Watanabe Masahito, Miyamoto Hitomaru, Okamoto Kazutoshi, Nakano Kazuaki, Matsunari Hitomi, Kazuki Kanako, Hasegawa Koki, Uchikura Ayuko, Takayanagi Shuko, Umeyama Kazuhiro, Hiramuki Yosuke, Kemter Elisabeth, Klymuik Nikolai, Kurome Mayuko, Kessler Barbara, Wolf Eckhard, Kazuki Yasuhiro, Nagashima Hiroshi
Meiji University International Institute for Bio-Resource Research, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
Department of Chromosome Biomedical Engineering, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan.
Mol Ther Nucleic Acids. 2023 Jul 23;33:444-453. doi: 10.1016/j.omtn.2023.07.021. eCollection 2023 Sep 12.
Mammalian artificial chromosomes have enabled the introduction of extremely large amounts of genetic information into animal cells in an autonomously replicating, nonintegrating format. However, the evaluation of human artificial chromosomes (HACs) as novel tools for curing intractable hereditary disorders has been hindered by the limited efficacy of the delivery system. We generated dystrophin gene knockout (-KO) pigs harboring the HAC bearing the entire human via a somatic cell cloning procedure (DYS-HAC-cloned pig). Restored human dystrophin expression was confirmed by immunofluorescence staining in the skeletal muscle of the DYS-HAC-cloned pigs. Viability at the first month postpartum of the DYS-HAC-cloned pigs, including motor function in the hind leg and serum creatinine kinase level, was improved significantly when compared with that in the original -KO pigs. However, decrease in systemic retention of the DYS-HAC vector and limited production of the DMD protein might have caused severe respiratory impairment with general prostration by 3 months postpartum. The results demonstrate that the use of transchromosomic cloned pigs permitted a straightforward estimation of the efficacy of the DYS-HAC carried in affected tissues/organs in a large-animal disease model, providing novel insights into the therapeutic application of exogenous mammalian artificial chromosomes.
哺乳动物人工染色体能够以自主复制、非整合的形式将极大量的遗传信息导入动物细胞。然而，作为治疗难治性遗传性疾病的新型工具，人类人工染色体（HACs）的评估受到递送系统有限功效的阻碍。我们通过体细胞克隆程序培育出了携带含有整个人类 的HAC的肌营养不良蛋白基因敲除（-KO）猪（DYS-HAC克隆猪）。通过免疫荧光染色在DYS-HAC克隆猪的骨骼肌中证实了人类肌营养不良蛋白表达的恢复。与原始-KO猪相比，DYS-HAC克隆猪产后第一个月的活力，包括后腿的运动功能和血清肌酸激酶水平，有显著改善。然而，DYS-HAC载体全身保留率的降低和DMD蛋白产量的有限可能导致产后3个月出现严重的呼吸功能障碍并伴有全身衰弱。结果表明，在大型动物疾病模型中，使用转染色体克隆猪能够直接评估受影响组织/器官中携带的DYS-HAC的功效，为外源性哺乳动物人工染色体的治疗应用提供了新的见解。
