Biology subjects

McHugh, N.

Publications and source records attributed to McHugh, N..

11 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

Organyl 5'-Phosphates in siRNA Guide Strands: Structure-Function Relationships Governing Anchoring in Argonaute 2 and Metabolic Stability

Efficient siRNA loading into Argonaute2 (AGO2) requires a 5'-phosphate (5'-P) on the guide strand, yet this group is vulnerable to metabolic degradation in vivo. Although numerous chemical mimics of 5'-P have been reported, structural principles governing AGO2 interactions with organyl substituents on the 5'-P remain unclear. Moreover, structural determinants of 5'-P mimic recognition by known degradative enzymes (principally phosphatases and 5'-exonucleases) are also poorly understood. The 5'-P binding site of the AGO2 MID domain contains a stack of aromatic residues (Y527/F811/Y815), presenting a structural basis for augmenting canonical anchoring interactions. Herein, we systematically synthesized and characterized a diverse panel of organyl 5'-phosphates (5'-POR; R = 35 variable substituents) as guide strand 5'-P mimics designed to engage this unique hydrophobic pocket. Among the compounds evaluated, 5'-POR guide strands bearing methyl (Me) or phenylpropargyl (PhPrp) substituents are well-tolerated by AGO2 in cells. Previously uncharacterized 5'-P mimics, including 5'-phosphorothioate (5'-PS), phenylpropargyl 5'-phosphorothioate (5'-PS-PhPrp), and 5'-mesylphosphoramidate (5'-MsPA), maintain comparable AGO2 compatibility. All examined 5'-P mimics are markedly resistant to phosphatase, while 5'-POR variants and 5'-PS-PhPrp are also resistant to 5'-exonuclease degradation due to masking a negative charge of 5'-P. A crystal structure of a 5'-PO-PhPrp guide strand loaded into AGO2 reveals an unexpected network of {pi}-{pi} interactions between the rigid phenylpropargyl group and the targeted hydrophobic pocket of the MID domain. Collectively, these findings expand the functional chemical space of 5'-P mimics and define new modes for metabolically stabilizing the guide strand 5'-end while augmenting AGO2 MID anchoring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/705631v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e6f88forg.highwire.dtl.DTLVardef@1c87dfaorg.highwire.dtl.DTLVardef@1c6e68corg.highwire.dtl.DTLVardef@14a1f60_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

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

Single-dose administration of therapeutic divalent siRNA targeting MECP2 prevents lethality for one year in an MECP2 duplication mouse model

AbstractMECP2 duplication syndrome (MDS) is a rare X-linked neurodevelopmental disorder caused by duplications of the dosage-sensitive methyl-CpG-binding protein 2 (MECP2) gene. Developing effective therapies for MDS is particularly challenging due to the variability in MECP2 expression among patients and the potential risk of inducing Rett syndrome through excessive pharmacological intervention. Reducing dosage to optimize silencing levels often compromises durability and necessitates increased dosing frequency. We present here a series of fully chemically modified small interfering RNAs (siRNAs) designed for both isoform-selective and total MECP2 silencing. Among these, we identify six lead siRNA candidates across two distinct chemical scaffolds, achieving targeted total MECP2 expression reductions ranging from 25% to 75%, sustained for at least four months following a single administration. The efficacy and safety of human ortholog silencing were evaluated using two mouse models with distinct levels of human MECP2 transgene expression. In the severe duplication model, a single dose of the total isoform-silencing siRNA fully rescued early mortality and behavioral impairments. Additionally, we show that the isoform-selective targeting strategy may be safer in mild cases of MDS where exaggerated pharmacology may lead to Rett Syndrome. Overall, this study introduces a series of preclinical candidates with the capacity to address the varying levels of MECP2 duplication encountered in clinical settings. Furthermore, it establishes a target selection strategy that may be applied to other dosage-sensitive gene imbalances. One Sentence SummaryTherapeutic siRNAs provide safe and durable modulation of MECP2 for the treatment of mild and severe MECP2 Duplication Syndrome.

neuroscience↗

bioRxiv2024

RNAi-mediated silencing of SOD1 profoundly extends survival and functional outcomes in ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition, with 20% of familial and 2-3% of sporadic cases linked to mutations in the cytosolic superoxide dismutase (SOD1) gene. Mutant SOD1 protein is toxic to motor neurons, making SOD1 gene lowering a promising approach, supported by preclinical data and the 2023 FDA approval of the GapmeR ASO targeting SOD1, tofersen. Despite the approval of an ASO and the optimism it brings to the field, the pharmacodynamics and pharmacokinetics of therapeutic SOD1 modulation can be improved. Here, we developed a chemically stabilized divalent siRNA scaffold (di-siRNA) that effectively suppresses SOD1 expression in vitro and in vivo. With optimized chemical modification, it achieves remarkable CNS tissue permeation and SOD1 silencing in vivo. Administered intraventricularly, di-siRNASOD1 extended survival in SOD1-G93A ALS mice, surpassing survival previously seen in these mice by ASO modalities, slowed disease progression, and prevented ALS neuropathology. These properties offer an improved therapeutic strategy for SOD1-mediated ALS and may extend to other dominantly inherited neurological disorders. One sentence summarySilencing SOD1 with chemically optimized divalent siRNA profoundly extends the lifespan of G93A mice and prevents neurodegenerative biochemical and behavioral phenotypes.

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

Single intravitreal administration of a tetravalent siRNA exhibits robust and efficient gene silencing in rodent and swine photoreceptors

Inherited retinal dystrophies caused by dominant mutations in photoreceptor-expressed genes, are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets photoreceptor-expressed genes. Here we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best photoreceptor accumulation. A single intravitreal administration induces multi-months efficacy in rodent and porcine retinas while showing a good safety profile. The data suggest that this configuration can treat retinal diseases caused by photoreceptor-expressed genes with 1-2 intravitreal injections per year.

neuroscience↗

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↗

bioRxiv2023

Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline deficient, high fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

molecular biology↗

bioRxiv2022

Silencing of ApoE with Divalent siRNAs Drives Activation of Immune Clearance Pathways and Improves Amyloid Pathology in Mouse Models of Alzheimer's Disease

The most common genetic risk factor for late-onset Alzheimers disease (AD) is the APOE4 allele, with evidence for gain- and loss-of-function mechanisms. ApoE knockout in mice abrogates AD phenotypes but causes severe atherosclerosis due to the role of liver ApoE in cholesterol homeostasis. Previous attempts to inhibit brain-specific ApoE with anti-sense oligonucleotides only modestly reduced ApoE expression and had no effect on amyloid burden in adult AD mice. Here, we optimized a divalent small interfering RNA (di-siRNA) to selectively and potently silence ApoE in the brain. Silencing brain ApoE in AD mice significantly reduced amyloid plaque formation without affecting systemic cholesterol levels, confirming that brain and liver APOE pools are spatially and functionally distinct. Mechanistically, APOE appears to be a scaffold for beta-amyloid aggregation that limits clearance by microglia. Di-siRNAs from this study can be taken to pre-clinical and clinical trials to accelerate development of AD-modifying therapies and establish siRNA-based modulation of ApoE as a viable path towards therapeutic development.

neuroscience↗