Uncovering mechanisms and developing novel therapeutic strategies for TBCD-related developmental and epileptic encephalopathy - Project Summary/Abstract Variants in the tubulin folding cofactor D (TBCD) gene result in a rare early-onset encephalopathy with neurodevelopmental and neurodegenerative features including developmental regression, epilepsy, microcephaly, hypotonia, and spasticity which progresses to immobilization, ventilation, and premature death. TBCD functions as a tubulin-specific chaperone, which plays a crucial role in regulating microtubule dynamics throughout neurodevelopment. Since first reported 7 years ago, modest progress has been made in understanding molecular and cellular mechanisms contributing to disease pathogenesis and therapeutic development. Our team has recently established and characterized the largest known cohort of induced pluripotent stem cell (iPSC) lines from a phenotypic spectrum of 5 TBCD patients, from whom we gathered corroborative clinical data. We show TBCD patient-derived cerebral organoids (COs) display a phenotypic spectrum of profound growth deficits, microtubule instability, and early neurodegeneration, highlighting the value of COs to recapitulate TBCD patient phenotypes. To compare the effect of different pathogenic variants on clinical, molecular, and biochemical disease progression in vivo, we have generated a novel allelic series of TBCD variant knock-in mouse models. We propose the central hypothesis that TBCD deficiency causes microtubule instability and cell-type specific dysfunction during critical milestones of neurodevelopment, which include molecular deficits in progenitor proliferation, migration, neuronal differentiation, and ultimately neurodegeneration. Through the use of novel transgenic mouse and complementary organoid models, we will test this hypothesis and in parallel establish a strong framework for developing a gene replacement therapy for patients with TBCD deficiency. In Aim 1, we will determine neuropathological mechanisms of TBCD variants using patient derived COs including through use of electrophysiology, single cell RNAseq, and CRISPR gene correction. In Aim 2, we will comprehensively investigate the molecular and clinical phenotypes of a novel allelic series of TBCD variant knock-in mouse models utilizing clinically relevant outcome measures such as CatWalk XT® gait analysis, MRI, and EEG. In Aim 3, we will determine the therapeutic efficacy of targeted AAV-mediated gene replacement by modulating the cell specificity, expression level, and neurodevelopmental stage in which functional TBCD is delivered. To enhance translation and validate that the lead construct provides comparable TBCD expression in the context of human brain cell types, we will treat previously established patient-derived COs with the lead AAV9-TBCD construct and assess for phenotypic rescue. Utilizing this rigorous and multidisciplinary approach, we aim to uncover molecular and cellular mechanisms underlying the profound deficits in TBCD-related developmental and epileptic encephalopathy; establish the first tractable in vitro and in vivo models of TBCD; and evaluate a highly translatable and potentially transformative AAV-mediated gene replacement therapy.
