Journal: bioRxiv

Article Title: Di-valent siRNA Mediated Silencing of MSH3 Blocks Somatic Repeat Expansion in Mouse Models of Huntington’s Disease

doi: 10.1101/2022.09.06.506795

Figure Lengend Snippet: (A) Chemical scaffold of fully modified siRNA utilized for in vitro screening. (B) MSH3 mRNA was measured in HeLa (red) & Neuro2a (blue) cells 72 hours post-treatment with 1.5 μM siRNA or Non-Targeting Control (NTC). UNT denotes untreated controls. Dose response results for MSH3_1000 (C) and MSH3_1438 (D) in HeLa (left), N2A cells (middle), and non-human primate (NHP) LLC-MK2 cells (right). Cells treated with siRNA at concentrations shown for 72 hours. For all analyses, mRNA levels were measured using the QuantiGene Singleplex assay and calculated as percentage of untreated.

Article Snippet: Equal amounts of protein (10 mg) were separated by SDS-PAGE and analyzed by western blot using antibodies to Huntingtin (1:2000, Ab1, aa1-17,( ) MSH3 (1:500, Santa Cruz) and b-tubulin (1:5000, Sigma), and GAPDH (1:10,000, Millipore) as previously described ( ).

Techniques: Modification, In Vitro, Control, Singleplex Assay

Journal: bioRxiv

Article Title: Di-valent siRNA Mediated Silencing of MSH3 Blocks Somatic Repeat Expansion in Mouse Models of Huntington’s Disease

doi: 10.1101/2022.09.06.506795

Figure Lengend Snippet: (A) Di-valent chemically modified siRNA structure. (B) Experimental setup in Hdh Q111 mice depicts bilateral intracerebroventricular injection of PBS, di-siRNA targeting a non-targeting control (NTC), di-siHTT_10150, or di-siMSH3_1000 with 125 μg siRNA per ventricle. Mice were injected at 12 weeks old and euthanized at 20 weeks old. (C) Msh3 mRNA (green) abundance and localization in striatum detected with RNAscope eight weeks post-injection following PBS (left) or siMSH3_1000 (center) treatment. Nuclei stained with DAPI (blue) (D) Quantification of nuclear (N) and cytoplasmic (C) foci per cell (striatum) in PBS (gray) or siMSH3_1000 (blue) treated animals. Analyzed by Kruskal-Wallis one-way ANOVA. (E) MSH3 protein expression and (F) mutant Htt (mutHTT) in striatum, cortex, and thalamus following treatment with PBS (gray), NTC (black), siHTT_10150 (red) or siMSH3_1000 (purple). Protein expression compared to NTC (one-way ANOVA with Dunnett’s multiple comparison test, *p <0.05, **p<0.01, or ***p<0.001). Each data point derives from striatum of one animal (N=4-6 animals per condition). (G) Representative fragment analysis of the expanded CAG locus in striatum of PBS, NTC, siMSH3_1000 and siHTT_10150 treated Hdh Q111 mice, eight weeks post-injection. Primers were reported in methods. (H) Somatic instability index calculated with a 5% signal-to-noise threshold as described in Methods. Each data point is one mouse. Instability index compared to PBS (one-way ANOVA with Dunnett’s multiple comparison test; *p <0.05, **p<0.01, or ***p<0.001).

Article Snippet: Equal amounts of protein (10 mg) were separated by SDS-PAGE and analyzed by western blot using antibodies to Huntingtin (1:2000, Ab1, aa1-17,( ) MSH3 (1:500, Santa Cruz) and b-tubulin (1:5000, Sigma), and GAPDH (1:10,000, Millipore) as previously described ( ).

Techniques: Modification, Injection, Control, RNAscope, Staining, Expressing, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Di-valent siRNA Mediated Silencing of MSH3 Blocks Somatic Repeat Expansion in Mouse Models of Huntington’s Disease

