DC2: Correction of splicing defects in inherited metabolic diseases with splice switching ASO (SS-ASO)

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Supervisors: Prof. L.R. Desviat and E. Richard

Host Institute: Universidad Autónoma de Madrid, Spain (www.uam.es)

Secondments planned: Astherna, The Netherlands; University College London, UK

Doctoral program: PhD in Biomedical Biosciences of Universidad Autonoma de Madrid

Anticipated starting date: September 1st, 2025

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Project description:

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The project focuses on splicing defects (exon skipping, pseudoexon insertion and others) in inherited metabolic disease (IMD) genes expressed in liver and resulting in neurological and/or multisystemic affectation, such as hyperphenylalaninemias (phenylketonuria, PKU) and organic acidemias (propionic acidemia). SS-ASO designed to block aberrant pseudoexon insertion increase transcript and protein levels, both in alleles with pseudoexon activating deep intronic variants and those with hypomorphic variants, thus can be a potential treatment strategy for different patients (doi: 10.1016/j.omtn.2023.102101; 10.1089/nat.2021.0066). In our group (www.cbm.uam.es/lab220), we have also identified vulnerable exons with weak 3’ and/or 5’ splice sites prone to exon skipping with different exonic variants, which are (mis) classified as missense or nonsense. SS-ASO targeting splice silencers may correct the exon skipping defect (doi: 10.1089/nat.2024.0014). In this project, the PhD student will use a combination of methods: bioinformatic analysis, minigenes, RNA affinity studies, CRISPR/Cas and transcript analysis to model and characterize splice defects identified in IMD patients and to identify splice regulatory sequences. In all cases, after the design of SS-ASO to correct  each type of splice defect, testing will be performed in cellular models, edited HepG2 cells, patient’s fibroblasts and/or hepatocyte-like cells derived from edited iPSCs. Transcript and protein analysis, enzymatic activity assays and targeted biochemical readouts will be performed to assess initial efficacy. Optimization based on SS-ASO walk across the target region and use of different chemistries will be performed to obtain a lead candidate SS-ASO for future testing in mouse models. Knowledge of the translatability into the clinic of the in vitro findings will be acquired during secondments.

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