Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria.

The PAH gene encodes the hepatic enzyme phenylalanine hydroxylase (PAH), and its deficiency, known as phenylketonuria (PKU), leads to neurotoxic high levels of phenylalanine. PAH exon 11 is weakly defined, and several missense and intronic variants identified in patients affect the splicing process. Recently, we identified a novel intron 11 splicing regulatory element where U1snRNP binds, participating in exon 11 definition. In this work, we describe the implementation of an antisense strategy targeting intron 11 sequences to correct the effect of PAH mis-splicing variants. We used an in vitro assay with minigenes and identified splice-switching antisense oligonucleotides (SSOs) that correct the exon skipping defect of PAH variants c.1199+17G>A, c.1199+20G>C, c.1144T>C, and c.1066-3C>T. To examine the functional rescue induced by the SSOs, we generated a hepatoma cell model with variant c.1199+17G>A using CRISPR/Cas9. The edited cell line reproduces the exon 11 skipping pattern observed from minigenes, leading to reduced PAH protein levels and activity. SSO transfection results in an increase in exon 11 inclusion and corrects PAH deficiency. Our results provide proof of concept of the potential therapeutic use of a single SSO for different exonic and intronic splicing variants causing PAH exon 11 skipping in PKU.

Spectral library search for improved TMTpro labelled peptide assignment in human plasma proteomics.

Clinical biomarker discovery is often based on the analysis of human plasma samples. However, the high dynamic range and complexity of plasma pose significant challenges to mass spectrometry-based proteomics. Current methods for improving protein identifications require laborious pre-analytical sample preparation. In this study, we developed and evaluated a TMTpro-specific spectral library for improved protein identification in human plasma proteomics. The library was constructed by LC-MS/MS analysis of highly fractionated TMTpro-tagged human plasma, human cell lysates, and relevant arterial tissues. The library was curated using several quality filters to ensure reliable peptide identifications. Our results show that spectral library searching using the TMTpro spectral library improves the identification of proteins in plasma samples compared to conventional sequence database searching. Protein identifications made by the spectral library search engine demonstrated a high degree of complementarity with the sequence database search engine, indicating the feasibility of increasing the number of protein identifications without additional pre-analytical sample preparation. The TMTpro-specific spectral library provides a resource for future plasma proteomics research and optimization of search algorithms for greater accuracy and speed in protein identifications in human plasma proteomics, and is made publicly available to the research community via ProteomeXchange with identifier PXD042546.