msh3  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Genetic or pharmacological disruption of the MSH3 Y245/K246 IDL binding pocket slows CAG repeat expansion

doi: 10.64898/2026.02.26.707948

Figure Lengend Snippet: (A) WT or MSH3 KO cells complemented with myc MSH3 6a, Y254S/K255E or E976A constructs were transduced with LV HTT exon 1 118 CAG. Repeat size was monitored over 40 days in culture using fragment analysis. WT cells show a smooth increase in CAG repeat size over time in culture. MSH3 KO cells do not support CAG repeat expansion (not shown). CAG repeat expansion is rescued by expression of the MSH3 6a construct (the full-length WT form) expressed at similar levels to the endogenous protein (with 0.1 ng/ml dox treatment). In contrast, cells expressing myc MSH3 Y254S/K255E or E976A mutants do not support CAG repeat expansion. (B) Histogram shows regression analysis of expansion curves in (A), rate CAG per day (mean ± standard error n=4, p values one-way ANNOVA). (C) Western blot showing expression of MutS proteins in MSH3 KO, WT and KO cells complemented with myc MSH3 6a, Y254S/K255E or E976A constructs. Complemented cells were treated with 0.1 ng/ml dox except the E976A line in which 1 ng/ml dox was used. Note the myc tagged MSH3 forms run at a higher molecular weight. (D) U2OS WT, MSH3 KO or MSH3 KO cells complemented myc MSH3 6a, Y254S/K255E or E976A were transduced with an EMAST frameshift reporter construct. This 16 tetranucleotide repeat construct measures MSH3 dependent MSI in cells, assayed by measuring NanoLuc signal normalised to a constitutive Luc signal. Results are expressed as NanoLuc/Luc2 normalised to WT cells (n=4 or 5 ± sd, p values one-way ANNOVA).

Article Snippet: Antibodies used were: MSH3 rabbit polyclonal (Proteintech, used for ChIP and some Westerns), MSH3 mouse monoclonal, MLH1 mouse monoclonal and MSH6 mouse monoclonal (BD Biosciences), MSH2 and PCNA rabbit (Cell Signaling Technology), and an actin mouse monoclonal (Sigma).

Techniques: Construct, Transduction, Expressing, Western Blot, Molecular Weight

Journal: bioRxiv

Article Title: Genetic or pharmacological disruption of the MSH3 Y245/K246 IDL binding pocket slows CAG repeat expansion

doi: 10.64898/2026.02.26.707948

Figure Lengend Snippet: (A) U2OS cell extracts from WT (+/+) MSH3 KO (-/-) or KO cells complemented with myc MSH3 6a or myc MSH3 Y254S/K255E (YSKE) constructs treated with 0.1 ng/ml dox were incubated O/N with MSH3 antibodies and protein A/G magnetic beads. Beads were isolated and washed with a magnetic device. Input (5%) proteins eluted from the beads were immunoblotted with the indicated antibodies. Similar levels of MSH2 were recovered in the MSH3 IP fraction from both WT MSH3, myc MSH3 6a and YSKE samples. MLH1 was not detected in the IP fractions under these conditions. (B) Quantification of MSH2/MSH3 binding from IP experiments normalized to WT MSH3 is shown in the histogram (n=3 ± sd). (C) U2OS cell extracts from MSH3 KO or KO cells complemented with myc MSH3 6a or myc MSH3 Y254S/K255E were incubated O/N with myc magnetic beads. Transgenes were over-expressed by approximately 8-fold with with 10 ng/ml dox. Beads were isolated and washed with a magnetic device. Input (5%) and proteins eluted from the beads were immunoblotted with the indicated antibodies. Similar levels of MSH2 were recovered in the myc IP fraction from both MSH3 6a and YSKE samples indicating the MSH3 YSKE mutant binds MSH2 normally. Low levels of MLH1 (green arrowhead) were consistently detected in myc MSH3 6a IP fractions. Note a non-specific band running slightly faster than MLH1 (red arrowhead) is detected in all of the IP fractions (D) Quantification normalized to MSH3 6a is shown in the histogram (n=3 ±sd).

Article Snippet: Antibodies used were: MSH3 rabbit polyclonal (Proteintech, used for ChIP and some Westerns), MSH3 mouse monoclonal, MLH1 mouse monoclonal and MSH6 mouse monoclonal (BD Biosciences), MSH2 and PCNA rabbit (Cell Signaling Technology), and an actin mouse monoclonal (Sigma).

