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.
