mdx mouse肌营养不良实验鼠课件

The treatment of muscular dystrophy (1) The treatment of pharmaceuticals (2) Gene therapy (3) Cell transplantation therapy Mesenchymal Stem Cell Transplant to Treat Duchenne Muscular Dystrophy The mdx mouse肌营养不 良实验鼠 These dystrophic mice have a point mutation that exchanged the base cytosine (C) in position 3,185 in exon 23 against a thymine (T). This converted the CAA codon, which normally codes for the amino acid glutamine, to the triplet TAA (in the mRNA it become UAA) which is a premature stop codon. n它是一种点突变实验鼠 ，基因突变发生在肌营 养不良蛋白23号外显子 的第3185号核苷酸上， 将本该编码为谷氨酸盐 氨基酸的CAA码，改变 成了TAA码，这是一个 停止合成蛋白质的密码 ，造成了点突变，使得 翻译蛋白质的过程提前 停止. The treatment of pharmaceuticals Studies on mdx mice have turned up one drug-an antibiotic called gentamicin(正大霉素) that causes the animals to synthesize dystrophin and protects them from muscle damage. Gentamicin cure in mdx mice nGentamicin is an antibiotic that causes the RNA translation mechanism in the ribosomes to ignore such a prema-ture stop codon, i.e. to read through it and produce a dystrophin molecule . nGentamicin injections into mdx mice led to the appearance of new dystrophin in up to 20% of the muscle fibers. And mice with this amount of dystrophin had ameliorated改善的 clinical symptoms. Gentamicin cure in humen nAbout 5% to 10% of Duchenne boys have a point mutation in their dystrophin gene which changed an amino acid code word into one of the three stop codons. In the mRNA, these codons become UGA, UAG, and UAA and cause the protein synthesis to shut down prematurely, before the new protein, in this case dystrophin, is ready. nSo ,we can use the same method to cure the muscular dystrophy in human being. Other discovery about this drug nThe researchers analyzed and discovered that gentamicin exists in 5 varieties with different structures. Although they all had the same antibiotic activity, their read through efficiency was quite distinct and their toxicity also. nThis means that gentamicin preparations would have to be carefully selected before they are used for clinical trials. The disadvantages of this drug nAlthough Gentamicin has the advantage of being a well known drug whose alternative use as a possible therapy for Duchenne muscular dystrophy . nAs read-through does not occur at the gene level but during protein synthesis in the ribosomes, treatment will have to be repeated periodically. nHowever, gentamicin retards减缓 the growth of the animals and in humans can cause serious side effects like inner ear and kidney damage. New chemical compound that is better than gentamicin nalmost one million chemical compounds were tested for their ability to cause read-through of premature stop codons. nThe most promising compound identified is PTC124 . It is about 50 times more efficient than gentamicin in restoring the dystrophin synthesis in mdx mice nit can be given orally口服, a very important advantage for a future therapy. Methods of Gene therapy n将外源的抗肌萎缩蛋白基因引入杜氏进 行性肌营养不良和贝克型进行性肌营养 不良患者的肌肉细胞. n用病毒、DNA质粒等作为载体将外源的 完整的或部分的抗肌萎缩蛋白导入杜氏 进行性肌营养不良和贝克型进行性肌营 养不良患者的肌肉细胞，但需要注意免 疫应答的问题。 腺病毒作为载体 Using virus as the vector n腺病毒为载体 n机理:病毒载体将自身粘附在肌肉细胞膜 上的一个特殊结构，这一结构被叫做腺 病毒受体（也被叫做舵手因为它引 导腺病毒与肌细胞膜结合），然后将它 所装载的抗肌萎缩蛋白基因通过肌细胞 膜渗透到肌肉细胞中，这一过程叫做卸 载。 