Fig 1: EGFP-reporter-treated miceHippocampus of a Mmut+/- mouse treated on DOL 1 with AAV9-CBA-GFP at 100× magnification (A–C). Cells are stained with nuclear DAPI+ stain in blue (all images), NeuN+ stain in red (A), GFP+ stain in green (B), and all three stains overlaid appear yellow-green (white arrow) (C). Following retro-orbital injection of PHP.eB-CBA-GFP of mice on DOL 0, the striatum (D) and choroid plexus (E) of an Mmut-/-;TgINS-MCK-Mmut mice, labeled MMA, at 200× magnification show strong GFP fluorescence in neuronal cells. (F) H-score of GFP positivity in MMA mice treated with PHP.eB-CBA-GFP reporter vector (p = 0.0071, n = 3). (G) Percent co-positivity of NeuN and GFP by brain region in Mmut+/- mice, labeled HET, or Mmut-/-;TgINS-MCK-Mmut mice treated with PHP.eB-CaMKII-GFP. No significant difference was found for any region when stratified by genotype (p = 0.8441, Mmut-/-;TgINS-MCK-Mmut mice [n = 4] versus Mmut+/- controls [n = 4]).
Fig 2: AAV8.MMUT.SV40 targeted to the Alb ATG leads to increased MMUT mRNA and MMUT protein expression and rescues Mmutp.R106C/p.R106C mice from disease lethality(A) Donor construct corresponding to the gRNA targeting the Alb start codon (Sa08). The left homology arm contains the Alb 5' UTR, including part of the albumin promoter. (B) Survival of untreated Mmutp.R106C/p.R106C and Mmutp.R106C/p.R106C administered 5e10 GC/pup AAV8.HLP.SaCas9.Sa08 + 2 × 1011 GC/pup AAV8.MMUT.SV40 at birth. The survival curve depicts the same control cohort of Mmutp.R106C/p.R106C and Mmutp.R106C/+ mice presented in Figure 1E. (C) Plasma methylmalonic acid concentrations for Mmutp.R106C/p.R106C treated with 5e10 GC/pup AAV8.HLP.SaCas9.Sa08 + 2 × 1011 GC/pup AAV8.MMUT.SV40 at 1, 2, 3, 6, and 9 months (terminal); concentrations from untreated Mmutp.R106C/p.R106C euthanized at birth were used as controls. (D) Weights of untreated Mmutp.R106C/p.R106C and untreated Mmut+/p.R106C compared with Mmutp.R106C/p.R106C treated with the gRNA targeting the Alb start codon and donor. Treated Mmutp.R106C/p.R106C were significantly larger than untreated Mmutp.R106C/p.R106C at both time points, and were not significantly different in size from Mmut+/p.R106C by age 1 month. (E) Liver sections from treated Mmutp.R106C/p.R106C mice euthanized at 9 months post-treatment were stained for MMUT mRNA. (F) Expression of the MMUT mRNA at 9 months in treated Mmutp.R106C/p.R106C mice was evenly distributed throughout the liver. The treated Mmut+/p.R106C control exhibited fewer cells with positive MMUT mRNA staining at the same time point. In (B), data were analyzed by log rank Mantel-Cox and Gehan-Breslow-Wilcoxon test. **p < 0.01, versus the untreated Mmutp.R106C/p.R106C group. In (C), plasma methylmalonic acid concentrations were analyzed by unpaired Student's t test as mean ± SD. Methylmalonic acid values were significantly reduced at 1 month in treated Mmutp.R106C/p.R106C mice compared with untreated neonatal Mmutp.R106C/p.R106C controls, and were further reduced for all later time points. ***p < 0.001, ****p < 0.0001, versus the untreated Mmutp.R106C/p.R106C group. In (D), weight values are expressed as mean ± SD. In (F), MMUT mRNA expression was quantified as percentage of hepatocytes stained positive and expressed as mean ± SEM. At 9 months edited Mmutp.R106C/p.R106C mice exhibited 52.89% ± 1.46% MMUT-positive cells in the liver. The treated Mmut+/p.R106C control exhibited 19.57% of hepatocytes with positive mRNA staining at this time point.