Techniques: Construct, Incubation, Magnetic Beads, Isolation, Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Genetic or pharmacological disruption of the MSH3 Y245/K246 IDL binding pocket slows CAG repeat expansion

doi: 10.64898/2026.02.26.707948

Figure Lengend Snippet: (A) Cell extracts from HTT exon 1 118Q transduced MSH3 KO cells or (-/-) or KO cells complemented with myc MSH3 6a or myc MSH3 Y254S/K255E (YSKE) were prepared for ChIP analysis and immunoprecipitated with anti MSH3, RNA PolII or control non-specific mouse IgG antibodies (NC). DNA from input and ChIP fractions was purified and probed with primers targeting the HTT exon 118Q CAG repeat in the construct (CAG set 3) or GFP primers downstream from this in the insertion cassette (HTT GFP). Input (5%) and ChIP fractions were analysed using TapeStation apparatus and software. MSH3 ChIP fractions from YSKE mutant contain less CAG repeat and HTT GFP DNA. Note the myc MSH3 cells were treated with 10 ng/ml dox which induces ∼8x MSH3 overexpression relative to WT cells. Plasmid DNA was amplified alongside the ChIP fractions as a positive control (PC) (B) Quantification of HTT GFP levels in ChIP fractions normalized to MSH3 -/- is shown (n=4 ± sd, p values one-way ANNOVA). (C) U2OS cell extracts from WT cells or KO cells complemented with myc MSH3 6a or myc MSH3 Y254S/K255E (YSKE) were prepared and incubated O/N with 2 CAG loopout oligos immobilized on streptavidin magnetic beads. Beads were isolated and washed with a magnetic device. Input (5%) and eluted proteins (CAG2 loopout) were immunoblotted with the indicated antibodies. Unconjugated beads (bead) were used as controls (WT sample only). Note the myc MSH3 cells were treated with 10 ng/ml dox which induces ∼8x MSH3 overexpression relative to WT cells (compare lanes +/+ and 6a). (D) Quantification of MSH3 levels in CAG2 loopout pull down fractions normalized to myc MSH3 6a is shown (n=4 ± sd, p values one-way ANNOVA). (E) U2OS cell extracts from WT cells, KO cells or KO cells complemented with myc MSH3 6a or myc MSH3 Y254S/K255E (YSKE) and treated with 0.1 ng/ml dox were prepared. These were incubated O/N with magnetic streptavidin beads conjugated to 2 CAG loopout oligos or empty beads. Input (5%) and eluted proteins (CAG2 loopout) were immunoblotted with the indicated antibodies. Equivalent levels of MSH3 and MSH2 were recovered in the pulldown fractions from WT and myc MSH3 6a indicating the WT and myc tagged MSH3 forms have similar activity. Proportionally less of the MSH2 input was pulled down compared to MSH3. Pull downs from myc MSH3 Y254S/K255E extracts contained only low levels of MSH3 and MSH2. MSH3 KO cell extracts (-/-) and unconjugated beads (bead, WT sample only) were used as controls. N.B. Lanes (-/-) and (+/+, bead) have been switched around in the middle (MSH2) panel to match the arrangement of the top (MSH3) panel.

Article Snippet: Antibodies used were: MSH3 rabbit polyclonal (Proteintech, used for ChIP and some Westerns), MSH3 mouse monoclonal, MLH1 mouse monoclonal and MSH6 mouse monoclonal (BD Biosciences), MSH2 and PCNA rabbit (Cell Signaling Technology), and an actin mouse monoclonal (Sigma).

Techniques: Immunoprecipitation, Control, Purification, Construct, Software, Mutagenesis, Over Expression, Plasmid Preparation, Amplification, Positive Control, Incubation, Magnetic Beads, Isolation, Activity Assay

Journal: bioRxiv

Article Title: Genetic or pharmacological disruption of the MSH3 Y245/K246 IDL binding pocket slows CAG repeat expansion

doi: 10.64898/2026.02.26.707948

Figure Lengend Snippet: (A) U2OS cell extracts from WT cells were treated with the indicated CP1 concentrations for 1 h prior to incubation with 2 CAG loopout oligos immobilized on streptavidin magnetic beads. Beads were isolated and washed with a magnetic device. Input (5%) and eluted proteins (CAG2) were immunoblotted with the indicated antibodies. MSH3 and MSH2 were recovered in the pulldown fractions in vehicle (DMSO) treated extracts. CP1 treatment reduced the recovery. (B) Quantification of MSH3 levels in CAG2 loopout pull downs normalized to DMSO control is shown in the (n=4 ± sd). (C) U2OS WT cells were treated for 24 h with CP1 at 0, 2.5 or 5 uM and cell extracts were prepared and incubated with 2 CAG loopout oligos immobilized on streptavidin magnetic beads as described in panel (A). MSH3 and MSH2 recovery in the bead fraction was reduced by CP1 treatment. Unconjugated beads were included as a control (E). ( D ) Quantification MSH3 levels in CAG2 loopout pull downs normalized to vehicle treated cells is shown (n=4 ± sd). ( E ) HD MSNs were treated with CP1 (2.5 and 5 µM) for 48 h, cell extracts were prepared and 2 CAG loopout oligo binding assays performed as in panel (A). ( F ) Quantification MSH3 levels in CAG2 loopout pull downs normalised to vehicle treated cells is shown (n=3 ± sd).

Article Snippet: Antibodies used were: MSH3 rabbit polyclonal (Proteintech, used for ChIP and some Westerns), MSH3 mouse monoclonal, MLH1 mouse monoclonal and MSH6 mouse monoclonal (BD Biosciences), MSH2 and PCNA rabbit (Cell Signaling Technology), and an actin mouse monoclonal (Sigma).

