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Metabolic profiling and in vivo applicable LNP‐mediated base editing. (A, B, C, and D) Metabolic analysis of differentiated hepatocyte‐like cell culture media detected by liquid chromatography‐mass spectrometry from different test conditions: Non‐edited ( n = 2 independent cell lines), Edited ( n = 1 cell line), and Control ( n = 1 cell line). After a 48‐h incubation, cells and media from five replicate wells per cell line and three replicate samples of the fresh media were sampled for metabolic analysis. Relative abundance of (A) intracellular oxalate, (B) media oxalate, (C) intarcellular pyridoxine, and (D) media pyridoxine in relation to the control lines. Each sample was normalized to the sum of the peak areas of 439 metabolites detected in the same sample. Data are represented as the mean with SD. Statistical significance is based on the Tukey test: p > 0.05 (ns, not significant), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). (E) The LNP mediated on‐target A to G editing from Sanger sequencing data of differentiated cells. Four doses of total RNA (250 ng, 500 ng, 1500 ng, and 2500 ng; synthesized single guide RNA and in vitro transcribed <t>ABE8e</t> mRNA in a 1:2 mass ratio) within LNP formulations were pipetted onto the culture media on day 19 of the differentiation. The media was changed 24 h after and the cells were collected for editing analysis five days after transfection.
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Metabolic profiling and in vivo applicable LNP‐mediated base editing. (A, B, C, and D) Metabolic analysis of differentiated hepatocyte‐like cell culture media detected by liquid chromatography‐mass spectrometry from different test conditions: Non‐edited ( n = 2 independent cell lines), Edited ( n = 1 cell line), and Control ( n = 1 cell line). After a 48‐h incubation, cells and media from five replicate wells per cell line and three replicate samples of the fresh media were sampled for metabolic analysis. Relative abundance of (A) intracellular oxalate, (B) media oxalate, (C) intarcellular pyridoxine, and (D) media pyridoxine in relation to the control lines. Each sample was normalized to the sum of the peak areas of 439 metabolites detected in the same sample. Data are represented as the mean with SD. Statistical significance is based on the Tukey test: p > 0.05 (ns, not significant), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). (E) The LNP mediated on‐target A to G editing from Sanger sequencing data of differentiated cells. Four doses of total RNA (250 ng, 500 ng, 1500 ng, and 2500 ng; synthesized single guide RNA and in vitro transcribed <t>ABE8e</t> mRNA in a 1:2 mass ratio) within LNP formulations were pipetted onto the culture media on day 19 of the differentiation. The media was changed 24 h after and the cells were collected for editing analysis five days after transfection.
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Metabolic profiling and in vivo applicable LNP‐mediated base editing. (A, B, C, and D) Metabolic analysis of differentiated hepatocyte‐like cell culture media detected by liquid chromatography‐mass spectrometry from different test conditions: Non‐edited ( n = 2 independent cell lines), Edited ( n = 1 cell line), and Control ( n = 1 cell line). After a 48‐h incubation, cells and media from five replicate wells per cell line and three replicate samples of the fresh media were sampled for metabolic analysis. Relative abundance of (A) intracellular oxalate, (B) media oxalate, (C) intarcellular pyridoxine, and (D) media pyridoxine in relation to the control lines. Each sample was normalized to the sum of the peak areas of 439 metabolites detected in the same sample. Data are represented as the mean with SD. Statistical significance is based on the Tukey test: p > 0.05 (ns, not significant), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). (E) The LNP mediated on‐target A to G editing from Sanger sequencing data of differentiated cells. Four doses of total RNA (250 ng, 500 ng, 1500 ng, and 2500 ng; synthesized single guide RNA and in vitro transcribed <t>ABE8e</t> mRNA in a 1:2 mass ratio) within LNP formulations were pipetted onto the culture media on day 19 of the differentiation. The media was changed 24 h after and the cells were collected for editing analysis five days after transfection.
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Metabolic profiling and in vivo applicable LNP‐mediated base editing. (A, B, C, and D) Metabolic analysis of differentiated hepatocyte‐like cell culture media detected by liquid chromatography‐mass spectrometry from different test conditions: Non‐edited ( n = 2 independent cell lines), Edited ( n = 1 cell line), and Control ( n = 1 cell line). After a 48‐h incubation, cells and media from five replicate wells per cell line and three replicate samples of the fresh media were sampled for metabolic analysis. Relative abundance of (A) intracellular oxalate, (B) media oxalate, (C) intarcellular pyridoxine, and (D) media pyridoxine in relation to the control lines. Each sample was normalized to the sum of the peak areas of 439 metabolites detected in the same sample. Data are represented as the mean with SD. Statistical significance is based on the Tukey test: p > 0.05 (ns, not significant), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). (E) The LNP mediated on‐target A to G editing from Sanger sequencing data of differentiated cells. Four doses of total RNA (250 ng, 500 ng, 1500 ng, and 2500 ng; synthesized single guide RNA and in vitro transcribed <t>ABE8e</t> mRNA in a 1:2 mass ratio) within LNP formulations were pipetted onto the culture media on day 19 of the differentiation. The media was changed 24 h after and the cells were collected for editing analysis five days after transfection.
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Metabolic profiling and in vivo applicable LNP‐mediated base editing. (A, B, C, and D) Metabolic analysis of differentiated hepatocyte‐like cell culture media detected by liquid chromatography‐mass spectrometry from different test conditions: Non‐edited ( n = 2 independent cell lines), Edited ( n = 1 cell line), and Control ( n = 1 cell line). After a 48‐h incubation, cells and media from five replicate wells per cell line and three replicate samples of the fresh media were sampled for metabolic analysis. Relative abundance of (A) intracellular oxalate, (B) media oxalate, (C) intarcellular pyridoxine, and (D) media pyridoxine in relation to the control lines. Each sample was normalized to the sum of the peak areas of 439 metabolites detected in the same sample. Data are represented as the mean with SD. Statistical significance is based on the Tukey test: p > 0.05 (ns, not significant), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). (E) The LNP mediated on‐target A to G editing from Sanger sequencing data of differentiated cells. Four doses of total RNA (250 ng, 500 ng, 1500 ng, and 2500 ng; synthesized single guide RNA and in vitro transcribed ABE8e mRNA in a 1:2 mass ratio) within LNP formulations were pipetted onto the culture media on day 19 of the differentiation. The media was changed 24 h after and the cells were collected for editing analysis five days after transfection.

