Discovery and Therapeutic Correction of Neurodevelopmental Splicing Defects in Fragile X Syndrome Using Human Brain Models

By Rachid Mazroui, PhD; Emeline Lelong, PhD; Samer Mohamed Hussein, PhD; and Karun Singh, PhD

Recent research shows that alternative RNA splicing, the process by which a single gene can produce multiple RNA versions, or isoforms, plays a critical role in brain development, neurogenesis, neuronal activity, and long-term brain function. Changes in RNA splicing have been linked to several neurological conditions, including amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, autism spectrum disorder, and intellectual disability. Importantly, recent studies show that widespread mis-splicing occurs in the brains of Fmr1-deficient mice, suggesting that abnormal RNA splicing during early brain development may contribute directly to Fragile X syndrome (FXS).

Although modern RNA sequencing methods have helped identify important transcriptional and translational changes in Fragile X syndrome, most previous studies relied on short-read sequencing technologies that cannot fully detect the many different isoforms produced in the developing brain. As a result, a large portion of RNA isoform diversity in FXS remains unexplored.

In this project, we will use human brain organoid models, laboratory-grown miniature brain-like structures derived from stem cells, to study how RNA splicing changes during early cortical development in Fragile X syndrome. Using an advanced technique called single-cell long-read RNA sequencing, we will identify specific RNA isoforms that are altered during early neurogenesis. Because the cerebral cortex is one of the brain regions most affected in Fragile X syndrome, understanding isoform diversity in this region is especially important.

This work will generate the first dataset of its kind describing isoform-level changes at single-cell resolution during early human cortical development in Fragile X syndrome. We will then test whether some of these altered isoforms can be corrected using antisense oligonucleotides, or ASOs, an emerging therapeutic approach that can adjust RNA processing directly.

Together, these studies may identify new treatment strategies aimed at correcting RNA splicing defects downstream of FMRP loss. By defining which RNA isoforms are altered early in brain development, this project could open a new path toward therapies that target the underlying biology of Fragile X syndrome.