Development of therapeutic strategies for Rett syndrome

雷特综合征是女性精神发育迟缓的最常见原因之一，由MECP2基因突变引起。该基因编码DNA结合蛋白MeCP2，它在基因表达的表观遗传控制中起着关键作用。令人惊讶的是，在有条件的MeCP2基因敲除小鼠中显示，MeCP2的重新表达可以逆转临床症状，表明Rett综合征是一种可治疗的疾病。此前，我们已经建立并鉴定了一个携带MECP2基因无义突变的小鼠模型，该突变最常见于Rett综合征患者(p.R168X)。这项研究项目的目的是开发治疗Rett综合征的策略，旨在重新表达或取代MeCP2。阅读无义突变：我们之前已经证明，通过各种氨基糖苷类药物的处理，可以在转基因细胞和Kockin小鼠的成纤维细胞中诱导MeCP2的显著表达。然而，由于化合物的毒性和血脑屏障的低渗透率，体内实验到目前为止还没有成功。为了克服这些问题，我们将使用新的化合物来增强通读性，并测试合成的氨基糖苷类化合物，这些化合物已经被改变，以允许更好地渗透血脑屏障。此外，我们将鞘内注射氨基糖苷类药物，从而绕过全身毒性和血脑屏障。此外，我们打算口服Ataluren(Translarna®)，这是一种小分子，最近被批准用于Duchenne肌营养不良症的直读治疗。蛋白质替代疗法：第二种疗法旨在直接取代MeCP2。由于MeCP2不能进入细胞，因此产生了一种融合蛋白，其中MeCP2蛋白与人类免疫缺陷病毒-1的蛋白转导结构域融合。为了绕过血脑屏障，融合蛋白将在鞘内注射。骨髓移植：使用敲门小鼠模型，我们能够证明，与之前报道的不同，骨髓移植不会延长雄性小鼠的存活时间。由于Rett综合征是一种几乎只影响女性的疾病，由于X-基因失活，女性在50%的细胞中表达正常的MECP2等位基因，现在将在雌性小鼠身上进行实验。

Rett syndrome, one of the most frequent causes of mental retardation in females, is caused by mutations in the MECP2 gene. The gene codes for the DNA binding protein MeCP2, which has a key role in the epigenetic control of gene expression. Surprisingly it was shown in conditional Mecp2 knockout mice that re-expression of Mecp2 can reverse the clinical symptoms indicating that Rett syndrome is a treatable disorder. Previously we have generated and characterized a mouse model that carries the nonsense mutation in the MECP2 gene most commonly found in patients with Rett syndrome (p.R168X). The aim of this research project is to develop therapeutic strategies for Rett syndrome that aim to re-express or replace MeCP2. Readthrough of nonsense mutations: We have shown previously that it is possible to induce significant expression of MeCP2 in transfected cells and in fibroblasts from the kockin mice by treatment with various aminoglycosides. Experiments in vivo, however, were so far unsuccessful due to the toxicity of the compounds and the low penetration of the blood brain barrier. To overcome these problems we will use novel compounds that enhance the readthrough and test synthetic aminoglycosides that have been altered to allow better penetration of the blood brain barrier. Moreover, we will administer the aminoglycosides intrathecally thereby circumventing the systemic toxicity and the blood brain barrier. Furthermore, we intend to treat the mice orally with Ataluren (Translarna®), a small molecule which was approved recently for readthrough therapy in Duchenne muscular dystrophy. Protein replacement therapy: The second therapeutic approach is aimed to replace MeCP2 directly. As MeCP2 cannot enter the cells a fusion protein was generated wherein the MeCP2 protein is fused to the protein transduction domain of the human immunodeficiency virus-1. To bypass the blood brain barrier the fusion protein will be given intrathecally. Bone marrow transplantation: Using the knockin mouse model we were able to show that other than previously reported, bone marrow transplantation does not lead to an extended survival in male mice. As Rett syndrome is a disorders that almost exclusively affects females, who due to the X-inactivation express a normal MECP2 allele in 50% of the cells, the experiments will now be carried out in female mice.