Techniques: Incubation, Magnetic Beads, Isolation, Control, Binding Assay

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-B) Still frames from an animation of untreated (A) and treated (B) HD patients are shown from example simulations of 3000 MSNs that depict somatic CAG repeat expansion in individual MSNs at 20-year intervals from 1-100 years. (A) Each point represents an untreated MSN and simulated CAG length (x-axis). The red line represents the 150 CAG threshold for transcriptionally healthy MSNs (<150 CAG; green) and dysregulated unhealthy MSNs (>150 CAG; red) progressing toward cell death (>300 CAGs). (B) Simulated MSNs are shown with SI-targeted therapy administration at 32 years old and 50% of MSNs targeted with 50% MSH3 knockdown. Targeted MSNs are circled in pink. (C) The percentage of untreated healthy MSNs (<150 CAGs) are tracked and plotted across a simulated lifetime (0-100 years). Shaded background panels represent the indicated HD-ISS stage, age (shown in red) when a HD-ISS stage is reached, and dotted lines represent a HD milestone event (measurable volumetric change and CAP-100 equivalent age). (D) A treated purple line is compared to the untreated grey line from (C) and tracks the percentage of healthy treated MSNs after administration at 32 years old. (E-G) The model tracks therapeutic benefit by determining the age and healthy MSN number coinciding with a HD landmark event (onset of striatum volume change, motor symptoms, or HD-ISS stage). (H) The model enables prediction of cUHDRS change from baseline between simulated treated (purple dotted line) and untreated Enroll-HD natural history cohorts (grey dotted line).

Article Snippet: RT-qPCR was performed using the TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems 4444432) and duplexed with primers and probes for mouse Msh3 Mouse (Applied Biosystems Mm00487756_m1) and mouse ActB ACTB (Applied Biosystems Mm00607939_s1).

Techniques: Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-C) Heatmaps depicting the simulated therapeutic benefit for a SI-lowering therapy in a range of treated patients with germline CAG repeat lengths from 37-60 CAGs (y-axis) and ages at treatment from 5-60 years of age (x-axis). The degrees of predicted therapeutic benefit as measured by years of delay to CAP-100 equivalent are depicted in the heatmaps (shown in the heatmap key; less than 5 years of benefit shown in grey, CAP-100 equivalent never reached shown in purple, and benefit between >5 and <50 years shown by the white to pink gradient). (A) Heatmap depicting the simulated range of therapeutic benefit achieved by targeting 25% of MSNs with 25% MSH3 knockdown per MSN. (B) Heatmaps depicting the range of therapeutic benefit with 50% MSH3 knockdown per MSN and a range of MSN targeting percentages (50%, 75%, and 100%). (C) Heatmaps depicting the range of therapeutic benefit with targeting 50% of MSNs and a range of MSH3 knockdown percentages (50%, 75%, and 100%) per MSN.

Article Snippet: RT-qPCR was performed using the TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems 4444432) and duplexed with primers and probes for mouse Msh3 Mouse (Applied Biosystems Mm00487756_m1) and mouse ActB ACTB (Applied Biosystems Mm00607939_s1).

Techniques: Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A) Study design for the nonhuman primate dose range finding study in rhesus macaques. (B) AAV vector biodistribution (vector genomes per diploid genome) in the globus pallidus (GP), caudate nucleus, and putamen of vehicle and treated rhesus macaques by droplet digital PCR (ddPCR) after bilateral intraparenchymal (IPa) GP administration of AAV-DB-3.miMSH3-06. (C) miMSH3 expression levels normalized to copies per µg of RNA by stem-loop RT-qPCR. (D) MSH3 mRNA levels normalized to TBP and relative to vehicle control by RT-qPCR. (E) MSH3 protein levels relative to vehicle control by Jess capillary-electrophoresis immunoassay. Data are shown as group mean (N=2) with data points from each animal. Panel A was created using BioRender.com.

Article Snippet: RT-qPCR was performed using the TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems 4444432) and duplexed with primers and probes for mouse Msh3 Mouse (Applied Biosystems Mm00487756_m1) and mouse ActB ACTB (Applied Biosystems Mm00607939_s1).

Techniques: Plasmid Preparation, Digital PCR, Expressing, Quantitative RT-PCR, Control, Electrophoresis

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-B) Representative RNAscope FISH confocal microscopy images of MSH3 mRNA puncta (green) in PPP1R1B+ MSNs (magenta) in the caudate (A) and putamen (B) of NHPs. Hoechst stain (blue) was used to counterstain nuclei. (C) Cytoplasmic MSH3 mRNA puncta count in PPP1R1B+ MSNs of the striatum (caudate and putamen) were determined with an automated cell segmentation and puncta counting algorithm. The percentages of cytoplasmic MSH3 mRNA knockdown (KD) levels in all MSNs profiled in the caudate and putamen relative to vehicle control are shown above each treatment group. Total MSN counts are displayed below each group. Data are shown as group mean (N=2) with data points from each animal.

