Journal: Cell
Article Title: In vivo prime editing rescues alternating hemiplegia of childhood in mice
doi: 10.1016/j.cell.2025.06.038
Figure Lengend Snippet: (A and B) Top: Correction of Atp1a3 mutations (red) to the wild type nucleotide (green) with co-installation of benign synonymous (silent) edits (blue). Bottom: Correction with RNA electroporation in mouse fibroblasts ( n = 3). Dots show individual replicates. (A) Correction in C57BL/6J Atp1a3 D801N c.2401A mouse primary fibroblasts. (B) Correction in E815K mouse primary fibroblasts. (C) D801N-PE-AAV9 (top) and E815K-PE-AAV9 (bottom) optimized components. (D) PE-AAV9 system encoding split-intein PE6c prime editor halves, epegRNA, ngRNA, and dsgRNA. Npu N or Npu C, Nostoc punctiforme intein N-terminal or C-terminal halves, respectively; P EFS , Promoter, elongation factor 1α short; P hU6 , Promoter, human U6 polymerase III; P mU6, Promoter, mouse U6 polymerase III; W3, minimized gamma portion of the woodchuck hepatitis virus post-transcriptional regulatory element; bGH, bovine growth hormone polyadenylation signal. (E) Mice were characterized by molecular, behavioral, and biometric readouts. (F–I) HTS quantification ( n = 4–7) from gDNA (F and G) and cDNA (H and I) of bulk and GFP + nuclei from D801N mice (F and H) or E815K mice (G and I) injected with PE-AAV9 (PE) or phosphate-buffer saline (PBS, vehicle). (J and K) Specific activity of Atp1a3 in hippocampal homogenates of PE- or vehicle-treated D801N mice (J, n =3–16) or E815K mice (K, n = 5–12). Two-way ANOVA with Tukey’s multiple comparisons. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. For (F)–(K), dots show values from individual mice with tissue collected at P28. For (A), (B), and (F)–(I), samples start from ~50% wild-type genotype heterozygous baseline (dotted line). See also .
Article Snippet: For the validation of TaqMan probes (species specificity or species cross-reaction), TaqMan probes were tested on mouse brain cDNA derived from wild type mice (brain tissue from JR #664, C57BL/6J, The Jackson Laboratory) and human cDNA derived from a mixture of human tissues including the CNS (Takara #636693).
Techniques: Electroporation, Virus, Injection, Saline, Activity Assay
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