Guerrero-Vargas Jacky M, Suarez-Garcia Diego A, Leal Andrés F, Diaz-Ariza Ivonne L, Pérez-Pérez León D, Espejo-Mojica Angela J, Alméciga-Díaz Carlos J
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá D.C. 110231, Colombia.
Nemours Children's Health, Wilmington, DE 19803, USA.
Pharmaceutics. 2025 May 9;17(5):628. doi: 10.3390/pharmaceutics17050628.
Tay-Sachs disease (TSD) is a neurodegenerative disorder caused by a deficiency in β-hexosaminidase A (HexA), which accumulates GM2 gangliosides, primarily in neurons. Currently, therapeutic options are limited, highlighting the need for new strategies such as gene therapy. Despite their effectiveness, viral vectors can elicit adverse immune responses; consequently, non-viral vectors are being explored as an alternative. We have previously investigated the use of CRISPR/Cas9 nickase (nCas9) as a potential tool for treating TSD. Here, we expanded our study by evaluating the PP6D5 polymer as a novel non-viral vector for delivering the CRISPR/nCas9 system to restore HexA activity. First, we evaluated the PP6D5-mediated CRISPR/nCas9 system's transfection efficiency in NIH-3T3 fibroblasts, U87MG astrocytoma, SHSY5Y neuroblastoma, and TSD fibroblasts. We then evaluated the potential of PP6D5 to correct the gene defect in TSD fibroblasts. The results showed that PP6D5 exhibited significantly higher transfection efficiency compared to lipofectamine 3000 in all tested cell models. In TSD fibroblasts, transfection with both HEXA and HEXB cDNAs increased the HexA activity levels by up to 7.4-fold, compared to a 3.2-fold increase in cells transfected only with HEXA cDNA after 15 days post-transfection. These levels were up to 4.5-fold higher than those observed in lipofectamine-mediated transfection. Additionally, PP6D5-mediated CRISPR/nCas9-based genome editing led to a significant reduction in the lysosomal mass of TSD fibroblasts. This study provides promising evidence for the use of the PP6D5 polymer as a non-viral vector for delivering CRISPR/nCas9-based gene therapy in TSD. The use of the PP6D5 polymer may offer some advantages that viral vectors cannot, such as a reduction in cytotoxicity and higher TE in difficult-to-transfect cell lines. Furthermore, this type of polymeric vector has not been extensively explored for gene therapy, making this study an important contribution to the development of non-viral delivery systems for the treatment of neurodegenerative diseases.
泰-萨克斯病（TSD）是一种神经退行性疾病，由β-己糖胺酶A（HexA）缺乏引起，导致GM2神经节苷脂蓄积，主要在神经元中。目前，治疗选择有限，这凸显了对基因治疗等新策略的需求。尽管病毒载体有效，但它们可引发不良免疫反应；因此，正在探索非病毒载体作为替代方案。我们之前研究了使用CRISPR/Cas9切口酶（nCas9）作为治疗TSD的潜在工具。在此，我们通过评估PP6D5聚合物作为一种新型非病毒载体来递送CRISPR/nCas9系统以恢复HexA活性，从而扩展了我们的研究。首先，我们评估了PP6D5介导的CRISPR/nCas9系统在NIH-3T3成纤维细胞、U87MG星形细胞瘤、SHSY5Y神经母细胞瘤和TSD成纤维细胞中的转染效率。然后，我们评估了PP6D5纠正TSD成纤维细胞基因缺陷的潜力。结果表明，在所有测试的细胞模型中，PP6D5的转染效率均显著高于Lipofectamine 3000。在TSD成纤维细胞中，与转染后15天仅转染HEXA cDNA的细胞中HexA活性水平增加3.2倍相比，同时转染HEXA和HEXB cDNA可使HexA活性水平提高多达7.4倍。这些水平比Lipofectamine介导的转染中观察到的水平高出多达4.5倍。此外，PP6D5介导的基于CRISPR/nCas9的基因组编辑导致TSD成纤维细胞的溶酶体质量显著降低。本研究为使用PP6D5聚合物作为非病毒载体在TSD中递送基于CRISPR/nCas9的基因治疗提供了有前景的证据。使用PP6D5聚合物可能具有一些病毒载体所没有的优势，例如降低细胞毒性以及在难以转染的细胞系中具有更高的转染效率。此外，这种类型的聚合物载体尚未被广泛用于基因治疗，因此本研究对开发用于治疗神经退行性疾病的非病毒递送系统做出了重要贡献。