Article Snippet: RT-qPCR was performed using the TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems 4444432) and duplexed with primers and probes for mouse Msh3 Mouse (Applied Biosystems Mm00487756_m1) and mouse ActB ACTB (Applied Biosystems Mm00607939_s1).

Techniques: RNAscope, Confocal Microscopy, Staining, Knockdown, Control

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-D) Heatmaps depicting the simulated range of therapeutic benefit for patients treated with an SI-lowering AAV gene therapy over a lifetime of 100 years. Each heatmap represents a range of patients with germline CAG lengths from 40-50 CAG repeats (y-axis) and age at treatment (x-axis). The degrees of predicted therapeutic benefit as measured by years of delay to CAP-100 equivalent are depicted in the heatmaps (shown in the heatmap key; less than 5 years of benefit shown in grey, CAP-100 equivalent was never reached shown in purple, and benefit between >5 and <50 years shown by the gradient from white to pink). (A) The range of therapeutic benefit achieved after simulating 50% of MSNs transduced and 50% MSH3 KD per transduced MSN. This threshold is denoted by black bordered heatmap cells in all four heatmaps. (B-D) Heatmaps depict the predicted range of therapeutic benefit for HD patients treated with the NHP DRF dose equivalents of AAV-DB-3.miMSH3 at the low dose (1.5×10 11 vg) (B) , mid dose (8.2×10 11 vg) (C) , and high dose (3.2×10 12 vg) (D) based on the RNAscope FISH MSH3 mRNA KD levels detected in the NHP caudate and putamen . CAG repeat length incidence is shown by the red to yellow color gradient column . (E) To characterize predicted therapeutic performance using cUHDRS across the observed percentages of MSH3 knockdown, we simulated these therapeutic performance levels in a hypothetical patient with 42 CAGs treated at the age of 46.

Article Snippet: RT-qPCR was performed using the TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems 4444432) and duplexed with primers and probes for mouse Msh3 Mouse (Applied Biosystems Mm00487756_m1) and mouse ActB ACTB (Applied Biosystems Mm00607939_s1).

Techniques: RNAscope, Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A) Study design for the pharmacology study in heterozygous HdhQ111 KI mice. (B) Msh3 mRNA levels in the striatum of untreated and treated HdhQ111 mice by RT-qPCR. Msh3 mRNA levels were normalized to ActB mRNA and shown as the group mean relative to vehicle control (N=7, 8 week untreated; N=7, 24 week untreated; N=9, vehicle; N=10, 5×10 9 vg; N=10, 1.5×10 10 vg; N=10, 5×10 10 vg; N=8, 1.5×10 11 vg; one-way ANOVA with Dunnett’s post hoc analysis, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (C) Fragment analysis traces shown in blue depict the intensity of CAG repeat length alleles in untreated, vehicle and AAV-DB-3.miMSH3-06 treated mice by PCR and capillary electrophoresis. (D) Somatic instability index in untreated and treated mice (N=10/group; one-way ANOVA with Dunnett’s post hoc analysis, **** p < 0.0001). Data are shown as the mean and error bars represent SD. The percent reduction in SI relative to vehicle control is shown above each treatment group. Panel A was created using BioRender.com.

Article Snippet: RT-qPCR was performed using the TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems 4444432) and duplexed with primers and probes for mouse Msh3 Mouse (Applied Biosystems Mm00487756_m1) and mouse ActB ACTB (Applied Biosystems Mm00607939_s1).

Techniques: Quantitative RT-PCR, Control, Electrophoresis

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-B) Still frames from an animation of untreated (A) and treated (B) HD patients are shown from example simulations of 3000 MSNs that depict somatic CAG repeat expansion in individual MSNs at 20-year intervals from 1-100 years. (A) Each point represents an untreated MSN and simulated CAG length (x-axis). The red line represents the 150 CAG threshold for transcriptionally healthy MSNs (<150 CAG; green) and dysregulated unhealthy MSNs (>150 CAG; red) progressing toward cell death (>300 CAGs). (B) Simulated MSNs are shown with SI-targeted therapy administration at 32 years old and 50% of MSNs targeted with 50% MSH3 knockdown. Targeted MSNs are circled in pink. (C) The percentage of untreated healthy MSNs (<150 CAGs) are tracked and plotted across a simulated lifetime (0-100 years). Shaded background panels represent the indicated HD-ISS stage, age (shown in red) when a HD-ISS stage is reached, and dotted lines represent a HD milestone event (measurable volumetric change and CAP-100 equivalent age). (D) A treated purple line is compared to the untreated grey line from (C) and tracks the percentage of healthy treated MSNs after administration at 32 years old. (E-G) The model tracks therapeutic benefit by determining the age and healthy MSN number coinciding with a HD landmark event (onset of striatum volume change, motor symptoms, or HD-ISS stage). (H) The model enables prediction of cUHDRS change from baseline between simulated treated (purple dotted line) and untreated Enroll-HD natural history cohorts (grey dotted line).

