Patient-derived models of <i>UBA5-</i>associated encephalopathy identify defects in neurodevelopment and highlight potential therapeutic avenues.

Abstract

<i>UBA5</i> encodes for the E1 enzyme of the UFMylation cascade, which plays an essential role in endoplasmic reticulum (ER) homeostasis. The clinical phenotypes of <i>UBA5</i>-associated encephalopathy include developmental delays, epilepsy, and intellectual disability. To date, there is no humanized neuronal model to study the cellular and molecular consequences of <i>UBA5</i> pathogenic variants. We developed and characterized patient-derived cortical organoid cultures from two patients with compound heterozygous variants in <i>UBA5</i>. Both shared the same missense variant, which encodes a hypomorphic allele (p.A371T), along with a nonsense variant (p.G267* or p.A123fs*4). Single-cell RNA sequencing of 100-day organoids identified defects in GABAergic interneuron development. We demonstrated aberrant neuronal firing and reduction in size of patient-derived organoids. Mechanistically, we showed that ER homeostasis is perturbed along with an exacerbated unfolded protein response pathway in engineered U87-MG cells and patient-derived organoids expressing <i>UBA5</i> pathogenic variants. We also assessed two potential therapeutic modalities that augmented UBA5 protein abundance to rescue aberrant molecular and cellular phenotypes. We assessed SINEUP, a long noncoding RNA that augments translation efficiency, and CRISPRa, a modified CRISPR-Cas9 approach to augment transcription efficiency to increase UBA5 protein production. Our study provides a humanized model that allows further investigations of <i>UBA5</i> variants in the brain and highlights promising approaches to alleviate cellular aberrations for this rare, developmental disorder.