Biology subjects

Aronin, N.

Publications and source records attributed to Aronin, N..

13 recordsLinked to original sources

bioRxiv2026

Mismatch repair dissection by in vivo RNAi reveals dose-dependent modulators of somatic instability and proteome remodeling in Huntingtons disease

Human and mouse genetics have established mismatch repair (MMR) as a central mediator of somatic repeat expansion, a key pathogenic process in Huntingtons disease (HD) and related disorders. How individual MMR components function within the intact mammalian brain and interact with broader cellular networks remains poorly understood. We screened more than 500 chemically stabilized siRNAs targeting 10 MMR genes and used interventional RNAi in the Q111 HD mouse model to systematically dissect MMR function in vivo. MSH3 and PMS1 emerged as the most dose-sensitive regulators of somatic expansion but displayed markedly different effects on proteome stability. Quantitative proteomics generated an in vivo atlas of MMR component abundance and cross-regulation in the mammalian CNS, uncovering extensive connectivity between DNA repair, transcriptional regulation, chromatin remodeling, and mitochondrial biology. Together, these findings establish a systems-level framework linking MMR biology to neuronal function and offer mechanistic insight into selective neuronal vulnerability in HD.

neuroscience↗

bioRxiv2026

C57BL/6 BAC-CAG Huntingtons disease mice show somatic CAG expansion and responses to small interfering RNAs comparable to the FVB strain

Huntingtons disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion in the huntingtin (HTT) gene, with longer repeats linked to earlier onset. Somatic CAG expansion, particularly in the striatum, contributes to disease progression and is influenced by HTT biology and genetic modifiers. Modulating somatic expansion is emerging as a promising approach to slow or prevent HD, and mouse models have been crucial for preclinical testing of different therapeutic strategies. The BAC-CAG model, developed on the FVB strain, has been used to study somatic expansion of human expanded HTT. However, comparisons with other key HD mouse models have been limited by differences in genetic background, as many other models are on the C57BL/6 strain. The BAC-CAG model has now been developed on a C57BL/6 background. To determine whether the C57BL/6 BAC-CAG model can be used to study and modulate somatic expansion, we compared CAG expansion in mice on C57BL/6 or FVB backgrounds, with and without intraventricular divalent small interfering RNAs (siRNA) targeting HD modifiers MutS homolog 3 (MSH3) and HTT. Both strains exhibited robust, comparable somatic expansion over two months, which was blocked by MSH3-, but not HTT-, targeted siRNA. RNA sequencing identified gene expression differences primarily in pseudogenes, with no differences in endogenous Htt, human HTT, or mismatch repair genes. These results demonstrate that BAC-CAG mice on a C57BL/6 background exhibit somatic CAG expansion comparable to the validated FVB strain, providing a model to study and preclinically test therapies targeting somatic expansion in HD.

neuroscience↗

bioRxiv2025

Early reduction and impaired targeting of myelin-associated glycoprotein to myelin membranes in Huntington disease

BackgroundHuntingtons disease (HD) is a hereditary life-threatening disease marked by progressive neuronal loss and atrophy of grey matter structures, particularly the caudate putamen. Brain imaging studies have revealed that the degradation of the white matter occurs many years prior to symptomatic onset and neuronal loss, suggesting that the decay of brain white matter is an active contributor to the disease progression. However, the mechanisms by which the HD mutation triggers white matter loss is not well understood. MethodsWestern blot, immunohistochemistry, and electron microscopy were conducted to assess white matter pathology and explore the relevant mechanisms in CAG140 knock-in mice, which express the HD protein in the same way as patients suffering from HD and thus biologically replicate HD in human. ResultsWestern blot analysis of proteins localized at different layers of the myelin coat revealed that the myelin-associated glycoprotein (MAG), which is localized at the innermost layer of the myelin coat and essential for maintaining the periaxonal space and the integrity of the myelin sheath, manifested as an early and progressive decline in HD mouse caudate putamen. The loss of MAG was detected at myelinated axons and in fiber bundles in HD mouse brains at an age when the abundance of myelinated axons was normal. Fluorescence immunohistochemical studies found that MAG labeling was concentrated in the soma of a subset of oligodendrocytes, which expressed breast carcinoma amplified sequence 1, a marker for new oligodendrocytes. While their abundance was normal, new oligodendrocytes in HD mouse caudate appeared to be impeded in acquiring the expression of MAG and in targeting MAG away from perinuclear punctate structures to processes, signs of impaired maturation. Compared with those in wildtype mouse brains, oligodendrocytes in HD mouse brains had a reduced abundance of small vesicles whereas an increased abundance of large punctate structures in the perinuclear region, implying defective generation of small vesicles transporting MAG from large punctate structures in the soma to processes. The MAG-containing perinuclear punctate structures were negative for proteins specifying trans-Golgi networks, early endosomes, or exosomes but had a minor portion labeled with lysosome-associated membrane protein 1, indicating that the structures where MAG accumulates in the soma are derived from the late endosomal lysosomal compartment. ConclusionsOur study suggests that the decay of the brain white matter in Huntingtons disease involves a deficit in trafficking of myelin-associated glycoprotein, preventing its proper delivery from the soma of oligodendrocytes to myelin-forming processes.