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-C) Heatmaps depicting the simulated therapeutic benefit for a SI-lowering therapy in a range of treated patients with germline CAG repeat lengths from 37-60 CAGs (y-axis) and ages at treatment from 5-60 years of age (x-axis). The degrees of predicted therapeutic benefit as measured by years of delay to CAP-100 equivalent are depicted in the heatmaps (shown in the heatmap key; less than 5 years of benefit shown in grey, CAP-100 equivalent never reached shown in purple, and benefit between >5 and <50 years shown by the white to pink gradient). (A) Heatmap depicting the simulated range of therapeutic benefit achieved by targeting 25% of MSNs with 25% MSH3 knockdown per MSN. (B) Heatmaps depicting the range of therapeutic benefit with 50% MSH3 knockdown per MSN and a range of MSN targeting percentages (50%, 75%, and 100%). (C) Heatmaps depicting the range of therapeutic benefit with targeting 50% of MSNs and a range of MSH3 knockdown percentages (50%, 75%, and 100%) per MSN.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A) Study design for the nonhuman primate dose range finding study in rhesus macaques. (B) AAV vector biodistribution (vector genomes per diploid genome) in the globus pallidus (GP), caudate nucleus, and putamen of vehicle and treated rhesus macaques by droplet digital PCR (ddPCR) after bilateral intraparenchymal (IPa) GP administration of AAV-DB-3.miMSH3-06. (C) miMSH3 expression levels normalized to copies per µg of RNA by stem-loop RT-qPCR. (D) MSH3 mRNA levels normalized to TBP and relative to vehicle control by RT-qPCR. (E) MSH3 protein levels relative to vehicle control by Jess capillary-electrophoresis immunoassay. Data are shown as group mean (N=2) with data points from each animal. Panel A was created using BioRender.com.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: Plasmid Preparation, Digital PCR, Expressing, Quantitative RT-PCR, Control, Electrophoresis

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-B) Representative RNAscope FISH confocal microscopy images of MSH3 mRNA puncta (green) in PPP1R1B+ MSNs (magenta) in the caudate (A) and putamen (B) of NHPs. Hoechst stain (blue) was used to counterstain nuclei. (C) Cytoplasmic MSH3 mRNA puncta count in PPP1R1B+ MSNs of the striatum (caudate and putamen) were determined with an automated cell segmentation and puncta counting algorithm. The percentages of cytoplasmic MSH3 mRNA knockdown (KD) levels in all MSNs profiled in the caudate and putamen relative to vehicle control are shown above each treatment group. Total MSN counts are displayed below each group. Data are shown as group mean (N=2) with data points from each animal.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: RNAscope, Confocal Microscopy, Staining, Knockdown, Control

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A-D) Heatmaps depicting the simulated range of therapeutic benefit for patients treated with an SI-lowering AAV gene therapy over a lifetime of 100 years. Each heatmap represents a range of patients with germline CAG lengths from 40-50 CAG repeats (y-axis) and age at treatment (x-axis). The degrees of predicted therapeutic benefit as measured by years of delay to CAP-100 equivalent are depicted in the heatmaps (shown in the heatmap key; less than 5 years of benefit shown in grey, CAP-100 equivalent was never reached shown in purple, and benefit between >5 and <50 years shown by the gradient from white to pink). (A) The range of therapeutic benefit achieved after simulating 50% of MSNs transduced and 50% MSH3 KD per transduced MSN. This threshold is denoted by black bordered heatmap cells in all four heatmaps. (B-D) Heatmaps depict the predicted range of therapeutic benefit for HD patients treated with the NHP DRF dose equivalents of AAV-DB-3.miMSH3 at the low dose (1.5×10 11 vg) (B) , mid dose (8.2×10 11 vg) (C) , and high dose (3.2×10 12 vg) (D) based on the RNAscope FISH MSH3 mRNA KD levels detected in the NHP caudate and putamen . CAG repeat length incidence is shown by the red to yellow color gradient column . (E) To characterize predicted therapeutic performance using cUHDRS across the observed percentages of MSH3 knockdown, we simulated these therapeutic performance levels in a hypothetical patient with 42 CAGs treated at the age of 46.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: RNAscope, Knockdown

Journal: bioRxiv

Article Title: Computational modeling and preclinical validation support targeting somatic instability for Huntington’s disease treatment

doi: 10.64898/2026.01.06.697909

Figure Lengend Snippet: (A) Study design for the pharmacology study in heterozygous HdhQ111 KI mice. (B) Msh3 mRNA levels in the striatum of untreated and treated HdhQ111 mice by RT-qPCR. Msh3 mRNA levels were normalized to ActB mRNA and shown as the group mean relative to vehicle control (N=7, 8 week untreated; N=7, 24 week untreated; N=9, vehicle; N=10, 5×10 9 vg; N=10, 1.5×10 10 vg; N=10, 5×10 10 vg; N=8, 1.5×10 11 vg; one-way ANOVA with Dunnett’s post hoc analysis, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (C) Fragment analysis traces shown in blue depict the intensity of CAG repeat length alleles in untreated, vehicle and AAV-DB-3.miMSH3-06 treated mice by PCR and capillary electrophoresis. (D) Somatic instability index in untreated and treated mice (N=10/group; one-way ANOVA with Dunnett’s post hoc analysis, **** p < 0.0001). Data are shown as the mean and error bars represent SD. The percent reduction in SI relative to vehicle control is shown above each treatment group. Panel A was created using BioRender.com.

