MeCP2 reactivation from the inactive X chromosome as treatment for Rett syndrome

从失活的 X 染色体重新激活 MeCP2 作为雷特综合征的治疗方法

基本信息

批准号：
10826905
负责人：
Antonio Bedalov
金额：
$ 84.9万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-15 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10826905
关键词：
Adult Age Alleles Animal Model Animals Brain Breathing Cells Cessation of life Chimeric Proteins Chromatin Clinical Trials DNA Methylation Data Development Disease Electrophysiology (science)Enterobacteria phage P1 Cre recombinase Epigenetic Process Exhibits Face Female Future Gene Mutation Genes Genetic Genetic Transcription Heterozygote Hindlimb Histones Human Immune Immune response In Vitro Injections Knockout Mice Lead Life Link Longevity Loss of Heterozygosity Measures Mediating Methods Methyl-CpG-Binding Protein 2 Methylation Modeling Mus Mutation Neurodevelopmental Disorder Neurologic Deficit Neuronal Differentiation Neuronal Dysfunction Neurons Patients Pattern Phenotype Proteins Public Health Reporter Repression Research Rett Syndrome Severities Symptoms Testing Therapeutic Time Tissues Transgenic Mice Transgenic Model Transgenic Organisms Untranslated RNA Viral X Chromosome X Inactivation bisulfite sequencing demethylation detection sensitivity early childhood effective therapy experimental study genomic locus girls human embryonic stem cell in vivo limb movement loss of function mutation male methylome mouse model nanoluciferase neonatal death neonatal encephalopathy neuronal cell body novel promoter restoration reverse genetics success tool transcriptome transcriptome sequencing treatment strategy vector whole genome

Adult Age Alleles Animal Model Animals Brain Breathing Cells Cessation of life Chimeric Proteins Chromatin Clinical Trials DNA Methylation Data Development Disease Electrophysiology (science)Enterobacteria phage P1 Cre recombinase Epigenetic Process Exhibits Face Female Future Gene Mutation Genes Genetic Genetic Transcription Heterozygote Hindlimb Histones Human Immune Immune response In Vitro Injections Knockout Mice Lead Life Link Longevity Loss of Heterozygosity Measures Mediating Methods Methyl-CpG-Binding Protein 2 Methylation Modeling Mus Mutation Neurodevelopmental Disorder Neurologic Deficit Neuronal Differentiation Neuronal Dysfunction Neurons Patients Pattern Phenotype Proteins Public Health Reporter Repression Research Rett Syndrome Severities Symptoms Testing Therapeutic Time Tissues Transgenic Mice Transgenic Model Transgenic Organisms Untranslated RNA Viral X Chromosome X Inactivation bisulfite sequencing demethylation detection sensitivity early childhood effective therapy experimental study genomic locus girls human embryonic stem cell in vivo limb movement loss of function mutation male methylome mouse model nanoluciferase neonatal death neonatal encephalopathy neuronal cell body novel promoter restoration reverse genetics success tool transcriptome transcriptome sequencing treatment strategy vector whole genome