Journal: Journal of Inherited Metabolic Disease

Article Title: Genetic Correction of the Most Common Mutation Causing Primary Hyperoxaluria Restores Enzyme Localization and Oxalate Metabolism

doi: 10.1002/jimd.70122

Figure Lengend Snippet: Metabolic profiling and in vivo applicable LNP‐mediated base editing. (A, B, C, and D) Metabolic analysis of differentiated hepatocyte‐like cell culture media detected by liquid chromatography‐mass spectrometry from different test conditions: Non‐edited ( n = 2 independent cell lines), Edited ( n = 1 cell line), and Control ( n = 1 cell line). After a 48‐h incubation, cells and media from five replicate wells per cell line and three replicate samples of the fresh media were sampled for metabolic analysis. Relative abundance of (A) intracellular oxalate, (B) media oxalate, (C) intarcellular pyridoxine, and (D) media pyridoxine in relation to the control lines. Each sample was normalized to the sum of the peak areas of 439 metabolites detected in the same sample. Data are represented as the mean with SD. Statistical significance is based on the Tukey test: p > 0.05 (ns, not significant), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). (E) The LNP mediated on‐target A to G editing from Sanger sequencing data of differentiated cells. Four doses of total RNA (250 ng, 500 ng, 1500 ng, and 2500 ng; synthesized single guide RNA and in vitro transcribed ABE8e mRNA in a 1:2 mass ratio) within LNP formulations were pipetted onto the culture media on day 19 of the differentiation. The media was changed 24 h after and the cells were collected for editing analysis five days after transfection.

Article Snippet: In vitro transcription of ABEmax and ABE8e: The purified ABEmax [ ] or ABE8e [ ] IVT plasmid (Addgene: 201676 and 171761) was linearized by SfiI restriction (Thermo Fisher Scientific; FD1824).

Techniques: In Vivo, Cell Culture, Liquid Chromatography, Mass Spectrometry, Control, Incubation, Sequencing, Synthesized, In Vitro, Transfection