Article Snippet: Total RNA was extracted from NHP brain samples using the Quick-DNA/RNA Miniprep Plus kit (Zymo Research D7003) and quantified with the Qubit BR Assay kit (Life Technologies Q10210). cDNA was generated from 500 ng total RNA with Maxima H minus reverse transcriptase (Thermo Scientific EP0752) and random hexamers (Life Technologies SO142). qPCR was performed with Luna Primer Probe 2X Master Mix (NEB M3004) including primer probe sets for rhesus MSH3 (Applied Biosystems Rh00989001_m1) and TBP (Applied Biosystems Rh00427620_m1).

Techniques: Quantitative RT-PCR, Control, Electrophoresis

Journal: Nucleic Acids Research

Article Title: RNA/DNA-binding protein TDP43 regulates DNA mismatch repair genes with implications for genome stability

doi: 10.1093/nar/gkaf920

Figure Lengend Snippet: Differential expressions of MMR genes in iPSC-derived neuronal cells and their regulation by TDP43. ( A ) WB images and histogram illustrate a quantitative comparison of MMR expression in control and siTDP43-treated human iPSC-derived neural lineage progenitor stem cells (NPSCs). Quantitation of protein levels normalized to that of GAPDH. ( B ) Schematic of iPSC, NPSC, and terminally differentiated motor neurons (iMN) utilized in this study. ( C ) IF images revealing the expression of TDP43; and MLH1, MSH2, MSH3, MSH6, and PMS2, and nuclear DNA (DAPI) after siTDP43-mediated KD of TDP43 in iPSC-derived iMNs. ( D ) WB images show the expression of select MMR factors across the following states of cellular differentiation: iPSC, NPSC, and iMN. Error bars indicate mean ± SEM from three independent experiments. Significance values (P-values) are as follows P > 0.5 (ns), P <0.5 (*), P <0.001 (***) and P <0.0001 (****).

Article Snippet: Primary antibodies used: MLH1 (Cell Signaling #4256), MSH2 (Cell Signaling #2017), MSH3 (Protein Tech #22393-1-AP), MSH6 (Protein Tech #18120-1-AP), PMS2 (ABClonal #A6947), GAPDH (Cell Signaling #2118), \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\gamma$\end{document} H2AX (Cell Signaling #2577), H2AII (Cell Signaling #2578), TDP43 (Protein Tech #10782–2-AP), FLAG (Sigma #F1804), cleaved-caspase-3 (Cell Signaling #9664), and PARP-1 (Cell Signaling #9542).

Techniques: Derivative Assay, Comparison, Expressing, Control, Quantitation Assay, Cell Differentiation

Journal: Nucleic Acids Research

Article Title: RNA/DNA-binding protein TDP43 regulates DNA mismatch repair genes with implications for genome stability

doi: 10.1093/nar/gkaf920

Figure Lengend Snippet: Mouse models of TDP43–ALS display an altered MMR expression phenotype. ( A ) Schematic of the construct used to generate TDP43-ΔNLS expression under the UBC promoter in C57BL6 mice. ( B ) WB of cortical brain tissue lysate from three control and three transgenic (Tg) mice are shown. ( C ) Histograms showing fold change in immunoblot band intensity of averaged Tg samples relative to control indicate significant increases in MSH2, MSH3, and trends toward increased MSH6 in TDP43-ΔNLS mice. ( D ) Schematic of the construct used to generate moderate OE of WT murine TDP43 (mTDP43) under the PrP promoter in FVB/NJ mice. ( E ) Representative immunoblots of cortical brain tissue lysate from control and Tg mice are shown for male and female mice. ( F and G ) Histograms of fold change in immunoblot band intensity of averaged Tg samples relative to control indicate significant increases in MLH1, MSH2, and MSH3 expression. ( H ) WB and quantitative histogram of insoluble protein isolates from control and Guam ALS-affected human postmortem CNS tissue. Significance values ( P -values) are as follows: P >0.5 (ns), P <0.5 (*), P <0.01 (**), P <0.001 (***). Error bars indicate standard error mean ± SD, derived from at least three biological and technical replicates.

Article Snippet: Primary antibodies used: MLH1 (Cell Signaling #4256), MSH2 (Cell Signaling #2017), MSH3 (Protein Tech #22393-1-AP), MSH6 (Protein Tech #18120-1-AP), PMS2 (ABClonal #A6947), GAPDH (Cell Signaling #2118), \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\gamma$\end{document} H2AX (Cell Signaling #2577), H2AII (Cell Signaling #2578), TDP43 (Protein Tech #10782–2-AP), FLAG (Sigma #F1804), cleaved-caspase-3 (Cell Signaling #9664), and PARP-1 (Cell Signaling #9542).