Using virus as the vector n在肌肉组织成熟后，不再分裂时，这些 受体的量被大大调低，因此当肌肉细胞 在分裂时，用腺病毒作为载体递送抗肌 萎缩蛋白基因的效率较高。 Using virus as the vector n用腺相关病毒作为载体进行基因治疗 n优势在于移植基因的效率比普通的腺病 毒高 n其劣势在于无法将整条的外源的抗肌萎 缩蛋白全部送入杜氏进行性肌营养不良 患者的肌肉细胞，不得不将抗肌萎缩蛋 白基因截短，使无法发挥出全部的功能 。 Using virus as the vector n贝克型进行性肌营养不良患者的肌肉组 织退化的速度之所以大大慢于杜氏进行 性肌营养不良，就是因为在这些患者的 肌肉细胞中，尚存有截短的抗肌萎缩蛋 白。 n利用截短的蛋白虽然无法完全治愈他们 的疾病，但是可以将其转变为较良性的 贝克型进行性肌营养不良 The comparison between Becker and Duchenne The experimental evidence about this method n正常的抗肌萎缩蛋白基因两个端点，富含半 光氨酸区域和中心的杆状区域这四个区域 The experimental evidence about this method n抗肌萎缩蛋白的中心杆状区域被截短， 不会明显的失去抗肌萎缩蛋白对肌肉组 织的保护功能。这样,一种大约只有正常 的抗肌萎缩蛋白一半大的被截短的抗肌 萎缩蛋白，基本保持了正常抗肌萎缩蛋 白的功能。 The experimental evidence about this method n截短的抗肌萎缩蛋白基因通过腺相关病毒送 入杜氏进行性肌营养不良实验鼠，停止甚至 部分逆转了这些实验鼠肌肉组织的退化. n这些抗肌萎缩蛋白虽然比自然发生的贝克型 进行性肌营养不良患者肌肉细胞内的抗肌萎 缩蛋白还要短一些，但是功能却更强。 有的 甚至产生相当的正常的抗肌萎缩蛋白. Plasmid-mediated gene therapy The advantages of plasmid: n(1) it doesnt contain any protein. n(2) it doesnt trigger any problems of immune rejection Plasmid-mediated gene therapy nUsing the gene engineering nPlasmid-mediated gene therapy can restore dystrophin expression in skeletal muscle in the mdx mouse Plasmid-mediated gene therapy nUsing a phage integrase(C31)-it can mediate the integration of suitably modified plasmids into the mammalian genome. nUsing a luciferase荧光素酶 expression plasmid-we monitored plasmid gene expression in living mice by bioluminescence imaging Plasmid-mediated gene therapy nplasmid-mediated dystrophin expression in mdx mouse was enhanced by integration nIn the presence of integrase, dystrophin expression was distributed along a much greater length of individual fibers 关于免疫应答问题 n由于质粒不包含任何蛋白,使研究者更加 清晰地了解免疫系统是否会对这些被导 入的外源抗肌萎缩蛋白基因进行攻击 n而在用病毒载体或者细胞载体递送抗肌 萎缩蛋白基因的方法，有时较难分辨免 疫系统攻击的究竟是被导入的抗肌萎缩 蛋白基因本身，还是基因载体。 Summary Pharmaceuticals: gentamicin PTC124 Vector: virus as vector plasmid as vector Gene replacement therapy nThe gene which induce the muscular dystrophy is called dystrophin. nIt can express one kind of protein (dystrophin), which play a key role in muscular dystrophy. Gene therapy nAt the present time, there is no treatment to stem the degenerative changes that occur with any of the muscular dystrophy. nHowever, patients can rely on the development of gene therapy . nGene therapy involves giving a person a normal gene to replace a defective or missing gene Exon skipping can change a DMD into a BMD nWe know that the reasons of the DMD are gene deleting, gene replicating and gene mutant . nSo we can induce gene splicing. nSplice sites are specific sequences at the borders of exons and introns which are essential for the correct removal of the non-coding intron sequences from the pre-mRNA. The splicing itself is accomplished by spliceosomes, a complex of many small RNAs and proteins whose details and interplay are still not completely understood. intro n exon For example nNow, exon 45 of the dystrophin gene is the single most frequently deleted exon in Duchenne boys causing an out-of -frame mRNA. Experiment nIn laboratory experiments with a culture of muscle cells from a Duchenne patient with an exon 45 deletion, exon 46 could be skipped. Experiment nHowever, if both exons 45 and 46 are missing simultaneously, the reading frame is not disturbed.