Centro de Terapia Gênica do Hospital de Clinicas de Porto Alegre, R. Ramiro Barcelos 2350, 90035-903 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Ciências Farmacêuticas da Universidade Federal do Rio Grande do Sul (UFRGS), Faculdade de Farmácia, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil.
Centro de Terapia Gênica do Hospital de Clinicas de Porto Alegre, R. Ramiro Barcelos 2350, 90035-903 Porto Alegre, RS, Brazil; Programa de Pós-Graduação em Genética e Biologia Molecular da Universidade Federal do Rio Grande do Sul (UFRGS), Departamento de Genética, Campus do Vale, Av. Bento Gonçalves, 9500, 91501-970 Porto Alegre, RS, Brazil.
J Control Release. 2018 Oct 28;288:23-33. doi: 10.1016/j.jconrel.2018.08.031. Epub 2018 Aug 28.
Mucopolysaccharidosis type I (MPS I) is a multisystemic disorder caused by the deficiency of alpha-L-iduronidase (IDUA) that leads to intracellular accumulation of glycosaminoglycans (GAG). In the present study we aimed to use cationic liposomes carrying the CRISPR/Cas9 plasmid and a donor vector for in vitro and in vivo MPS I gene editing, and compare to treatment with naked plasmids. The liposomal formulation was prepared by microfluidization. Complexes were obtained by the addition of DNA at +4/-1 charge ratio. The overall results showed complexes of about 110 nm, with positive zeta potential of +30 mV. The incubation of the complexes with fibroblasts from MPS I patients led to a significant increase in IDUA activity and reduction of lysosomal abnormalities. Hydrodynamic injection of the liposomal complex in newborn MPS I mice led to a significant increase in serum IDUA levels for up to six months. The biodistribution of complexes after hydrodynamic injection was markedly detected in the lungs and heart, corroborating the results of increased IDUA activity and decreased GAG storage especially in these tissues, while the group that received the naked plasmids presented increased enzyme activity especially in the liver. Furthermore, animals treated with the liposomal formulation presented improvement in cardiovascular parameters, one of the main causes of death observed in MPS I patients. We conclude that the IDUA production in multiple organs had a significant beneficial effect on the characteristics of MPS I disease, which may bring hope to gene therapy of Hurler patients.
黏多糖贮积症 I 型（MPS I）是一种多系统疾病，由α-L-艾杜糖苷酸酶（IDUA）缺乏引起，导致糖胺聚糖（GAG）在细胞内积累。本研究旨在使用携带 CRISPR/Cas9 质粒和供体载体的阳离子脂质体进行体外和体内 MPS I 基因编辑，并与裸质粒治疗进行比较。脂质体制备通过微流化处理进行。通过添加 DNA 至+4/-1 电荷比获得复合物。总体结果表明复合物的粒径约为 110nm，具有+30mV 的正 ζ 电位。将复合物与 MPS I 患者的成纤维细胞孵育可显著提高 IDUA 活性并减少溶酶体异常。对新生 MPS I 小鼠进行脂质体复合物的水力注射可使血清 IDUA 水平在长达六个月的时间内显著升高。水力注射后复合物的生物分布在肺部和心脏中明显检测到，这与 IDUA 活性增加和 GAG 储存减少尤其是在这些组织中的结果相符，而接受裸质粒的组则表现出肝酶活性增加。此外，用脂质体制剂治疗的动物心血管参数得到改善，这是 MPS I 患者死亡的主要原因之一。我们得出结论，多个器官中 IDUA 的产生对 MPS I 疾病的特征具有显著的有益影响，这可能为 Hurler 患者的基因治疗带来希望。
