Goldyne Savad Institute of Gene Therapy, Hadassah Medical Center, The Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel.
Institute for Physiological Chemistry, Medical Faculty, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
J Neuromuscul Dis. 2024;11(5):905-917. doi: 10.3233/JND-240056.
GNE Myopathy is a unique recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, caused by mutations in the GNE gene which is a key enzyme in the biosynthesis of sialic acid. To date, the precise pathophysiology of the disease is not well understood and no reliable animal model is available. Gne KO is embryonically lethal in mice.
To gain insights into GNE function in muscle, we have generated an inducible muscle Gne KO mouse. To minimize the contribution of the liver to the availability of sialic acid to muscle via the serum, we have also induced combined Gne KO in liver and muscle.
A mouse carrying loxp sequences flanking Gne exon3 was generated by Crispr/Cas9 and bred with a human skeletal actin (HSA) promoter driven CreERT mouse. Gne muscle knock out was induced by tamoxifen injection of the resulting homozygote GneloxpEx3loxp/HSA Cre mouse. Liver Gne KO was induced by systemic injection of AAV8 vectors carrying the Cre gene driven by the hepatic specific promoter of the thyroxine binding globulin gene.
Characterization of these mice for a 12 months period showed no significant changes in their general behaviour, motor performance, muscle mass and structure in spite of a dramatic reduction in sialic acid content in both muscle and liver.
We conclude that post weaning lack of Gne and sialic acid in muscle and liver have no pathologic effect in adult mice. These findings could reflect a strong interspecies versatility, but also raise questions about the loss of function hypothesis in Gne Myopathy. If these findings apply to humans they have a major impact on therapeutic strategies.
GNE 肌病是一种独特的隐性神经肌肉疾病，其特征为成人发病、进行性缓慢的远端和近端肌肉无力，由 GNE 基因突变引起，该基因是唾液酸生物合成的关键酶。迄今为止，该疾病的确切病理生理学机制尚不清楚，也没有可靠的动物模型。Gne KO 在小鼠中胚胎致死。
深入了解 GNE 在肌肉中的功能，我们生成了一种可诱导的肌肉 Gne KO 小鼠。为了最大限度地减少肝脏通过血清中唾液酸向肌肉的可用性的贡献，我们还诱导了肝脏和肌肉中的联合 Gne KO。
通过 Crispr/Cas9 生成了携带 Gne 外显子 3 侧翼loxP 序列的小鼠，并与携带人骨骼肌肌动蛋白（HSA）启动子驱动的 CreERT 小鼠杂交。通过注射他莫昔芬诱导产生的纯合子 GneloxpEx3loxp/HSA Cre 小鼠的肌肉 Gne KO。通过注射携带甲状腺素结合球蛋白基因的肝特异性启动子驱动的 Cre 基因的 AAV8 载体诱导肝脏 Gne KO。
对这些小鼠进行了 12 个月的特征描述，尽管肌肉和肝脏中的唾液酸含量显著降低，但它们的一般行为、运动表现、肌肉质量和结构均无明显变化。
我们得出结论，断奶后肌肉和肝脏中缺乏 Gne 和唾液酸在成年小鼠中没有病理影响。这些发现可能反映了强烈的种间多功能性，但也对 GNE 肌病的功能丧失假说提出了质疑。如果这些发现适用于人类，它们将对治疗策略产生重大影响。
