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

CTNNB 1综合征是一种以智力残疾为特征的失能性发育障碍，全球 发育迟缓（运动、语言）和运动残疾（躯干肌张力减退、远端肌张力减退） 张力亢进、痉挛状态、肌力降低）。孩子们有痉挛性步态或无法行走，严重的 影响他们的生活质量。他们的父母优先考虑他们能够走路。目前没有治疗方法 available.该综合征是由CTNNB 1（β-连环蛋白）单倍不足引起的， 导致部分或完全缺失的变体。因为这是一种单基因遗传疾病，我们提出了第一个测试， 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β-连环蛋白水平并显著纠正表型。我们将测试我们的 使用CTNNB 1杂合性的两种临床前模型，我们的体内小鼠系和人iPSC 衍生的肌管，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 开发了下一代肌肉嗜性AAV以在杂合子模型中表达CTNNB 1/β-连环蛋白。新 研究报道，用这些AAV的一种治疗提供安全、有效和持久的转基因表达， 在小鼠和非人灵长类动物体内骨骼肌和体外人肌管中， 治疗小鼠中两种不同神经肌肉肌病的表型。我们的CTNNB 1全杂合子 小鼠表现出类似于儿童CTNNB 1综合征的关键特征的运动和认知障碍。 相对于野生型，运动障碍包括运动学习、协调和握力降低 同窝出生的我们还发现het肌肉中的分子变化包括调节蛋白质水平的改变， 收缩性、松弛性和工作肌肉的力量。此外，我们发现肌肉来源的 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Aim 1研究 将测试CTNNB 1的直接转基因表达是否会提高CTNNB 1 het小鼠中的β-连环蛋白水平 体内骨骼肌和人肌管。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β-连环蛋白水平，单次给药后改善水平的测试持续时间 治疗，无不良反应。我们的目标是增加小鼠和人类het中的het-cat水平， 肌细胞，以类似于野生型同窝仔和同基因回复突变体对照的正常基线范围 肌管。目标2将测试体内和体内运动能力的统计学显著改善。 基因治疗处理的小鼠和人HET模型中的分子变化。我们将使用定量行为 测定、免疫印迹和RT-qPCR。我们的研究将首次深入了解 基因治疗用于治疗由CTNNB 1单倍不足引起的病理生理变化。这些关键 在两个临床前模型中进行的概念验证研究将为治疗策略的设计提供信息， 改变CTNNB 1综合征患儿的获益。

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.