Fig 3: Targeting an AAV8.IRES.MMUT cassette into Alb intron 1 results in MMUT mRNA expression and improves clinical outcomes in Mmutp.G715V/p.G715V(A) Schematic of the donor construct corresponding to the Sa37 gRNA targeting intron 1 of Alb. (B) Plasma methylmalonic acid concentrations for Mmutp.G715V/p.G715V administered AAV8.HLP.SaCas9.Sa37 + AAV8.IRES.MMUT.SV40 at birth, compared with untreated Mmutp.G715V/p.G715V controls at 1, 2, 3, 6, and 9 months (terminal). At 1 month, no reduction in plasma methylmalonic acid was observed in treated versus untreated mice. At 2 months, a significant difference was observed in methylmalonic acid in untreated versus treated mice which persisted. (C) Weights for Mmutp.G715V/p.G715V treated with 5e10 GC/pup AAV8.HLP.SauCas9.Sa37 + 2 × 1011 GC/pup AAV8.IRES.MMUT.SV40 compared with untreated Mmutp.G715V/p.G715V and untreated Mmut+/p.G715V mice at 1 month and 45 days, and 2, 3, and 6 months. Treated Mmutp.G715V/p.G715V were comparable in size with heterozygous Mmut+/p.G715V controls for all time points. The growth data shown from these experiments include time points for which we had relevant untreated Mmutp.G715V/p.G715V and Mmut+/p.G715V control data. (D) Liver sections from treated Mmutp.G715V/p.G715V were stained for MMUT mRNA using a transgene-specific probe. (E) Expression of MMUT mRNA in treated Mmutp.G715V/p.G715V at 9 months. Treated Mmutp.G715V/p.G715V exhibited an even distribution of positively stained cells throughout the liver. One Mmut+/p.G715V littermate control treated with 5e10 GC/pup AAV8.HLP.SauCas9.Sa37 + 2 × 1011 GC/pup AAV8.IRES.MMUT.SV40 exhibited significantly fewer positively stained hepatocytes at 9 months. In (B), plasma methylmalonic acid concentrations were analyzed by unpaired Student's t test as mean ± SD. Methylmalonic acid values at 1 month in treated Mmutp.G715V/p.G715V mice (690.5 ± 130.9 µM) were comparable with untreated Mmutp.G715V/p.G715V (820.3 ± 92.6 µM), but were significantly reduced in treated mice for later time points. *p < 0.05, **p < 0.01, ***p < 0.001, versus the untreated Mmutp.G715V/p.G715V group. In (C), weight values are expressed as mean ± SD and analyzed by unpaired Student's t test. In (E), MMUT mRNA expression was quantified as percentage of hepatocytes stained positive and expressed as mean ± SEM. At 9 months, edited Mmutp.G715V/p.G715V mice exhibited 41.85% ± 4.90% MMUT-positive cells in the liver. The treated Mmut+/p.G715V control exhibited 9.69% of hepatocytes with positive mRNA staining at this time point.
Fig 4: Hepatic mRNA expression of MMUT in a pilot study. (A-C) Representative RISH images of AAV-Alb-2A-MMUT–treated mice. Cells positive for MMUT mRNA are stained brown. The scale bar is 2 mm, and the insert is a 10× magnification. (A) A 2-month treated Mmut–/–;TgINS-MCK-Mmut mouse liver. (B) A 15-month treated Mmut–/–;TgINS-MCK-Mmut mouse liver. (C) A 13-month treated Mmut+/- WT control mouse liver. (D) Percentage of liver area stained for MMUT mRNA in treated animals; MMA mice at 2 months posttreatment (n = 3), 13-15 months posttreatment (n = 5), or WT mice posttreatment (n = 5; *P < 0.005, one-way ANOVA). All error bars are ± the SEM.
Fig 5: Improvement in disease-related metabolites and growth in MMA mice after AAV44.9-CBA-Mmut treatmentMmut-/-MCK-Mmut+ received 7 × 1012 vg/kg of AAV44.9-CBA-Mmut at 1 month of life by retro-orbital delivery. (A) Methylmalonic acid levels were measured in the plasma and compared with pretreatment levels. (**p < 0.004, one-way ANOVA with Kruskal-Wallis test). (B) Methylcitrate levels were measured in the plasma and compared with pretreatment levels. (*p < 0.04, one-way ANOVA with Kruskal-Wallis test). (C) Growth in mice before and after AAV44.9-CBA-Mmut treatment in female Mmut-/-MCK-Mmut+ relative to untreated Mmut-/-MCK-Mmut+ and wild-type (WT) mice. (D) Growth in mice before and after AAV44.9-CBA-Mmut treatment in male Mmut-/-MCK-Mmut+ relative to untreated Mmut-/-MCK-Mmut+ and WT mice (*p < 0.05, **p < 0.005, two-way ANOVA). Error bars = ± SEM.
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