Techniques: Expressing, Construct, Control, Transgenic Assay, Western Blot, Derivative Assay

Journal: bioRxiv

Article Title: Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntington’s disease pathogenesis in mice

doi: 10.1101/2025.06.24.661398

Figure Lengend Snippet: ( a ) In vivo study design and treatment paradigm. 2-month-old wild-type (WT) or Q111 mice were treated with artificial CSF (aCSF), or divalent siRNA programmed with sequences targeting a non-targeting control (NTC), MSH3, HTT, or MSH3 and HTT , and euthanized at 12 months old. ( b ) Divalent siRNA scaffold and ( c ) chemical modification pattern key. ( d, e) Endpoint protein levels in WT striatum: ( d ) WT HTT, ( e ) MSH3. ( f-h ) Endpoint protein levels in Q111 striatum: ( f ) WT HTT, ( g ) mutant HTT, ( h ) MSH3. ( i ) Representative striatum fragment analysis curves for baseline (2 months Q111 untreated) or Q111 mice (12 months old) treated with artifical CSF (aCSF), non-targeting control (NTC) siRNA, HTT siRNA, MSH3 siRNA, or MSH3+HTT siRNA combination. ( j,k ) Somatic instability index quantified from fragment analysis curves in ( j ) striatum or ( k ) medial cortex. WT cohorts: aCSF, n=12; NTC, n=10; MSH3 siRNA, n=12; HTT siRNA, n=12; MSH3+HTT siRNA, n=12 mice. Q111 cohorts: aCSF, n=12; NTC, n=10; MSH3 siRNA, n=10; HTT siRNA, n=9; MSH3+HTT, n=11 mice. Statistics are one-way ANOVA with Dunnett’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Western blots were performed as previously described (Sapp et al., 2020) using antibodies to HTT (aa1-17, 1:2000, DiFiglia et al., 1995), Msh3 (1:500, Santa Cruz sc-271079), HTT1a (5ug/ml, P90 1B12, Coriell Institute), Gapdh (1:10000, MilliporeSigma), and B-actin (1:5000, MilliporeSigma).

Techniques: In Vivo, Control, Modification, Mutagenesis

Journal: bioRxiv

Article Title: Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntington’s disease pathogenesis in mice

doi: 10.1101/2025.06.24.661398

Figure Lengend Snippet: ( a-g ) Striatal sections probed with EM48 immunohistochemistry. Representative images shown. The top panel is 40x, and the bottom panel is an area within a boxed region of interest. White arrow indicates representative EM48+ focal puncta. ( a ) WT aCSF ( b ) 2-month-old untreated Q111 ( c ) aCSF treated Q111 ( d ) NTC treated Q111 ( e ) MSH3 siRNA treated Q111 ( f ) HTT siRNA treated Q111 ( g ) MSH3+HTT combination siRNA treated Q111 ( h ) Representative histological characterization of puncta (top) or diffuse (bottom) EM48+ mHTT events ( i ) Quantified puncta (right) or diffuse (right) events per mouse totaled from three 40x images across three 40 µM sections (nine images counted per mouse). Each dot is average from one mouse. ( j ) mHTT puncta (left) or diffuse (right) quantified over each cohort. Same data as panel i, but each dot is puncta or diffuse EM48+ event from one 40x image, not averaged per mouse. ( k ) Percentage of focal puncta to total EM48+ events. Each dot is the average of nine slides from one mouse. ( l ) Percentage of diffuse EM48+ events to total EM48+ events. Each dot is the average of nine slides from one mouse. Total EM48+ events are defined as focal plus diffuse events. n=4 mice/group strained and quantified. Statistics are one-way ANOVA with Holm-Šídák’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Western blots were performed as previously described (Sapp et al., 2020) using antibodies to HTT (aa1-17, 1:2000, DiFiglia et al., 1995), Msh3 (1:500, Santa Cruz sc-271079), HTT1a (5ug/ml, P90 1B12, Coriell Institute), Gapdh (1:10000, MilliporeSigma), and B-actin (1:5000, MilliporeSigma).

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntington’s disease pathogenesis in mice

doi: 10.1101/2025.06.24.661398

Figure Lengend Snippet: Striatal immunofluorescence of PHP1 (green) co-stained with DARPP32 (purple) and DAPI (blue). Representative images of 12-month-old ( a ) aCSF-treated WT ( b ) aCSF treated Q111 ( c ) NTC treated Q111 ( d) MSH3 siRNA treated Q111 ( e ) HTT siRNA treated Q111 ( f ) MSH3+HTT combination siRNA treated Q111 mice. Quantification of ( g ) average aggregate count per field of view, ( h ) PHP1+ area per ROI, ( i ) average aggregate size. n=4 mice/group imaged and quantified. Each dot summarizes events over six regions of interest, covering three striatal slices in one mouse. Statistics are one-way ANOVA with Šídák’s multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Western blots were performed as previously described (Sapp et al., 2020) using antibodies to HTT (aa1-17, 1:2000, DiFiglia et al., 1995), Msh3 (1:500, Santa Cruz sc-271079), HTT1a (5ug/ml, P90 1B12, Coriell Institute), Gapdh (1:10000, MilliporeSigma), and B-actin (1:5000, MilliporeSigma).

