Journal: Nucleic Acids Research

Article Title: RNA editing for the treatment of alpha-1 antitrypsin deficiency

doi: 10.1093/nar/gkaf1518

Figure Lengend Snippet: RNA-seq identifies gene expression changes after treatment with SERPINA1-994. ( A ) Gene expression changes in 40 genes identified as inappropriately upregulated in the mouse model . Genes are ranked by amount of increased or decreased expression in liver after treatment with SERPINA1-994 (compared with PBS treated animals). ( B ) KEGG pathway enrichment analysis of downregulated genes in the SERPINA1-994 treated group compared with PBS, showing decreasing expression of genes in cancer-associated pathways. ( C ) Expression changes for specific cancer-associated genes ( Akt, Egfr , and Myc ) at baseline and after treatment with PBS or SERPINA1-994. Data are presented as mean ± SEM ( n = 5).

Article Snippet: The following Taqman probes were used—human SERPINA1: Hs01097800_m1 (Thermo Fisher Scientific), mouse HPRT control: Mm.PT.39a.22214828 (IDT).

Techniques: RNA Sequencing, Gene Expression, Expressing

Journal: Nucleic Acids Research

Article Title: RNA editing for the treatment of alpha-1 antitrypsin deficiency

doi: 10.1093/nar/gkaf1518

Figure Lengend Snippet: SERPINA1 editing with optimized GalNAc-AIMer. ( A ) Schematic of SERPINA1 AIMers used in this work with legend for chemical modifications. ( B ) Percentage SERPINA1 mRNA editing for increasing concentrations of GalNAc-AIMer and unconjugated AIMer in heterozygous primary human (MZ) hepatocytes. Data are presented as mean ± SEM ( n = 5). Absolute EC 75 and 95% confidence intervals (CI) are indicated. ( C ) Percentage of SERPINA1 mRNA editing for increasing concentrations of GalNAc-AIMer in primary mouse hepatocytes derived from NSG-PiZ mice. Data are presented as mean ± SEM ( n = 4). Absolute EC 50 and 95% CIs are indicated. For panels (B) and (C), error bars that are not visible are smaller than the plotted point. N3U, N-3-uridine; 2′-OMe, 2′-O-methyl.

Article Snippet: The following Taqman probes were used—human SERPINA1: Hs01097800_m1 (Thermo Fisher Scientific), mouse HPRT control: Mm.PT.39a.22214828 (IDT).

Techniques: Derivative Assay

Journal: Nucleic Acids Research

Article Title: RNA editing for the treatment of alpha-1 antitrypsin deficiency

doi: 10.1093/nar/gkaf1518

Figure Lengend Snippet: A single dose of SERPINA1-994 directs RNA editing and increases serum AAT levels. ( A ) Experimental regimen with subcutaneous dosing indicated by arrowhead, weekly serum collection and necropsy points (5 mice/time point) indicated by arrows. ( B ) Percentage of SERPINA1 mRNA editing at 7- or 35-days post-dose for the indicated treatment. Data are presented as mean ± SEM ( n = 5). Welch’s one-way ANOVA with post-hoc test versus PBS at day 35 allowing heteroscedasticity. For panel (B), P = 0.0 indicates that calculated P values were below the limit of the program (2.2 × 10 −16 ). ( C ) Serum AAT protein levels in NSG-PiZ mice treated with PBS or SERPINA1-994 over the 35-day experiment. Time 0 (baseline) is pre-dose. Data are presented as mean ± SEM ( n = 10, d0, d7; n = 5, d14–35). Two-way mixed ANOVA with post-hoc tests versus PBS per time point. ( D ) Concentration of SERPINA1-994 in liver. Data are presented as mean ± SEM ( n = 5).

Article Snippet: The following Taqman probes were used—human SERPINA1: Hs01097800_m1 (Thermo Fisher Scientific), mouse HPRT control: Mm.PT.39a.22214828 (IDT).

Techniques: Concentration Assay

Journal: Nucleic Acids Research

Article Title: RNA editing for the treatment of alpha-1 antitrypsin deficiency

doi: 10.1093/nar/gkaf1518

Figure Lengend Snippet: SERPINA1-994 restores functional AAT protein in serum. ( A ) Serum AAT protein levels in NSG-PiZ mice treated with PBS or SERPINA1-994 over 13 weeks. Dosing is indicated by black arrows. After three loading doses, 7-weeks-old NSG-PiZ mice were treated biweekly. Data are presented as mean ± SEM ( n = 5). Two-way mixed ANOVA with post-hoc test comparing PBS to SERPINA1-994 per time point. The highest P- value is shown; most P- values were below the limit of the program (2.2 × 10 −16 ). ( B ) Percentage of Z-AAT and M-AAT in serum as assessed by mass spectrometry from pre-dose baseline (time 0) to 13 weeks after treatment with PBS or SERPINA1-994. Data are presented as mean ± SEM ( n = 5). Two-way mixed ANOVA with post-hoc test comparing M-AAT isoform percentage in PBS versus SERPINA1-994 per time point. ( C ) Inhibition of neutrophil elastase in serum from mice at pre-dose baseline (time 0) or after 13 weeks of treatment with PBS or SERPINA1-994. Data are presented as mean ± SEM ( n = 5). Two-way mixed ANOVA with post-hoc test comparing PBS to SERPINA1-994 per time point. For panel (C), P = 0.0 indicates that calculated P- values were below the limit of the program (2.2 × 10 −16 ).

