Leal Andrés Felipe, Alméciga-Díaz Carlos Javier
Institute for the Study of Inborn Errors of Metabolism, Faculty of Science, Pontificia Universidad Javeriana, Bogotá D.C., 110231, Colombia.
Gene Ther. 2023 Feb;30(1-2):107-114. doi: 10.1038/s41434-022-00344-3. Epub 2022 May 18.
Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder (LSD) caused by mutations in gene encoding for GALNS enzyme. Lack of GALNS activity leads to the accumulation of glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate. Although enzyme replacement therapy has been approved since 2014 for MPS IVA, still there is an unmet medical need to have improved therapies for this disorder. CRISPR/Cas9-based gene therapy has been tested for several LSDs with encouraging findings, but to date it has not been assayed on MPS IVA. In this work, we validated for the first time the use of CRISPR/Cas9, using a Cas9 nickase, for the knock-in of an expression cassette containing GALNS cDNA in an in vitro model of MPS IVA. The results showed the successful homologous recombination of the expression cassette into the AAVS1 locus, as well as a long-term increase in GALNS activity reaching up to 40% of wild-type levels. We also observed normalization of lysosomal mass, total GAGs, and oxidative stress, which are some of the major findings regarding the pathophysiological events in MPS IVA. These results represent a proof-of-concept of the use of CRISPR/Cas9 nickase strategy for the development of a novel therapeutic alternative for MPS IVA.
黏多糖贮积症IVA型（MPS IVA）是一种溶酶体贮积症（LSD），由编码GALNS酶的基因突变引起。GALNS活性缺乏导致糖胺聚糖（GAGs）硫酸角质素和硫酸软骨素6的积累。尽管自2014年以来酶替代疗法已被批准用于治疗MPS IVA，但对于这种疾病仍有未满足的医疗需求，需要改进治疗方法。基于CRISPR/Cas9的基因疗法已在几种溶酶体贮积症中进行了测试，结果令人鼓舞，但迄今为止尚未在MPS IVA上进行检测。在这项工作中，我们首次验证了使用CRISPR/Cas9（使用Cas9切口酶）在MPS IVA体外模型中敲入包含GALNS cDNA的表达盒。结果显示表达盒成功同源重组到AAVS1位点，并且GALNS活性长期增加，达到野生型水平的40%。我们还观察到溶酶体质量、总GAGs和氧化应激的正常化，这些是关于MPS IVA病理生理事件的一些主要发现。这些结果代表了使用CRISPR/Cas9切口酶策略开发MPS IVA新型治疗替代方案的概念验证。
