Fig 2: AAV-Alb-2A-MMUT longitudinal dosage study in Mmut–/–;TgINS-MCK-Mmut mice. (A) Survival curve of Mmut–/–;TgINS-MCK-Mmut mice treated with 2.5e12 VG/pup (n = 4) or 2.5e11 VG/pup (n = 18) of AAVDJ-Alb-2A-MMUT versus untreated Mmut–/–;TgINS-MCK-Mmut mice (n = 42; *P < 0.05). Some mutants treated with 2.5e11 VG/pup were sacrificed at 3 (n = 5), 6 (n = 5), 8 (n = 1), and 9 months (n = 2) for terminal assays. They are censored in the survival curve at the day of euthanization. (B) Plasma methylmalonic acid levels of Mmut–/–;TgINS-MCK-Mmut mice treated with 2.5e12 VG/pup (n = 4, 3, 0, 2, 2, 2, 2, and 2), with 2.5e11 VG/pup treated (n = 18, 18, 15, 7, 9, 9, 4, and 1), or untreated Mmut–/–;TgINS-MCK-Mmut mice (n = 5, 11, 10, 5, 7, 5, 7, and 7; P < 0.03 and 1, 2, 3, 4, 5, 8, and 10 months, one-way ANOVA). (C) Weights of Mmut–/–;TgINS-MCK-Mmut mice (MMA) treated with 2.5e12 VG/pup (n = 4, 3, 0, 2, 2, 2, 2, and 2), with 2.5e11 VG/pup (n = 16, 17, 7, 9, 4, 9, 4, and 1), or untreated (n = 18, 25, 18, 8, 5, 12, 6, and 4; *P < 0.05, unpaired t test). (D) Immunoblotting of hepatic MMUT (85 kDa) and ß-actin (42 kDa) expression in WT (+), untreated MMA (-), MMA mice treated with 2.5e11 VG/pup at 3, 6, 8, 9, and 10 months (m), or treated with 2.5e12 VG/pup at 11 months. (E) Quantification of hepatic MMUT protein in 2.5e11 VG/pup and 2.5e12 VG/pup treated MMA mice expressed as a percentage of MMUT WT expression and normalized to ß-actin (*P < 0.0001, unpaired t test and one-way ANOVA). (F) Propionic oxidation improves in both treatment groups beginning at 6 months compared to untreated controls, but the oxidation in treated mice never reaches WT levels. Oxidation measurements are displayed for each dose: 2.5e12 VG/pup (n = 1, 2, and 1) and 2.5e11 (n = 1, 1, 3, 1, 1, 2, 2, and 1). Positive control range was defined from 5 assayed WT mice, and negative control range was defined from 5 assayed untreated MMA mice.

Fig 3: MMUT mRNA and MMUT protein expression in livers of treated mice. Representative MMUT RISH and MMUT protein immunohistochemistry staining of serial liver sections in treated mice. Cells positive for mRNA or protein expression are stained brown. The scale bar is 1 mm, and the insert is a 10× magnification. (A) A Mmut–/–;TgINS‐MCK‐Mmut mouse 3 months posttreatment (2.5e11 VG). (B) A Mmut–/–;TgINS‐MCK‐Mmut mouse 9 months posttreatment (2.5e11 VG). (C) A Mmut–/–;TgINS‐MCK‐Mmut mouse 10 months posttreatment (2.5e11 VG). (D) A Mmut–/–;TgINS‐MCK‐Mmut mouse 11 months posttreatment (2.5e12 VG). (E) A Mmut+/–;TgINS‐MCK‐Mmut (WT phenotype, 2.5e12 VG) mouse 11 months posttreatment.

Fig 4: MMUT protein expression in mice treated with 1×1014 vg/kg mLB-001 at PND 1.(A) Plasma ALB-2A levels remained stable in all heterozygous (Mmut+/-) animals while showing an exponential increase over time in all MMA (Mmut-/-) mice. (B) Immunoblot analysis of MMUT protein in the liver tissues. Vehicle-treated heterozygous and MMA mice were used as positive and negative controls for protein expression. β-actin was used as a loading control. (C-E) Immunohistochemistry analysis of MMUT protein expression in livers of three mLB-001-treated MMA mice sacrificed at 1.5- (C), 3- (D), and 6- (E) months post-dosing. The scale bars represent 400 μm.

Fig 5: No signs of HCC detected following AAV treatment. Representative H&E and MMUT RISH staining of serial liver sections in treated mice. (A) A Mmut–/– mouse 13 months posttreatment (8.58e11 VG). Areas with little MMUT RNA staining show a normal MMA hepatocyte phenotype; no sign of HCC present. Both images taken at 40×, scale bar is 20 µm. (B) A Mmut–/–;TgINS-MCK-Mmut mouse 15 months posttreatment (1.9e12 VG). Areas with significant MMUT RNA staining show a normal microscopic appearance. Both images taken at 40×, scale bar is 20 µm. (C) A Mmut–/–;TgINS-MCK-Mmut mouse 15 months posttreatment (8.58e11 VG). Areas that border regions of corrected and uncorrected hepatocytes again show normal hepatocyte phenotypes for MMA or healthy tissues. At these borders, there is no sign of HCC. Both images taken at 10×, scale bar is 100 µm. (D) A magnification of (C). Both images taken at 40×, scale bar is 20 µm. (E) Incidence of HCC in WT and MMA mice treated with AAV-Alb-2A-MMUT versus historical HCC samples.( 9 ) Abbreviations: CBA, chicken beta actin; GFP, green fluorescent protein; TBG, thyroxine-binding globulin.