Antisense oligonucleotides (ASOs) have been shown to effectively rescue defects in preclinical Timothy syndrome models, a neurodevelopmental disorder associated with significant morbidity and mortality. Stanford University researchers developed an ASO therapy that corrects a splicing error that results in the expression of a gain-of-function variant in a subunit of a voltage-gated calcium channel that is widely expressed in the developing brain.

The ASO restored the cellular and functional deficiencies that normally affect cortical neurons in this neurodevelopmental disorder in cortical organoids and forebrain assembloids derived from Timothy syndrome patients. These findings were translated into an in vivo model, in which human stem cell-derived cortical organoids were implanted and integrated into the rat brain. These experiments show a new genetic rescue strategy for a devastating neurodevelopmental disorder.

This research article, “Antisense oligonucleotide therapeutic approach for Timothy syndrome” was published in Nature.

A spliced gain-of-function variant

Timothy syndrome type 1 affects several organ systems and is one of the most common genetic causes of autism spectrum disorder and epilepsy. The neurodevelopmental disorder is caused by a gain-of-function missense variant in CACNA1C, a subunit of the L-type voltage-gated calcium channel CaV1.2 that is widely expressed in the developing and adult nervous systems. Common CACNA1C variants have also been strongly associated with other neuropsychiatric disorders, such as schizophrenia, bipolar disorder, and attention deficit hyperactivity disorder.

Neurons from Timothy syndrome type 1 patients have significantly higher levels of the CACNA1C splice form with exon 8A compared to control neurons. CACNA1C splicing is developmentally regulated in mice and humans, shifting exon utilization from exon 8A to 8 during early development. The inclusion of either of these mutually exclusive spliced exons has been shown to result in channel isoforms with similar electrophysiological properties. These findings suggest that decreasing the inclusion of the 8A isoform of CACNA1C could be a therapeutic strategy for Timothy syndrome type 1.

Preclinical potential of ASOs for Timothy Syndrome

ASOs are short oligonucleotides that can bind to target RNAs, activate cytoplasmic degradation of target RNAs, or regulate the splicing of pre-messenger RNAs within the cell. Several ASOs targeting splicing have progressed from the laboratory to the clinic as therapeutic options, including those for spinal muscular atrophy and Duchenne muscular dystrophy. 

Co-lead authors Xiaoyu Chen, PhD, and Fikri Birey, PhD, developed an ASO-based intervention to decrease exon-8A inclusion in neural cells from three individuals with Timothy syndrome type 1.

After identifying ASOs that inhibit exon 8a splicing in a dose- and time-dependent manner, Stanford University researchers tested candidate ASOs on human cortical neurons in two- or three-dimensional cultures derived from Timothy syndrome type 1 patients. This rescued both delayed channel inactivation and the defect in depolarization-induced calcium elevation. The ASO-mediated switch from CACNA1C exon 8A to 8 successfully rescued defects in patient-derived cortical organoids and migration in forebrain assembloids.

Chen and Birey used a transplantation model developed recently in the Sergiu P. Pașca, MD, lab to test the ASO therapy’s effectiveness in vivo. In this setup, human stem cell-derived cortical organoids are transplanted into the somatosensory cortex of newborn athymic rats, where they grow and develop mature cell types that integrate into sensory and motivational circuits. Chen, Birey, and colleagues from the Pașca lab discovered that intrathecal injection of an ASO into rats transplanted with human Timothy syndrome type 1 cortical organoids resulted in a robust downregulation of exon-8A. This was accompanied by the rescue of depolarization-induced calcium defects and aberrant activity-dependent dendritic morphology.

These studies show how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies for reversing disease-relevant neural pathophysiology. When taken together, these studies show a new genetic rescue strategy for a devastating neurodevelopmental disorder.

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