TY - JOUR
T1 - Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria
AU - Martínez-Pizarro, Ainhoa
AU - Álvarez, Mar
AU - Dembic, Maja
AU - Lindegaard, Caroline A.
AU - Castro, Margarita
AU - Richard, Eva
AU - Andresen, Brage S.
AU - Desviat, Lourdes R.
PY - 2024/6/1
Y1 - 2024/6/1
N2 - 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.
AB - 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.
KW - antisense oligonucleotides
KW - CRIPSR/Cas
KW - knock-in cell model
KW - phenylketonuria
KW - splicing
KW - Oligonucleotides, Antisense/genetics
KW - Introns/genetics
KW - Humans
KW - Gene Editing/methods
KW - Alternative Splicing/genetics
KW - CRISPR-Cas Systems/genetics
KW - Phenylalanine Hydroxylase/genetics
KW - RNA Splicing/genetics
KW - Phenylketonurias/genetics
KW - Exons/genetics
U2 - 10.1089/nat.2024.0014
DO - 10.1089/nat.2024.0014
M3 - Journal article
C2 - 38591802
AN - SCOPUS:85190523672
SN - 2159-3337
VL - 34
SP - 134
EP - 142
JO - Nucleic Acid Therapeutics
JF - Nucleic Acid Therapeutics
IS - 3
ER -