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项目摘要

Project Summary Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that mainly manifests in girls without effective treatment. RTT is caused by loss-of-function mutations of the Methyl CpG-binding Protein 2 gene (MECP2) on the X chromosome. The majority of RTT patients are females heterozygous for MECP2 mutation, in which random X chromosome inactivation (XCI) during development leaves ~50% of neurons without functional MeCP2 protein, thereby creating cell-autonomous neuronal dysfunction. Corresponding mutations in hemizygous males lead to severe neonatal encephalopathy and early death. Mice carrying null alleles of Mecp2 closely mimic symptoms seen in patients, including irregular breathing, stereotypical limb movements and shortened lifespan, and are thus faithful models of RTT. Male Mecp2-null mice start exhibiting symptoms as early as 30-60 days of age, with only half of the animals surviving beyond 75 days. Heterozygous females have a near-normal lifespan with neurologic deficits that are delayed (~6 months) and highly variable in severity. A major breakthrough in RTT research was the demonstration that RTT-like symptoms in adult mice can be reversed by genetic or viral restoration of MeCP2 protein. Thus, reactivation of the silenced wild type (WT) allele of MECP2 from the inactive X chromosome (Xi) presents an exciting therapeutic opportunity that attacks the root cause of this disease by restoring MeCP2 function. Our preliminary data demonstrate that targeted demethylation of the MECP2 promoter is sufficient to reactivate MECP2 from the Xi in human RTT ESCs and neurons in vitro as well as in vivo in the RTT mouse brain. This was accomplished using a dCas9-Tet1 protein targeted to the MECP2 locus via sgRNA, a DNA methylation editing tool pioneered by Dr. Shawn Liu. We hypothesize that reactivation of MECP2 from the Xi will rescue RTT-associated phenotypes in mice. We have developed two new transgenic mouse models (1,2) and two novel methods of delivery of epigenetic editors (3,4) for MECP2 reactivation in vivo including: 1) Xi-linked Mecp2-NanoLuciferase-tdTomato dual reporter mice, which enables high sensitivity detection and quantification of Mecp2 reactivation; 2) MeCP2-null heterozygous female model of severe RTT with exclusive inactivation of the X-chromosome harboring the WT Mecp2 for measuring reactivation-induced rescue which circumvents evaluating delayed and variable phenotypes in Mecp2 heterozygous with random XCI; 3) Cre recombinase-dependent dCas9-Tet1 transgenic line enables efficient and tissue-specific DNA methylation editing in vivo; and 4) dCasMini-Tet1, in which the bulky dCas9 (4.1 kb) is replaced with a compact dCasMini (1.6 kb) for delivery of a methylation editor via a single AAV9 vector. This combination of transgenic models to measure reactivation efficacy (1) and rescue (2) with those that enable efficient in vivo editing via genetic means (3) and single vector AAV9-mediated delivery (4) comprise a state-of- the-art tool kit to evaluate the in vivo feasibility of a MECP2 reactivation strategy for treatment of RTT and other X-linked disorders in females.

项目摘要 RETT综合征（RTT）是一种严重的X连锁神经发育障碍，主要在没有的女孩中表现有效的治疗。 RTT是由甲基CpG结合蛋白2基因的功能丧失突变引起的（MECP2）在X染色体上。大多数RTT患者是女性用于MECP2突变的杂合子，其中在发育过程中随机X染色体灭活（XCI）叶有约50％的神经元没有功能性MECP2蛋白，从而产生细胞自主神经元功能障碍。相应的突变半合子雄性导致严重的新生儿脑病和早期死亡。携带MECP2无效等位基因的小鼠在患者中看到的紧密模仿症状，包括不规则的呼吸，刻板印象的肢体运动和寿命缩短，因此是RTT的忠实模型。雄性mecp2-null小鼠开始表现出症状早在30-60天，只有一半的动物存活超过75天。杂合雌性有具有延迟（〜6个月）且严重程度高的神经功能缺陷的近期寿命。一个 RTT研究中的主要突破是成年小鼠中RTT样症状的证明是通过MECP2蛋白的遗传或病毒恢复而反转。因此，沉默的野生型（WT）等位基因的重新激活来自非活动X染色体（XI）的MECP2构成了激动人心的治疗机会通过恢复MECP2功能而导致该疾病的原因。我们的初步数据证明了目标 MECP2启动子的去甲基化足以使人类RTT ESC中的XI重新激活MECP2，并且 RTT小鼠脑中的体外神经元以及体内。这是使用DCAS9-TET1蛋白完成的通过SGRNA针对MECP2基因座，Sgrna是Shawn Liu博士开创的DNA甲基化编辑工具。我们假设从XI中重新激活MECP2将挽救小鼠中与RTT相关的表型。我们有开发了两种新的转基因小鼠模型（1,2）和两种新型表观遗传编辑器的递送方法（3,4）对于体内MECP2重新激活，包括：1）XI连接的MECP2-NANANONONONONONONOLUCIFERASE-TDTTOMATO双重报道小鼠，这可以使MECP2重新激活的高灵敏度检测和定量； 2）mecp2-null杂合重度RTT的女性模型，具有X染色体的独家灭活，该X染色体带有WT MECP2 测量重新激活引起的救援，该救援绕过评估MECP2中延迟和可变表型的救援随机XCI杂合子； 3）CRE重组酶依赖性DCAS9-TET1转基因线使得有效和组织特异性的DNA甲基化编辑体内； 4）DCASMINI-TET1，其中笨重的DCAS9（4.1 Kb）为由紧凑的dcasmini（1.6 kb）取代，用于通过单个AAV9载体传递甲基化编辑器。这转基因模型的结合以测量重新激活功效（1）和拯救（2）与能够通过遗传均值（3）和单载体AAV9介导的递送（4）的有效体内编辑（4） ART工具套件以评估MECP2重新激活策略用于治疗RTT和其他其他的体内可行性女性的X连锁疾病。