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

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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.