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Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and <t>novobiocin</t> 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.
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Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and <t>novobiocin</t> 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.
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Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and <t>novobiocin</t> 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.
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Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and <t>novobiocin</t> 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.
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Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and <t>novobiocin</t> 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.
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Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and <t>novobiocin</t> 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.
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Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and <t>novobiocin</t> 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.
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Image Search Results


Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and novobiocin 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.

Journal: Molecular Therapy. Nucleic Acids

Article Title: Enhancement of therapeutic transgene insertion for treatment of murine phenylketonuria

doi: 10.1016/j.omtn.2025.102813

Figure Lengend Snippet: Inhibition of NHEJ and MMEJ increases efficacy of homology-directed recombination in vivo (A) Serum Phe concentrations in untreated mice (mean = 2,134 ± 197 μM, n = 6) and animals treated with Cas9 and RT AAV vectors with Vanbiocin (vanillin 100 mg/kg and novobiocin 50mg/kg) (mean = 631 ± 211 μM, n = 6). Animals received Vanbiocin treatment at 5 days of age for 3 days. Black dashed line indicates a therapeutic threshold of 360 μM. Data are shown as means ± SD. ∗∗∗∗ p < 0.0001 by paired t test. (B) Insertion frequency of the mPah cDNA (mean = 9.71% ± 3.62%) in treated animals, as determined by qPCR. (C) PAH enzyme activity in Vanbiocin-treated mice (mean = 6.14% ± 1.15%) measured as percentage of wild-type PAH activity. (D) Anti-PAH (Red), anti-lectin (green), and nuclear stain (blue) in treated animals. Scale bars, 100 μm. (E) Coat color rescue of wild-type C57BL/6, untreated Dexon1 mice, and Vanbiocin-treated mice. Baseline serum Phe concentrations without sapropterin provided. (F) Serum Phe concentrations in Vanbiocin-treated animals were measured over a 5-day course of daily treatment with 100 mg/kg sapropterin dihydrochloride, administered via oral gavage 6 h prior to retro-orbital serum collection. Black dashed line indicates a therapeutic threshold of 360 μM. p < 0.1188, ∗ p < 0.0242, and ∗∗ p < 0.0037 by one-way ANOVA. (G) Serum Phe concentrations in the progeny (mean = 2,124, n = 4) of Vanbiocin-treated female post-weaning. Dunnett’s multiple comparison used with all ANOVAs. Data are shown as means ± SD.

Article Snippet: Novobiocin sodium salt (Thermo Fisher Scientific, Waltham, MA) was dissolved in 400 μL of DMSO and heated in a 50°C water bath before slowly adding 600 μL of saline preheated to 50°C.

Techniques: Inhibition, In Vivo, Activity Assay, Staining, Comparison