫酸软骨素6 - 硫酸酯（C6S）等糖胺聚糖（GAGs）降解受损。此前，我们成功开发并验证了一种基于CRISPR/nCas9的基因疗法（GT），分别在人MPS IVA成纤维细胞和MPS IVA小鼠的AAVS1和ROSA26位点插入表达盒。在本研究中，我们扩展了我们的方法，以评估基于CRISPR/nCas9的GT在编辑人CD34 +细胞以介导MPS IVA成纤维细胞交叉校正方面的有效性。用靶向AAVS1位点的CRISPR/nCas9系统对CD34 +细胞进行电穿孔。评估了nCas9介导的靶向供体模板插入以及CRISPR/nCas编辑的CD34 +细胞的干性。此外，将MPS IVA成纤维细胞与CRISPR/nCas编辑的CD34 +细胞共培养以评估交叉校正。基于CRISPR/nCas9的基因编辑不影响CD34 +细胞的干性，但确实导致GALNS酶水平超过生理水平。共培养后，MPS IVA成纤维细胞的GALNS酶活性显著增加，同时溶酶体质量减少、促氧化剂谱改善、线粒体质量恢复以及促凋亡和促炎谱改善。这些结果表明我们基于CRISPR/nCas9的GT在编辑CD34 +细胞以介导交叉校正方面的潜力。