Article Snippet: The following Taqman probes were used—human SERPINA1: Hs01097800_m1 (Thermo Fisher Scientific), mouse HPRT control: Mm.PT.39a.22214828 (IDT).

Techniques: Functional Assay, Mass Spectrometry, Inhibition

Journal: Nucleic Acids Research

Article Title: RNA editing for the treatment of alpha-1 antitrypsin deficiency

doi: 10.1093/nar/gkaf1518

Figure Lengend Snippet: Improved liver pathology with repeated SERPINA1-994 dosing. ( A ) Percentage of SERPINA1 transcript in liver edited at 13 weeks. Data are presented as mean ± SEM ( n = 5), Welch’s t- test. ( B ) Concentration of AIMer detected in mouse liver after dosing. Data are presented as mean ± SEM ( n = 5). ( C ) In situ hybridization visualizing edited SERPINA1 transcripts in liver of PBS or SERPINA1-994-treated mice at 13 weeks. Mutant (Z) mRNA (light blue), edited (M) mRNA (violet), and DAPI-stained nuclei (dark blue) are shown. ( D ) Quantification of SERPINA1 mRNA (relative to Hprt mRNA) in mouse liver at baseline (time 0) and after 13 weeks of treatment. Data are presented as mean ± SEM ( n = 5); one-way ANOVA with Tukey HSD post-hoc test. ( E ) Diameter of PAS-D-positive globules at baseline and after 13 weeks of treatment. Dots represent diameters from the 40 largest globules per mouse across all five mice in each group. Horizontal lines indicate mean globule diameter with stats by Kruskal–Wallis with Dunn post-hoc test. ( F ) Representative images from mouse liver showing globules. ( G ) Number of mitotic figures at baseline and after 13 weeks of treatment. Dots represent the mean number of mitotic figures per 10 medium power field (MPF) in each animal. Data are presented as mean ± SEM ( n = 5); one-way ANOVA with Tukey’s HSD post-hoc test. ( H ) Median lobular inflammation (based on grades 0–4) at baseline and after 13 weeks of treatment. Data are presented as median grade for each mouse ( n = 5); Kruskal–Wallis with Dunn post-hoc test. ( I ) Representative images show lobular inflammation. The scale bars in all panels are 100 μm.

Article Snippet: The following Taqman probes were used—human SERPINA1: Hs01097800_m1 (Thermo Fisher Scientific), mouse HPRT control: Mm.PT.39a.22214828 (IDT).

Techniques: Concentration Assay, In Situ Hybridization, Mutagenesis, Staining

Journal: Nucleic Acids Research

Article Title: RNA editing for the treatment of alpha-1 antitrypsin deficiency

doi: 10.1093/nar/gkaf1518

Figure Lengend Snippet: Limited off-target editing by SERPINA1-994 in the human hepatocyte transcriptome. ( A ) Total RNA coverage and sequence of the Z transcript (magenta, top panel) in mouse liver after treatment PBS (left) or SERPINA1-994 (right). Editing to form M transcript (purple, top right panel) is shown. ( B ) Total RNA coverage versus percent editing for all potential edit sites identified after treatment of healthy human hepatocytes with 10 or 100 nM SERPINA1-994. SERPINA1 editing is not detected, as the hepatocytes lack the Z mutation. ( C ) Total RNA coverage versus percent editing for all potential edited sites identified after treatment of healthy human hepatocytes with 10 or 100 nM UGP2-42.

Article Snippet: The following Taqman probes were used—human SERPINA1: Hs01097800_m1 (Thermo Fisher Scientific), mouse HPRT control: Mm.PT.39a.22214828 (IDT).

Techniques: Sequencing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: RNA editing for the treatment of alpha-1 antitrypsin deficiency

doi: 10.1093/nar/gkaf1518

Figure Lengend Snippet: SERPINA1-994 decreases polymeric AAT accumulation in human iPSC-derived ZZ hepatocytes. ( A ) Images of cultured iPSCs after 14 days of treatment with the indicated AIMer. AAT polymer staining (green) and DAPI staining of nuclei (blue) are shown. The scale bars are 100 μm. ( B ) Quantification of polymeric AAT after 5, 10, or 14 days of treatment with increasing concentrations (0, 1.25, 2.5, 5.0, or 10 µM) of the indicated AIMer. Data are presented as mean ± SEM ( n = 6). White-adjusted two-way ANOVA with post-hoc comparisons to untreated allowing heteroscedasticity. ( C ) SERPINA1 RNA editing after 5, 10, or 14 days of treatment. Data are presented as mean ± SEM ( n = 4–6). White-adjusted two-way ANOVA with post-hoc comparisons to untreated allowing for heterscedasticity. ( D ) Quantification of nuclei per field after 5, 10, or 14 days. Data are presented as mean ± SEM ( n = 6). Two-way ANOVA with post-hoc comparisons to untreated. For all panels, P = 0.0 indicates that calculated P- values were below the limit of the program (2.2 × 10 −16 ). ns: nonsignificant.

