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残疾为特征的无行为能力发育障碍， 发育迟缓(运动、语言)和运动障碍(躯干肌张力减退、远端肌肉 高张力、痉挛、肌力下降)。孩子们步态痉挛或无法行走，情况严重。 影响他们的生活质量。他们的父母优先考虑他们是否会走路。目前没有任何治疗方法 可用。该综合征是由cTnnb1(-连环蛋白)单倍体不足引起的，这是一种新的致病因素 导致部分或全部缺失的变体。因为这是一种单基因疾病，我们建议进行第一次测试， 作为概念验证，基因治疗可以提供有效和安全的治疗结果。我们建议 基因治疗将改善降低的-连环蛋白水平，并显著纠正表型。我们将测试我们的 使用CTNNB1杂合性的两种临床前模型--我们的活体小鼠系和人IPSC--的假说 衍生肌管，一种与这种综合征的运动障碍有关的人类细胞类型。我们将使用新的 开发的下一代肌嗜性AAVs可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AAVs的一种治疗方法可提供安全、有效和持久的转基因表达 在小鼠和非人灵长类骨骼肌体内和体外人肌管中，并显著 治疗小鼠两种不同的神经肌肉疾病的表型。我们的CTNNB1全球杂合子 小鼠表现出类似于儿童CTNNB1综合征的关键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、协调性和握力的降低。 一窝产仔。我们还发现HET肌肉中的分子变化由调节蛋白质水平的变化组成 工作肌肉的收缩、松弛和力量。此外，我们发现肌肉衍生的水平降低 正常运动神经元突触前终末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CTNNB1het小鼠的-catenin水平 活体骨骼肌和人体肌管。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，提高单次后检测持续时间的水平 治疗，且无不良反应。我们的目标是提高小鼠和人类HET中-CAT的水平 肌肉细胞，以接近野生型窝仔和同基因回复对照的正常基线范围 分别为肌管。AIM 2将测试在体内和体内运动能力的统计显著改善 基因治疗的小鼠和人het模型中的分子变化。我们将使用数量行为 检测、免疫印迹和RT-qPCR。我们的研究将为我们提供对 CTNNB1单倍体缺陷引起的病理生理改变的基因治疗。这些关键因素 两个临床前模型的概念验证研究将为提供生命的治疗策略的设计提供信息 改变CTNNB1综合征儿童的福利。