Activities of MMA mice injected with vehicle or mLB-001.

Animals were injected with vehicle or 5×1013 vg/kg mLB-001 at 8 weeks of age. At 5 months post-dosing, two animals, one from each treatment group, were removed from their home cages and placed together in a freshly prepared cage. The video was recorded within 2 minutes of the animal placement in the cage. (GIF)</p

Effect of high protein diet challenge on MMA mice treated with vehicle or 5×10¹³ vg/kg mLB-001 at 8 weeks of age.

(A) Study design. Animals were maintained on the 12% protein diet and dosed with vehicle or mLB-001 at 8 weeks of age. At 3 months post-dosing, the diet was switched to the standard chow with 21% protein content and then to a 40% protein diet at 4 months post-dosing. Necropsy was performed at 6 months post-dosing when the mice were approximately 8 months old. (B) Kaplan-Meier survival curves of MMA mice show that the mLB-001-treated group survived longer than the vehicle-treated group (P = 0.052, Log-rank test). (C) Change in body weight of MMA mice in response to the high protein diet challenge. The body weight of each animal was normalized to its weight at the introduction of the 21% protein diet. Females and males were combined due to lack of significant difference. Animal numbers at each timepoint are indicated above the graph. * P 0.05, **** P 0.0001, mixed-effects analysis with multiple comparisons between vehicle- and mLB-001-treated groups at the indicated timepoints. (D) Circulating methylmalonic acid in the mLB-001-treated animals maintained at low levels but elevated in the vehicle-treated animals in response to high protein diet challenge. *** P 0.001, **** P 0.0001, mixed-effects analysis with multiple comparisons between vehicle- and mLB-001-treated groups at the indicated timepoints. (E) Plasma ALB-2A levels remained stable in the mLB-001-treated heterozygous animals while demonstrating exponential increases over time in the MMA mice.</p

Body weight of MMA mice and their heterozygous littermates.

(A) Animals were dosed with vehicle or 1×1014 vg/kg mLB-001 on PND 1. Nursing dams were kept on the standard 21% protein diet until PND 14, when the diet was switched to a 12% protein diet. Pups were weaned on PND 28 and maintained on the 12% protein diet. At 3 months of age, all surviving animals were switched to the standard chow with 21% protein content and then to a 40% protein diet at 4 months of age. Animals were monitored until 6 months of age. (B) Animals were dosed with vehicle, 2.5×1013, 5×1013 or 1×1014 vg/kg mLB-001 on PND 1. Other procedures are the same as for (A). (C) Animals were maintained on the 12% protein diet and dosed with vehicle or 5×1013 vg/kg mLB-001 at 8 weeks of age. At 3 months post-dosing, the diet was switched to the standard chow with 21% protein content and then to a 40% protein diet at 4 months post-dosing. Animals were monitored until 8 months of age. (TIF)</p

MMUT holo-mutase activity in liver.

MMA mice (-/-) or heterozygous mice (+/-) were treated with 1×1014 vg/kg mLB-001 at PND 1 (D1) or 5×1013 vg/kg mLB-001 at 8 weeks of age (W8). Terminal samples were analyzed at 6 months post-dosing. The mutase activity unit is defined as the amount of the enzyme that converted methylmalonyl CoA to succinyl CoA (μmol/min), and the data were normalized by the total protein in the liver lysates. (TIF)</p

Raw data used to generate figures.

(PDF)</p

Analyses of terminal samples from animals treated with vehicle or mLB-001 at 6-month post-dosing.

MMA mice (-/-) or heterozygous mice (+/-) were treated with 1×1014 vg/kg mLB-001 at PND 1 (D1) or 5×1013 vg/kg mLB-001 at 8 weeks of age (W8). Terminal samples were analyzed at 6 months post-dosing. (A) Percentage of integrated albumin alleles in liver. (B) Fused mRNA expression in liver. (C) Circulating ALB-2A level. **** P 0.0001, Student’s t-test.</p

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.</p

Immunohistochemistry analysis of Ki67 in 10-week-old mice.

Heterozygous (Mmut+/-) and MMA (Mmut-/-) mice were fed standard chow (21% protein content). Animals were killed at 7–10 weeks of age, and liver tissues were subjected to immunohistochemistry analysis for Ki67. Ten 0.4 mm2 regions were randomly selected from each tissue section and Ki67-positive cells were counted by staff blinded to the sample identity. A representative sample of each is shown. * P t-test. The scale bars represent 100 μm. (TIF)</p

Kaplan-Meier survival curves of MMA mice on a transient low protein diet.

Nursing dams were on a 21% protein diet (standard chow) until pups were PND 14. Randomly selected litters were provided with a 12% protein diet between PND 14 and 49 and then returned to the 21% protein diet. The rest of the litters were on a 21% protein diet throughout their life span. Color-matched arrows on top represent the time periods in which the % protein diets were provided. All pups were weaned on PND 28. Log-rank test demonstrated a significantly better survival of MMA mice provided with the 12% protein diet (P 0.01), indicating that a transient, low protein diet can offer significant benefit to the MMA mice, although the effect is only temporary. (TIF)</p

Correlations of terminal parameters in MMA mice treated with mLB-001 on PND 1.

Animals were killed at 6 months of age and terminal samples were analyzed for liver genomic DNA integration, liver fused mRNA level, liver MMUT expression by immunohistochemistry, plasma ALB-2A by ELISA and methylmalonic acid by LC-MS/MS. Straight lines represent the best fit to the data by simple linear regression. In these mice, 1000 μg/mL ALB-2A was equivalent to 5% of total albumin. * P 0.05, ** P 0.01, *** P P 0.0001, F-test for non-0 slope hypothesis. (TIF)</p