Extended Data Fig. 7: Generation and characterization of the Idua-W401X(TGA) mouse model.
From: An engineered UGA suppressor tRNA gene for disease-agnostic AAV delivery
(a) Schematics showing a portion of the mouse Idua gene. The sequences of both the wildtype (WT) allele and the knock-in (KI) allele are shown. In the KI allele, the G-to-A mutation is colored in red, and the resulting TGA nonsense mutation is underlined. The Sanger sequencing chromatograph for the KI allele is shown at the bottom, with the G-to-A mutation highlighted in red background. (b) Bar graph showing the iduronidase (IDUA) enzymatic activity levels in the liver and heart from WT (Idua+/+) and homozygous KI (IduaKI/KI) mice. Data are presented as percentage of the WT levels. (c) Bar graph showing the Idua mRNA levels in the liver and heart from WT and homozygous KI mice normalized to Tbp mRNA levels. (d) Determination of the amino acid residues incorporated by sup-tRNA-mediated readthrough at codon 401 by mass spectrometry. A schematics showing the workflow is shown on the left, and a pie chart showing detected amino acids and their percentages is shown on the right. (e) Schematics showing the workflow of in vitro assessment of IDUA variants with different residues at codon 401 for their expression and enzyme activities. (f) Western blot image showing the expression levels of IDUA variants with a FLAG tag at the C-terminus in patient fibroblasts transfected with individual mIdua cDNA constructs. The amino acid residues encoded by codon 401 are labelled with three-letter and single-letter abbreviations. X denotes the mIdua cDNA construct with a TGA at codon 401. (g) Quantification of the anti-FLAG signal intensity in the western blot as shown in (f). Data are normalized to the WT mIdua cDNA encoding Trp at codon 401. (h) IDUA activity in patient fibroblasts transfected with various mIdua cDNA constructs. (i) Specific IDUA activity (that is, IDUA activity normalized to protein expression level) from various mIdua cDNA constructs. Data are normalized to the Trp construct as 100%. In b and c, each dot represents an individual animal (n = 3 or 4). In f-i, each lane or dot represents a biological repeat (n = 3). Data are presented as mean and standard deviation. Statistical analysis was performed with two-sided t-test in b and c, and one-way ANOVA followed by Dunnett’s multiple comparisons test in i. Schematic in e created in BioRender. Zou, H. (2025) https://BioRender.com/dqjni51.
