Chieppa M N, Perota A, Corona C, Grindatto A, Lagutina I, Vallino Costassa E, Lazzari G, Colleoni S, Duchi R, Lucchini F, Caramelli M, Bendotti C, Galli C, Casalone C
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Torino, Italy.
Neurodegener Dis. 2014;13(4):246-54. doi: 10.1159/000353472. Epub 2013 Oct 23.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of treating ALS.
肌萎缩侧索硬化症（ALS）是一种致命的神经退行性疾病，有两种临床难以区分的形式：散发性（SALS）和家族性（FALS），后者与几种基因突变有关，大多以显性方式遗传。近20%的FALS形式与铜/锌超氧化物歧化酶（SOD1）基因突变有关。ALS的研究依赖于转基因模型，特别是携带人SOD1基因第93位密码子由甘氨酸转换为丙氨酸（G93A）的小鼠。尽管G93A转基因小鼠已广泛应用于临床试验和基础研究，但最近众多可靠来源对其能否忠实地再现人类疾病的适用性提出了质疑。此外，由于猪在解剖学、生理学和生物化学特征上比啮齿动物更接近人类，科学界已经预见猪是一种有吸引力的替代非人灵长类动物的人类疾病模型。在此基础上，我们通过体外转染培养的体细胞并结合体细胞核移植（SCNT）制备了首个猪ALS模型。为实现这一目标，我们开发了一种SOD1（G93A）（超氧化物歧化酶1在甘氨酸93-丙氨酸处突变）载体，能够在原代成年雄性猪成纤维细胞（PAF）中促进高且稳定的转基因表达。转染、克隆筛选和转基因表达水平评估后，选择的SOD1（G93A）PAF集落用作SCNT程序中的核供体。SOD1（G93A）胚胎被移植到受体母猪体内，妊娠足月。共有5只仔猪在人工饲养和断奶后存活并正常发育，成年。初步分析揭示了转基因在猪组织中的整合和hSOD1（G93A）表达，360°表型特征分析正在进行。我们相信，我们的SOD1（G93A）猪将在啮齿动物模型的基础研究与治疗ALS的现实之间提供一座重要的桥梁。