Techniques: Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntington’s disease pathogenesis in mice

doi: 10.1101/2025.06.24.661398

Figure Lengend Snippet: (a) Experimental design and treatments. ( b-f ) Striatal HTT protein species quantified using homogenous time-resolved fluorescence detected from respective antibody donor (D) or acceptor (A) pairs across time points and treatment groups. ( b ) Full-length total HTT (d: MAB2166-Tb, a: CHDI-1414-d2) ( c) Soluble full-length mHTT (excludes HTT1a) (d: MAB2166-Tb, a: 4C9-488) ( d ) Total soluble mHTT (d: 2B7-Tb, a: MW1-d2) ( e ) Soluble HTT1a (d: 2B7-Tb, a: 11G2-d2) ( f ) Aggregated HTT1a (d:4C9-Tb, a:11G2-d2). Statistics are one-way ANOVA with Dunnett’s multiple comparisons versus NTC. Mice per group at two-month timepoint: WT n=6, Q111 n=6; Mice per group at 11-month endpoint: untreated WT n=7, NTC siRNA Q111 n=8, MSH3 siRNA Q111 n=7, HTT siRNA Q111 n=7, MSH3+HTT siRNA Q111 n=6. ( g ) Quantified western blot probing for HTT1a using antibody 1B12 in the striatum of the cohort of Q111 mice presented in . Statistics are one-way ANOVA with all comparisons and Tukey’s multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Western blots were performed as previously described (Sapp et al., 2020) using antibodies to HTT (aa1-17, 1:2000, DiFiglia et al., 1995), Msh3 (1:500, Santa Cruz sc-271079), HTT1a (5ug/ml, P90 1B12, Coriell Institute), Gapdh (1:10000, MilliporeSigma), and B-actin (1:5000, MilliporeSigma).

Techniques: Fluorescence, Western Blot, Comparison

Journal: bioRxiv

Article Title: Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntington’s disease pathogenesis in mice

doi: 10.1101/2025.06.24.661398

Figure Lengend Snippet: ( a ) Volcano plot of the differential gene expression analysis performed using DESeq2 between 12-month-old vehicle (aCSF-NTC) Q111 versus WT mice. Red indicates significant gene expression change. Significance determined by fold change > 1.5, FDR < 0.05. ( b ) Gene ontology biological processes (GOBP) pathway enrichment of the differentially expressed genes between vehicle Q111 and WT mice. ( c-e ) Overall gene reversal percentage of Q111 mice treated with ( c ) HTT siRNA ( d ) MSH3 siRNA or ( e ) MSH3+HTT siRNA combination compared to vehicle Q111 mice. Color indicates the degree of reversal classified as full, partial, super, exacerbation, or negligible. ( f-i ) Prevention, reversal, recuse (PuRR) classification of the degree of gene expression reversal. Number of genes differentially expressed or reversed by siRNA treatment in the ( f ) whole HD signature or ( h ) select STR266 HD signature genes across treatment cohorts as defined by Marchionini et al. 2022 . ( g-i ) classification of the degree of reversal for the ( g ) whole HD signature or ( i ) STR266 top gene list. Degree of gene expression reversal color coded as: Blue-full reversal (F-Rev); green-partial reversal (P-Rev); orange-super reversal (S-Rev); red-exacerbation (Exac); gray-negligible reversal (N-Rev). Transcriptomics performed on the same mouse cohorts presente d in Figures 1-3 . N=9-12 mice per treatment group.

Article Snippet: Western blots were performed as previously described (Sapp et al., 2020) using antibodies to HTT (aa1-17, 1:2000, DiFiglia et al., 1995), Msh3 (1:500, Santa Cruz sc-271079), HTT1a (5ug/ml, P90 1B12, Coriell Institute), Gapdh (1:10000, MilliporeSigma), and B-actin (1:5000, MilliporeSigma).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntington’s disease pathogenesis in mice

doi: 10.1101/2025.06.24.661398

Figure Lengend Snippet: Model of HTT species across time with ( a ) artificial CSF or non-targeting control siRNA, ( b ) HTT siRNA, ( c ) MSH3 siRNA, ( d ) MSH3+HTT siRNA combination. Total full-length mutant HTT (red), soluble HTT1a (green), diffuse mHTT aggregates (blue), or mHTT inclusions (purple).

Article Snippet: Western blots were performed as previously described (Sapp et al., 2020) using antibodies to HTT (aa1-17, 1:2000, DiFiglia et al., 1995), Msh3 (1:500, Santa Cruz sc-271079), HTT1a (5ug/ml, P90 1B12, Coriell Institute), Gapdh (1:10000, MilliporeSigma), and B-actin (1:5000, MilliporeSigma).

Techniques: Control, Mutagenesis