Breaking: CRISPR Gene Activation Rescues Neuronal Defects in UBA5-Associated Encephalopathy

“We felt it was important to study UBA5-related disease in human cells, and specifically in human neuronal cells. Our goal was to identify disease-relevant changes in a cellular model and then test therapeutic approaches”Heather Mefford

Scientists at St. Jude Children's Research Hospital have developed the first patient-derived neuronal model for UBA5-associated encephalopathy, a rare developmental and epileptic encephalopathy characterised by severe developmental delay, hypotonia, spasticity, microcephaly, and epilepsy. This research provides critical insights into disease mechanisms and evaluates therapeutic strategies targeting gene expression.

»We felt it was important to study UBA5-related disease in human cells, and specifically in human neuronal cells. Our goal was to identify disease-relevant changes in a cellular model and then test therapeutic approaches,« explains Heather Mefford, MD, PhD, and senior author of the study.

UBA5-associated encephalopathy results from biallelic pathogenic variants in the UBA5 gene, which encodes an E1 activating enzyme essential for ufmylation, a ubiquitin-like post-translational modification process that regulates endoplasmic reticulum (ER) homeostasis. While previous studies relied on non-neuronal human cells or animal models, this study establishes cortical organoids from induced pluripotent stem cells of patients with UBA5 pathogenic variants, enabling investigation of human-specific neurodevelopmental effects.

“It was important for us to generate a patient-derived relevant neuronal model like organoids to test out potential therapies”Helen Chen

»Neurodevelopmental disorders are challenging to study in a laboratory setting, and rare paediatric disorders are even more so,« notes co-lead author Helen Chen, PhD. »Therefore, it was important for us to generate a patient-derived relevant neuronal model like organoids to test out potential therapies. We are highly appreciative of the opportunity to work closely with patients and the advocacy group and including them to be part of our study.«

The researchers generated cortical organoids from two patients with compound heterozygous UBA5 variants – each carrying the p.A371T hypomorphic variant plus a nonsense variant (p.G267* or p.A123fs*4). Single-cell RNA sequencing revealed a significant reduction in GABAergic interneuron populations in patient-derived organoids, with GABAergic marker expression markedly decreased. Patient organoids were consistently 25% smaller than controls and exhibited abnormal neuronal activity patterns – increased firing rates but decreased network burst frequency.

“We are energized by our findings, especially the promising results using SINEUP and CRISPRa as potential therapies. Our next step is to pinpoint the therapeutic window while establishing potential delivery methods”Helen Chen

Molecular analyses demonstrated perturbed ER homeostasis in cells with UBA5 pathogenic variants, resulting in exacerbated unfolded protein response pathway activity. The researchers tested two approaches to augment UBA5 expression: SINEUP, a long non-coding RNA that enhances translation, and CRISPRa, a modified CRISPR-Cas9 system that increases transcription. Both methods achieved modest 1.5-2 fold increases in UBA5 protein abundance, successfully restoring ER homeostasis, and treatment with SINEUP transiently rescued aberrant neuronal activity in patient-derived organoids.

»We are energized by our findings, especially the promising results using SINEUP and CRISPRa as potential therapies,« says Helen Chen. »Our next step is to pinpoint the therapeutic window while establishing potential delivery methods.«

These therapeutic strategies capitalise on the unique genetic profile of most UBA5 patients. Over 65% of patients share the p.A371T variant, which retains partial function. Since healthy individuals homozygous for p.A371T show no disease symptoms, increasing the abundance of this hypomorphic allele represents a promising therapeutic approach.

The research was conducted by Helen Chen and Heather Mefford at St. Jude Children's Research Hospital, and it was published in Science Translational Medicine on 7 May, 2025.