A new DMD model with a humanized glycome

描述(由申请人提供)：在这项资助中，我们建议定义一种重要的人类特有的糖基化变化，即N-羟基神经氨酸(Neu5Gc)的丢失，在Duchenne肌营养不良症(DMD)和肢体Girdle肌营养不良症2D中的作用。Neu5Gc是唾液酸的一种形式，由于人类CMAH基因的失活突变，所有人类中都没有这种唾液酸。相比之下，Neu5Gc在心脏和骨骼肌中是一种丰富的唾液酸形式，在除人类之外的几乎所有其他哺乳动物中，包括类人猿。为了评估人类CMAH在DMD中的作用，我们创造了Cmah-/-mdx小鼠，有效地人源化了小鼠糖基化的这一方面。几十年来，MDX小鼠一直被研究为DMD的模型，DMD是一种相对常见的最终致命的X连锁神经肌肉疾病。尽管在几乎所有的肌肉细胞中，MDX小鼠都模仿DMD中有缺陷的Dstrophin基因的丢失，但MDX小鼠并没有表现出与儿科疾病相当的小鼠症状。患有DMD的儿童通常在12岁时表现为行走能力丧失，随后出现呼吸和/或心力衰竭，通常在生命的第三个十年死亡。相比之下，MDX小鼠在正常寿命接近尾声之前，这些相同的特征几乎没有变化，与野生型动物相比，正常寿命通常只减少1-2个月。与MDX动物相比，与野生型相比，cmah-/-mdx小鼠在8个月大时表现出88%的横隔肌力量不足和66%的心脏小梁肌力不足，其中一半的动物在11个月前死亡。与MDX相比，这类小鼠也表现出明显的行走障碍。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适当的人类遗传变化的小动物模型中，这些表型的早期和强有力的呈现将是翻译研究的一大财富，以确定DMD和其他人类疾病的治疗方法。在这项建议的目标1中，我们将研究Neu5Gc缺乏导致的功能丧失以及功能免疫方面的获得，因为它们与Cmah/-mdx小鼠的疾病严重程度有关。在目标2中，我们将设计治疗方法来抵消由于缺失cmah而导致的疾病严重程度的增加，这些治疗方法可以转化为对肌营养不良患者的治疗。在目标3中，我们将研究Cmah在第二个肌营养不良小鼠模型中的作用，该模型是肢体Girdle肌营养不良2D模型。这些实验将调查Cmah缺乏导致疾病严重程度增加的原因，评估Cmah缺乏在改变疾病方面的普遍性，并开发可以改善DMD和LGMD2D患者肌肉营养不良的人类CMAH缺失的治疗方法。

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.