Defining the Potential of Gene Therapy to Correct Motor Disabilities of CTNNB1 Syndrome Using in Vivo Mouse and in Vitro Human Cell Models

利用体内小鼠和体外人类细胞模型确定基因疗法纠正 CTNNB1 综合征运动障碍的潜力

• 批准号: 10809254
• 负责人: Michele H. Jacob
• 金额: $ 45.38万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-25 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10809254
• 关键词: Adverse effects; Behavior; Binding; Biological Assay; CTNNB1 gene; Cancer Etiology; Capsid; Cell model; Child; Childhood; Chronic; Clinical Trials; Cognitive; Data; Developmental Delay Disorders; Disease; Distal; Dose; Duchenne muscular dystrophy; Enzyme Inhibition; Exhibits; Family; Felis catus; Gene Mutation; Generations; Genes; Glycogen Synthase Kinases; Goals; Half-Life; Hand Strength; Heterozygote; Human; Impaired cognition; In Vitro; Intellectual functioning disability; K ATPase; Language; Learning; Leg; Life; Liver; Measures; Medical; Messenger RNA; Modeling; Molecular; Monitor; Motor; Motor Neurons; Mus; Muscle; Muscle Cells; Muscle Fibers; Muscle Hypertonia; Muscle hypotonia; Myopathy; National Institute of Neurological Disorders and Stroke; Neurons; Nonprofit Organizations; Outcome; PF4 Gene; Parents; Pathogenicity; Patients; Performance; Pharmacotherapy; Phenotype; Pitt-Hopkins syndrome; Pre-Clinical Model; Presynaptic Terminals; Proteins; Quality of life; Relaxation; Reporting; Research Personnel; Risk Reduction; Safety; Signal Transduction; Signaling Molecule; Skeletal Muscle; Spastic Gait; Syndrome; Testing; Therapeutic; Toxic effect; Variant; Viral; Viral Genes; Walking; Western Blotting; Wheelchairs; behavior test; cancer risk; cell type; cognitive disability; design; developmental disease; disability; gene therapy; human model; improved; in vivo; induced pluripotent stem cell; inhibitor; insight; loss of function; motor impairment; motor learning; mouse model; muscle strength; neuromuscular; next generation; nonhuman primate; reduced muscle strength; response; small molecule; small molecule inhibitor; spasticity; therapeutic evaluation; therapeutically effective; therapy outcome; transcription factor; transduction efficiency; transgene expression; young adult

CTNNB1 syndrome is an incapacitating developmental disorder characterized by intellectual disabilities, global developmental delays (motor, language) and motor disabilities (truncal muscle hypotonia, distal muscle hypertonia, spasticity, reduced muscle strength). The children have spastic gait or inability to walk, severely impacting their quality of life. Their parents prioritize their being able to walk. No treatments are currently available. The syndrome is caused by CTNNB1 (-catenin) haploinsufficiency due to de novo pathogenic variants causing partial or complete deletions. Because it is a monogenetic disorder, we propose the first tests, as proof-of-concept, that gene therapy can provide effective and safe therapeutic outcomes. We propose that gene therapy will improve the reduced -catenin levels and significantly remedy the phenotypes. We will test our hypothesis using two preclinical models of CTNNB1 heterozygosity, our in vivo mouse line and human iPSC derived myotubes, a human cell type relevant to the motor disabilities of this syndrome. We will use newly developed, next-generation muscle tropic AAVs to express CTNNB1/-catenin in the heterozygote models. New studies report that one treatment with these AAVs provides safe, effective and long-lasting transgene expression in mouse and non-human primate skeletal muscles in vivo and human myotubes in vitro, and significantly remedies the phenotypes of two different neuromuscular myopathies in mice. Our CTNNB1 global heterozygote mouse exhibits motor and cognitive impairments that resemble key features of CTNNB1 syndrome in children. The motor impairments include reduced motor learning, coordination, and grip strength, relative to wildtype littermates. We also find molecular changes in the het muscle consisting of altered levels of proteins that regulate contractility, relaxation, and force in working muscle. Further, we find reduced levels of a muscle-derived retrograde signaling factor required for normal motor neuron presynaptic terminal maturation. Our Aim 1 studies will test whether direct transgene expression of CTNNB1 will improve -catenin levels in the CTNNB1 het mouse skeletal muscle in vivo and human myotubes. Our dose-response tests will identify the lowest viral doses that increase the reduced -catenin levels in the het models, test duration of the improved levels after the single treatment, and absence of adverse effects. Our goal is to increase -cat levels in the mouse and human het muscle cells, to resemble the normal baseline ranges of wildtype littermates and isogenic revertant control myotubes, respectively. Aim 2 will test for statistically significant improvements in motor capabilities in vivo and in molecular changes in the gene therapy treated mouse and human het models. We will use quantitative behavior assays, immunoblotting and RT-qPCR. Our studies will provide the first insights into the efficacy and safety of gene therapy for remedying pathophysiological changes caused by CTNNB1 haploinsufficiency. These critical proof-of-concept studies in two preclinical models will inform the design of therapeutic strategies to provide life altering benefits to children with CTNNB1 syndrome.

