Figure 4.

In vivo base editing of Pah^enu2 mouse corrected the Pah(F263S) mutation and normalized Phe metabolism

(A) Schematic maps showing the expression cassettes in AAV8 vector genomes. Hepatocyte-specific human TBG promoter was used to drive the expression of protein components of the base editor to ensure the base editing was limited to hepatocyte. (B) Time line of the in vivo experiments. Postnatal day 2 (P2) mice were fused with AAV8-miniSaCBE-PLUS-PKU or AAV8-miniSaCBE-PLUS-ctrl with dosage indicated in (C) (n = 3 mice for each group). Genotyping, sample collection, and analysis were performed at indicated time points. (C) In vivo editing efficiency of PKU target site in liver genomic DNA and mRNA determined by HTS (n = 3 mice for each group). Values represent mean ± SD. Asterisks indicate statistically significant differences in editing efficiencies observed between high-dose AAV8-miniSaCBE-PLUS-PKU-injected mice and low-dose AAV8-miniSaCBE-PLUS-PKU-injected mice. (D) Editing of 8 predicted off-target sites (OTs) in liver genomic DNA. OTs were predicted according to their sequence similarity to PKU target site¹⁶ (n = 3 mice for each group). Values represent mean ± SD. Asterisks indicate statistically significant differences in editing efficiencies observed between high-dose AAV8-miniSaCBE-PLUS-PKU-injected mice and AAV8-miniSaCBE-PLUS-ctrl-injected mice. (E) Blood Phe levels from male (top) and female (bottom) PKU mice were determined every 2 weeks from 8 to 24 weeks (n = 3 mice for each group). Values represent mean ± SD. (F) Body weight growth charts of male (top) and female (bottom) PKU mice. Compared with untreated or AAV8-miniSaCBE-PLUS-ctrl-treated PKU mice, AAV8-miniSaCBE-PLUS-PKU-treated mice showed slightly relieved growth retardation (n = 3 mice for each group). Values represent mean ± SD. (G) AAV8-miniSaCBE-PLUS-PKU treatment rescue hypopigmented phenotype. High-dose (5 × 10¹¹ GC) AAV8-miniSaCBE-PLUS-PKU (PKU+5 × 10¹¹) completely restored normal pigmentation in both male (3 of 3, n = 3) and female (3 of 3, n = 3) PKU mice. Low-dose AAV8-miniSaCBE-PLUS-PKU (PKU+1 × 10¹¹) partially rescued hypopigmentation in male PKU mice (3 of 3, n = 3). (H) AAV8-miniSaCBE-PLUS-PKU treatment reduced urinary Phe metabolites in male (top) and female (bottom) PKU mice.