A new DMD model with a humanized glycome

描述（由申请人提供）：在这项授权中，我们建议定义一个重要的人类特异性糖基化变化的作用，N-羟乙酰神经氨酸（Neu 5Gc）的损失，在杜氏肌营养不良症（DMD）和肢带肌营养不良症2D。Neu 5Gc是唾液酸的一种形式，由于人CMAH基因的失活突变，所有人都不存在。相比之下，Neu 5Gc是除人类以外几乎所有其他哺乳动物（包括类人猿）心脏和骨骼肌中丰富的唾液酸形式。为了评估人CMAH在DMD中的作用，我们创建了Cmah-/-mdx小鼠，实际上使小鼠糖基化的这一方面人源化。mdx小鼠作为DMD的模型已经研究了几十年，DMD是一种相对常见且最终致命的X连锁神经肌肉疾病。尽管在几乎所有的肌肉细胞中，mdx小鼠都模拟了DMD中有缺陷的基因dystrophin的丢失，但mdx小鼠并没有表现出与儿科疾病相当的小鼠表现。患有DMD的儿童通常在12岁时表现出心律失常，随后是呼吸和/或心力衰竭，并且通常在生命的第三个十年中死亡。相比之下，mdx小鼠在这些相同的特征上几乎没有变化，直到接近它们的正常寿命结束，其通常相对于野生型动物仅减少1-2个月。与mdx动物相比，Cmah-/-mdx小鼠在8个月大时，与野生型相比，显示出88%的膈肌强度缺陷和66%的心脏小梁肌强度缺陷，其中一半的动物在11个月大时死亡。相对于mdx，这些小鼠还显示出显著受损的amplitude。这些表型是DMD发病率和死亡率的主要驱动因素，在小鼠基因组中携带遗传上适当的人类样变化的小动物模型中早期和稳健地呈现这些表型将是转化研究的巨大资产，以确定DMD和其他人类疾病的疗法。在本提案的目的1中，我们将研究Neu 5Gc缺乏症的功能丧失以及功能免疫方面的获得，因为它们与Cmah-/-mdx小鼠中疾病的严重程度有关。在目标2中，我们将设计治疗方法来抵消Cmah缺失导致的疾病严重程度增加，这可以转化为肌营养不良症患者的治疗方法。在目标3中，我们将研究Cmah在第二种肌营养不良小鼠模型中的作用，即Sgca-/-肢带肌营养不良2D模型。这些实验将研究Cmah缺乏导致疾病严重程度增加的原因，评估Cmah缺乏在改变疾病中的普遍性，并开发治疗方法以抵消人CMAH的损失，从而改善DMD和LGMD 2D患者的肌营养不良症。

DESCRIPTION (provided by applicant): In this grant, we propose to define the role of an important human-specific change in glycosylation, the loss of N-glycolylneuraminic acid (Neu5Gc), in Duchenne muscular dystrophy (DMD) and in Limb Girdle Muscular Dystrophy 2D. Neu5Gc is a form of sialic acid that is absent in all humans due to an inactivating mutation in the human CMAH gene. By contrast, Neu5Gc is an abundant form of sialic acid in heart and skeletal muscles in almost all other mammals besides humans, including the great apes. To assess the role of human CMAH in DMD, we have created Cmah-/-mdx mice, in effect humanizing this aspect of mouse glycosylation. The mdx mouse has been studied for several decades as a model for DMD, a relatively common and ultimately fatal X- linked neuromuscular disorder. Despite mimicking loss of dystrophin, the gene defective in DMD, in almost all muscle cells, mdx mice do not show the mouse equivalent of a pediatric presentation of disease. Children with DMD show loss of ambulation, usually by 12 years of age, followed by respiratory and/or cardiac failure and death usually in the third decade of life. mdx mice, by contrast, show little change in these same features until near the end of their normal lifespan, which is typically reduced by only 1-2 months relative to wild type animals. Cmah-/-mdx mice, by contrast to mdx animals, show an 88% deficit in diaphragm muscle strength and a 66% deficit in cardiac trabecular muscle strength, compared to wild type, by 8 months of age, with half of animals dying by 11 months. Such mice also show significantly impaired ambulation relative to mdx. The early and robust presentation of these phenotypes, which are the primary drivers of DMD morbidity and mortality, in a small animal model that carries a genetically appropriate human-like change in the mouse genome will be a great asset to translational research to identify therapies for DMD and other human diseases. In Aim 1 of this proposal, we will investigate both the loss of function aspects to Neu5Gc deficiency as well as gain of function immune aspects as they relate to disease severity in Cmah-/-mdx mice. In Aim 2, we will devise therapies to offset the increased disease severity resulting from deletion of Cmah that could be translated into therapies for muscular dystrophy patients. In Aim 3, we will study the role of Cmah in a second mouse model of muscular dystrophy, the Sgca-/- model of Limb Girdle Muscular Dystrophy 2D. These experiments will investigate the cause of increased disease severity resulting from Cmah deficiency, assess the generality of Cmah deficiency in altering disease, and develop therapies to offset the loss of human CMAH that could ameliorate muscular dystrophy in DMD and LGMD2D patients.