CTNNB1综合征是一种以智力障碍为特征的失能性发育障碍，全球范围内 发育迟缓（运动、语言）和运动障碍（躯干肌张力减退、远端肌肉 肌张力亢进、痉挛、肌力下降）。孩子们出现严重的痉挛步态或无法行走 影响他们的生活质量。他们的父母优先考虑他们能够行走。目前尚无治疗方法 可用的。该综合征是由新生致病性导致的 CTNNB1（-连环蛋白）单倍体不足引起的。 导致部分或完全删除的变体。因为它是一种单基因疾病，我们提出第一个测试， 作为概念验证，基因疗法可以提供有效且安全的治疗结果。我们建议 基因治疗将改善降低的-连环蛋白水平并显着纠正表型。我们将测试我们的 使用 CTNNB1 杂合性的两个临床前模型（我们的体内小鼠系和人类 iPSC）进行假设 衍生肌管，一种与该综合征的运动障碍相关的人类细胞类型。我们将使用新的 开发了下一代肌肉热带 AAV，以在杂合子模型中表达 CTNNB1/-连环蛋白。新的 研究报告称，使用这些 AAV 进行的一种治疗可提供安全、有效且持久的转基因表达 在小鼠和非人类灵长类动物体内骨骼肌和体外人类肌管中，并且显着 治疗小鼠两种不同神经肌肉肌病的表型。我们的 CTNNB1 全球杂合子 小鼠表现出类似于儿童 C​​TNNB1 综合征的主要特征的运动和认知障碍。 与野生型相比，运动障碍包括运动学习、协调性和握力下降 同窝的。我们还发现热肌中的分子变化包括调节蛋白质水平的改变 工作肌肉的收缩性、松弛性和力量。此外，我们发现肌肉来源的一种物质的水平降低 正常运动神经元突触前末梢成熟所需的逆行信号因子。我们的目标 1 研究 将测试 CTNNB1 的直接转基因表达是否会提高 CTNNB1 het 小鼠中的 β-连环蛋白水平 体内骨骼肌和人类肌管。我们的剂量反应测试将确定最低的病毒剂量 增加het模型中降低的-连环蛋白水平，单次后改善水平的测试持续时间 治疗，并且没有不良反应。我们的目标是提高小鼠和人类 het 中的 -cat 水平 肌肉细胞，类似于野生型同窝小鼠和同基因回复回复对照的正常基线范围 分别是肌管。目标 2 将测试体内和体内运动能力的统计显着改善 基因疗法治疗小鼠和人类 het 模型中的分子变化。我们将使用定量行为 分析、免疫印迹和 RT-qPCR。我们的研究将首次深入了解以下药物的功效和安全性： 用于补救 CTNNB1 单倍体不足引起的病理生理变化的基因治疗。这些关键的 两个临床前模型的概念验证研究将为设计提供生命的治疗策略提供信息 改变对 CTNNB1 综合征儿童的益处。