neuroscience↗

bioRxiv2025

Blocking somatic repeat expansion and lowering huntingtin via RNA interference synergize to prevent Huntingtons disease pathogenesis in mice

Huntingtons disease (HD) is a progressive neurodegenerative disorder with no approved therapies. Two major molecular drivers--somatic expansion of inherited CAG repeats and toxic mutant HTT (mHTT) variants--lead to neuronal dysfunction. Despite multiple trials, HTT-lowering strategies have not shown meaningful clinical benefit. Using therapeutic divalent siRNAs, we assessed the long-term impact of silencing MSH3 (a key regulator of somatic expansion), HTT, or both. In Q111 HD mice (>110 CAGs), which exhibit robust expansion, mHTT inclusions, and transcriptional dysregulation by 12 months, long-term MSH3 silencing blocked expansion, reduced inclusions, and reversed gene expression changes. HTT silencing alone had limited effect, but combined MSH3/HTT targeting synergistically eliminated inclusions and restored transcriptomic profiles. Parallel treatment in wild-type mice showed no toxicity, supporting the safety of long-term intervention. These findings position somatic expansion as a promising therapeutic target and demonstrate the potential of RNAi-based co-silencing of MSH3 and HTT as a disease-modifying strategy for HD.

neuroscience↗

bioRxiv2025

Lowering the HTT1a transcript as an effective therapy for Huntingtons disease

Lowering the levels of HTT transcripts has been a major focus of therapeutic development for Huntingtons disease (HD), but which transcript should be lowered? HD is caused by a CAG repeat expansion in exon 1 of the HTT gene, and the rate of somatic expansion of this CAG repeat throughout life is now known to drive the age of onset and rate of disease progression. As the CAG repeat expands, the extent to which the HTT mRNA is alternatively processed to generate the HTT1a transcript and highly aggregation-prone and pathogenic HTT1a protein increases. Several HTT-lowering modalities have entered clinical trials that either target both HTT and HTT1a together, or full-length HTT alone. We have developed siRNAs that target the Htt1a mouse transcript (634/486) and used these, together with a potent Htt-targeting siRNA (10150) to compare the efficacy of lowering either full-length Htt or Htt1a. zQ175 and wild-type mice were treated with 10150 or 634/486 alongside control groups at 2 months of age with treatment to 6 or 10 months, or at 6 months with treatment to 10 months. The siRNA potency and durability were most effective in the hippocampus. Whilst both strategies showed benefits, despite the greater potency of 10150, targeting Htt1a was more effective at delaying HTT aggregation and transcriptional dysregulation than targeting full-length Htt. These data support HTT-lowering strategies that are designed to target the HTT1a transcript, either alone, or together with lowering full-length HTT. One Sentence SummaryLowering HTT1a transcript levels delays the onset of molecular and neuropathological phenotypes in a knock-in mouse model of Huntingtons disease.

neuroscience↗

bioRxiv2025

Intrastriatal delivery of a zinc finger protein targeting the mutant HTT gene obviates lipid phenotypes in zQ175DN HD mice

Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntingtons disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT lowering and whether these changes can be measured in peripheral biofluids. We previously found that lipid changes that occur in brain with HD progression could be prevented by attenuating HTT transcription of the mutant allele in a genetic mouse model (LacQ140) with inducible whole body lowering. Here, we tested whether intrastriatal injection of a therapeutic capable of repressing the mutant HTT allele with expanded CAG can provide similar protection against lipid changes in HD mice with a deletion of neo cassette (zQ175DN). Wild-type or zQ175DN mice were injected with AAV9 bearing a cDNA for a zinc finger protein (ZFP) which preferentially targets mutant HTT (ZFP-HTT) to repress transcription (1). Proteins from brain tissues were analyzed using western blot, capillary electrophoresis, and nitrocellulose filtration methods. Lipid analyses of brain tissue and plasma collected from the same mice were conducted by liquid chromatography and mass spectrometry (LC-MS). Somatic instability (SI) index was assessed using capillary gel electrophoresis of PCR products and was shown to be impeded by HTT-ZFP. Lowering mHTT levels by 43% for 4 months prevented loss of total lipid content including subclasses sphingomyelin (SM), ceramide, phosphatidylethanolamine (PE) and others of caudate-putamen in zQ175DN mice. Moreover, LC-MS analysis of plasma demonstrated total lipid increases and lipid changes in monogalactosyl monoacylglyceride (MGMG) and certain phosphatidylcholine (PC) species were reversed with the therapy. In summary, our data demonstrate that analyzing lipid signatures of brain tissue and peripheral biofluids are valuable approaches for evaluating potential therapies in a preclinical model of HD. FundingCHDI Foundation, Dake Family Fund Disclosure statementThe authors have nothing to disclose. Author contributionsAI and KS extracted lipids and performed computational and statistical analysis; AB and CS collected plasma and brain tissues; SL and ES performed protein chemistry; KC maintained mouse colonies, RM performed stereotaxic injections, RB subcloned ZFP cDNAs and prepared virus, MSE, NA, MD, and KKG planned experiments and wrote manuscript.

neuroscience↗

bioRxiv2025

Detection of HTTex1p by western blot and immunostaining of HD human and mouse brain using neo-epitope antibody P90 highlights impact of CAG repeat expansion on its size, solubility, and response to MSH3 silencing

HTT1a was identified in human and mouse Huntingtons disease brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 of HTT that contributes to aggregate formation and neuronal dysfunction.1 Detection of the huntingtin exon 1 protein (HTT1a) has been accomplished with fluorescence-based reporter assays (Meso Scale Discovery, Homogeneous Time Resolved Fluorescence) and immunoprecipitation assays in Huntingtons disease knock-in mice but direct detection in homogenates by gel electrophoresis and western blot assay has been lacking. Subcellular fractions prepared from mouse and human Huntingtons disease brain were separated by gel electrophoresis and probed by western blot with neo-epitope monoclonal antibodies 1B12 and 11G2 directed to the C-terminal eight residues of HTT1a. In caudate putamen of an allelic series of 6 month old Huntingtons disease knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTT1a migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass smear in Q111, Q140 and Q175 mice but weakly in Q80 and not in WT mice or Q50 indicating a CAG repeat size threshold for detecting HTT1a. HTT1a immunoreactivity diminished if 1B12 and 11G2 antibodies were preincubated with an eight amino acid peptide containing the C-terminus of HTT1a but not with unrelated peptide sequence. Migration of HTT1a and its high molecular mass smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating Q111 mice with siRNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the high molecular mass smear indicating that the effects of curbing CAG repeat expansion were quantifiable. A prominent 56-60 kDa doublet detected by 1B12 and 11G2 antibodies in lysates from human Huntingtons disease brain was not blocked by preincubation with C-terminal HTT1a blocking peptide and also appeared in brains of Parkinsons disease patients. 1B12 and 11G2 antibodies did not immunoprecipitate HTT proteins from either Huntingtons disease mouse or human brain lysates using conditions that pulled down full length HTT with anti-HTT antibody 2B7. Altogether these data show that 11G2 and 1B12 antibodies can be used in western blot assays to track and quantify immunoreactive HTT1a levels, solubility, and subcellular localization in Huntingtons disease mouse brain. Abbreviated SummarySapp et al., report that pathogenic exon 1 protein HTT1a is detected in brain of mouse models of Huntingtons disease by direct western blot assay using monoclonal antibodies 11G2 and 1B12. Lowering MSH3 mRNA in the caudate putamen to prevent CAG repeat expansion reduced levels of HTT1a.

neuroscience↗

bioRxiv2024

Preventing acute neurotoxicity of CNS therapeutic oligonucleotides with the addition of Ca2+ and Mg2+ in the formulation

Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography (EEG) and electromyography (EMG) in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca2+ and Mg2+ up to molar equivalents of the phosphodiester (PO)/phosphorothioate (PS) bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establishes the importance of adding Ca2+ and Mg2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides.