Article Snippet: The following Taqman probes were used—human SERPINA1: Hs01097800_m1 (Thermo Fisher Scientific), mouse HPRT control: Mm.PT.39a.22214828 (IDT).

Techniques: Derivative Assay, Cell Culture, Polymer, Staining

Journal: Nucleic Acids Research

Article Title: RNA editing for the treatment of alpha-1 antitrypsin deficiency

doi: 10.1093/nar/gkaf1518

Figure Lengend Snippet: Concentration and distribution in NHP liver. ( A ) Schematic representation of dosing regimen. NHPs received a single 5 mg/kg subcutaneous dose of SERPINA1-994 on d0. Liver biopsies (B) and necropsy (N) were performed as indicated. The concentration of SERPINA1-994 in liver with respect to time after dosing is shown. Data are presented as mean ± SEM ( n = 3). ( B ) Visualization of SERPINA1-994 (pink) in liver 57 days post-dose using ViewRNA for PBS (top) and treated (bottom). The nucleus is counterstained with hematoxylin (blue). The scale bars are 100 μm.

Article Snippet: The following Taqman probes were used—human SERPINA1: Hs01097800_m1 (Thermo Fisher Scientific), mouse HPRT control: Mm.PT.39a.22214828 (IDT).

Techniques: Concentration Assay

Journal: Diabetes

Article Title: Glucagon Receptor Deficiency Causes Early-Onset Hepatic Steatosis

doi: 10.2337/db25-0209

Figure Lengend Snippet: iPSC differentiation to iPSC-derived hepatocytes. A : Summary of four-stage differentiation protocol from human iPSCs to definitive endoderm, hepatic progenitor, and hepatocytes over a 21-day period, using a combination of growth factors and small molecules indicated in blue. B : Bright-field microscopy using EVOS cell imaging system, depicting typical hepatocyte morphology with characteristic polyhedral appearance; GCGR WT (left), GCGR R225H/V368M (right); scale bar = 100 µm. C : Expression of canonical hepatocyte markers (albumin, HNF4A1, SERPINA1) in GCGR WT and R225H/V368M hepatocytes relative to the housekeeping gene HMBS ; n = 6–7 experiments; mean ± SEM.

Article Snippet: TaqMan probes (Thermo Fisher Scientific) were albumin: Hs00609411_m1; HNF4A: Hs00230853_m1; SERPINA1: Hs00165475_m1; AFP: Hs01040598_m1; HMBS: Hs00609296_g1; UBC: Hs05002522_g1; GCGR: Hs01026191_g1; G6PC: Hs00609178_m1.

Techniques: Derivative Assay, Microscopy, Imaging, Expressing

Journal: PLOS One

Article Title: Genetic drivers of liver cirrhosis: The role of SERPINA1 and PNPLA3 variants in disease onset and progression

doi: 10.1371/journal.pone.0333051

Figure Lengend Snippet: Extent and density of intrahepatic PASD positive aggregates according to SERPINA1 genotype.

Article Snippet: Genotyping of the SERPINA1 rs28929474 (PI Z) variant was conducted using the TaqMan SNP Genotyping Assay C_34508510_10, and the rs17580 (PI S) variant using Assay C_594695_20 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques:

Journal: PLOS One

Article Title: Genetic drivers of liver cirrhosis: The role of SERPINA1 and PNPLA3 variants in disease onset and progression

doi: 10.1371/journal.pone.0333051

Figure Lengend Snippet: Extent and density of intrahepatic PASD positive aggregates according to SERPINA1 and PNPLA3 genotype.

Article Snippet: Genotyping of the SERPINA1 rs28929474 (PI Z) variant was conducted using the TaqMan SNP Genotyping Assay C_34508510_10, and the rs17580 (PI S) variant using Assay C_594695_20 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques:

Journal: PLOS One

Article Title: Genetic drivers of liver cirrhosis: The role of SERPINA1 and PNPLA3 variants in disease onset and progression

doi: 10.1371/journal.pone.0333051

Figure Lengend Snippet: Extent and density of intrahepatic PASD positive aggregates according to SERPINA1 genotype.

Article Snippet: Genotyping of the SERPINA1 rs28929474 (PI Z) variant was conducted using the TaqMan SNP Genotyping Assay C_34508510_10, and the rs17580 (PI S) variant using Assay C_594695_20 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques:

Journal: PLOS One

Article Title: Genetic drivers of liver cirrhosis: The role of SERPINA1 and PNPLA3 variants in disease onset and progression

doi: 10.1371/journal.pone.0333051

Figure Lengend Snippet: Extent and density of intrahepatic PASD positive aggregates according to SERPINA1 and PNPLA3 genotype.

Article Snippet: Genotyping of the SERPINA1 rs28929474 (PI Z) variant was conducted using the TaqMan SNP Genotyping Assay C_34508510_10, and the rs17580 (PI S) variant using Assay C_594695_20 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: