FROM PATHOGENICITY ASSESSMENT TO PERSONALIZED RNA THERAPY: RHOBTB2 IN NEURODEVELOPMENT

RHOBTB2-related neurodevelopmental disorder (NDD) is caused by heterozygous missense variants in RHOBTB2 and is characterized by severe developmental and epileptic encephalopathy (DEE), profound intellectual disability (ID) and movement disorders. In our clinic, an individual with DEE, recurrent, intractable epileptic states and developmental delay has been identified, harboring a variant of unknown significance (VUS) in RHOBTB2. Based on reported variants, a Gain-of-Function (GoF) mechanism is hypothesized. In this study, we aim to establish the pathogenicity of the VUS through functional experiments and develop a personalized RNA therapy.
In our functional experiments, cDNA constructs will be used expressing either RHOBTB2 WT, published pathogenic variants or our identified VUS. Protein stability and turnover of the VUS will be assessed in HEK293T cells. In addition, we will study its effect on neural outgrowth and morphology using murine primary hippocampal neurons. Furthermore, the constructs are in utero electroporated in mice pups to assess the effect of the VUS on neural migration and epileptogenesis.
In parallel, we are developing a personalized RNA therapy using allele-specific gapmer antisense oligonucleotides (ASOs). Gapmer ASOs are short, chemically modified sequences of RNA and DNA that can bind and degrade complementary RNA targets. We have designed and tested ASOs targeting the patient variant, as well as single nucleotide polymorphisms (SNPs) in RHOBTB2 to downregulate the variant transcript.
Altogether, these functional experiments enable the pathogenicity assessment of the VUS and open avenues to tailored ASO therapy for this individual, which could possibly be extended to other affected individuals.