neuroscience↗

bioRxiv2024

mRNA nuclear clustering leads to a difference in mutant huntingtin mRNA and protein silencing by siRNAs in vivo

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the first exon of the huntingtin gene (HTT). Oligonucleotide therapeutics, such as short interfering RNA (siRNA), reduce levels of huntingtin mRNA and protein in vivo and are considered a viable therapeutic strategy. However, the extent to which they silence HTT mRNA in the nucleus is not established. We synthesized siRNA cross-reactive to mouse (wild-type) Htt and human (mutant) HTT in a di-valent scaffold and delivered to two mouse models of HD. In both models, di-valent siRNA sustained lowering of wild-type Htt, but not mutant HTT mRNA expression in striatum and cortex. Near-complete silencing of both mutant HTT protein and wild-type Htt protein was observed in both models. Subsequent fluorescent in situ hybridization (FISH) analysis shows that di-valent siRNA acts predominantly on cytoplasmic mutant HTT transcripts, leaving clustered mutant HTT transcripts in the nucleus largely intact in treated HD mouse brains. The observed differences between mRNA and protein levels, exaggerated in the case of extended repeats, might apply to other repeat-associated neurological disorders.

neuroscience↗

bioRxiv2023

A programmable dual-targeting di-valent siRNA scaffold supports potent multi-gene modulation in the central nervous system

Di-valent short interfering RNA (siRNA) is a promising therapeutic modality that enables sequence-specific modulation of a single target gene in the central nervous system (CNS). To treat complex neurodegenerative disorders, where pathogenesis is driven by multiple genes or pathways, di-valent siRNA must be able to silence multiple target genes simultaneously. Here we present a framework for designing unimolecular "dual-targeting" di-valent siRNAs capable of co-silencing two genes in the CNS. We reconfigured di-valent siRNA - in which two identical, linked siRNAs are made concurrently - to create linear di-valent siRNA - where two siRNAs are made sequentially attached by a covalent linker. This linear configuration, synthesized using commercially available reagents, enables incorporation of two different siRNAs to silence two different targets. We demonstrate that this dual-targeting di-valent siRNA is fully functional in the CNS of mice, supporting at least two months of maximal target silencing. Dual-targeting di-valent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g., Huntingtons disease: MSH3 and HTT; Alzheimers disease: APOE and JAK1) with similar potency to a mixture of single-targeting di-valent siRNAs against each gene. This work potentiates CNS modulation of virtually any pair of disease-related targets using a simple unimolecular siRNA.

molecular biology↗

bioRxiv2023

Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3- and 5-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3-exonuclease by [~]32-fold over PS backbone and >1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure ([~]6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

biochemistry↗

bioRxiv2022

Di-valent siRNA Mediated Silencing of MSH3 Blocks Somatic Repeat Expansion in Mouse Models of Huntington's Disease

Huntingtons Disease (HD) is a severe neurodegenerative disorder caused by expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS Homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify fully chemically modified short interfering RNA (siRNA) that robustly silence MSH3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of MSH3 effectively blocked CAG repeat expansion in striatum of two HD mouse models without impacting tumor-associated microsatellite instability. Our findings establish a novel paradigm for treating patients with HD and other repeat expansion diseases. One Sentence SummarySilencing MSH3 in the CNS of two models of Huntingtons disease using di-valent siRNA blocks disease-accelerating somatic expansion of CAG repeats.

molecular biology↗

bioRxiv2022

Chemical engineering of therapeutic siRNAs for allele-specific gene silencing in vivo in CNS

Small interfering RNAs (siRNAs) are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expression in vivo. We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS. Many genetically-defined neurodegenerative disorders are autosomal dominant favoring selective silencing of the mutant allele. In some cases, successful targeting of the mutant allele requires targeting of a single nucleotide polymorphism (SNP) heterozygosity. Using Huntingtons disease as a model, we demonstrate allele-specific RNAi-based silencing of gene expression in vivo and in neurons differentiated from HD patient-derived iPSCs. A series of in vitro screens, with chemical and thermodynamic optimization, identified compounds with >50-fold selectivity for the mutant HD-causing allele, based on a single nucleotide difference. The optimized compound exhibits selective silencing of mutant huntingtin (HTT) protein in patient derived cells and throughout the HD mouse brain, providing a demonstration of SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. The ability to target a disease-causing allele using RNAi-based therapies could be applied to a wide range of dominant CNS disorders, where maintenance of wild-type expression is essential.

neuroscience↗