doi: 10.1101/2022.09.06.506795

Figure Lengend Snippet: (A) Di-valent chemically modified siRNA structure including the chemical structure used. (B) BAC-CAG study plan, injecting groups at 12 weeks old: PBS, NTC, siHTT_10150, and siMSH3_1000. Mice were injected with 125 μg per ventricle of di-valent siRNA and were euthanized at 20 weeks. (C) MSH3 protein measured in PBS, NTC and siMSH3_1000 groups showing 40-50% silencing of the Msh3 protein in the striatum, cortex, and thalamus. (D) mutHTT protein expression of PBS, NTC and siHTT_10150 showing >90% silencing in the striatum, cortex and thalamus. (E) Representative fragment analysis of the expanded CAG locus in striatum of PBS, NTC, siMSH3_1000 and siHTT_10150 treated BAC-CAG mice 8 weeks post-injection. Primers reported in methods. (F) Somatic instability index calculated with a 5% signal-to-noise threshold as described in methods. Each data point is one mouse. Instability index compared to NTC (one-way ANOVA treatment with Dunnett’s multiple comparison test; *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Equal amounts of protein (10 mg) were separated by SDS-PAGE and analyzed by western blot using antibodies to Huntingtin (1:2000, Ab1, aa1-17,( ) MSH3 (1:500, Santa Cruz) and b-tubulin (1:5000, Sigma), and GAPDH (1:10,000, Millipore) as previously described ( ).

Techniques: Modification, Injection, Expressing, Comparison

Journal: bioRxiv

Article Title: Di-valent siRNA Mediated Silencing of MSH3 Blocks Somatic Repeat Expansion in Mouse Models of Huntington’s Disease

doi: 10.1101/2022.09.06.506795

Figure Lengend Snippet: (A) Di-valent chemically modified siRNA structure including the chemical structure used. (B) BAC-CAG study plan, injecting groups at 12 weeks old: PBS, NTC, siMSH3_1000, siMSH3_1468. Mice were injected with 125 μg per ventricle of di-valent siRNA and were euthanized at 28 weeks. (C) MSH3 mRNA measured in PBS, NTC, siMSH3_1000 and siMSH3_1468 groups showing 70% and 60% Msh3 mRNA silencing (respectively) in the striatum. (D) MSH3 protein measured in PBS, NTC, siMSH3_1000 and siMSH3_1468 groups showing 60% silencing of the Msh3 protein in the striatum with MSH3_1000 and 5% with siMSH3_1468. (E) Representative fragment analysis of the expanded CAG locus in striatum of PBS, NTC, siMSH3_1000 and siMSH3_1468 treated BAC-CAG mice 12 weeks post-injection. Primers reported in methods. (F) Somatic instability index calculated with a 5% signal-to-noise threshold as described in methods. Each data point is one mouse. Instability index compared to NTC (one-way ANOVA treatment with Dunnett’s multiple comparison test; *p <0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: Equal amounts of protein (10 mg) were separated by SDS-PAGE and analyzed by western blot using antibodies to Huntingtin (1:2000, Ab1, aa1-17,( ) MSH3 (1:500, Santa Cruz) and b-tubulin (1:5000, Sigma), and GAPDH (1:10,000, Millipore) as previously described ( ).

Techniques: Modification, Injection, Comparison

Journal: Nature Communications

Article Title: Integrated digital pathology and transcriptome analysis identifies molecular mediators of T-cell exclusion in ovarian cancer

doi: 10.1038/s41467-020-19408-2

Figure Lengend Snippet: The tumour-immune phenotypes for a vendor-procured collection (including both primary tumours and recurrent tumours, n = 84 samples) were predicted based on gene expression. The pattern of CD8 + T-cell infiltration and molecular features associated with excluded tumours were validated using immunohistochemistry and in situ hybridization (ISH) on FFPE tumour tissues. a Representative images of CD8 IHC (top), MHC-I-IHC (middle) and FAP ISH (bottom) are shown for the three tumour-immune phenotypes. b Percentage of CD8 staining over tumour/stroma area ( n = 72 samples), H scores for MHC-I ( n = 77 samples) and FAP expression in the tumour or the stroma ( n = 77 samples) were presented by the three-class tumour-immune phenotypes. c RNA-seq gene expression level, represented as Log 2 (RPKM+1) for CD8A , HLA-A and FAP , is presented across the three-class tumour-immune phenotypes. b , c Whiskers ranging from minima to maxima, median and 25–75% IQR shown by boxplots; each dot is a tumour sample (primary tumours and recurrent tumours are pooled). The statistical significance is displayed with the exact P values on the graphs and calculated with a Kruskal–Wallis test corrected for multiple comparisons (Dunn’s test). Source data are provided as a Source Data file.

Article Snippet: Single-plex FAP RNAscope ISH assay was designed, implemented and scored at Advanced Cell Diagnostics (Hayward, CA).

Techniques: Gene Expression, Immunohistochemistry, In Situ Hybridization, Staining, Expressing, RNA Sequencing