ASO silencing of a glycosyltransferase, Poglut1 , improves the liver phenotypes in mouse models of Alagille syndrome.

BACKGROUND AND AIMS: Paucity of intrahepatic bile ducts (BDs) is caused by various etiologies and often leads to cholestatic liver disease. For example, in patients with Alagille syndrome (ALGS), which is a genetic disease primarily caused by mutations in jagged 1 ( JAG1) , BD paucity often results in severe cholestasis and liver damage. However, no mechanism-based therapy exists to restore the biliary system in ALGS or other diseases associated with BD paucity. Based on previous genetic observations, we investigated whether postnatal knockdown of the glycosyltransferase gene protein O -glucosyltransferase 1 ( Poglut1) can improve the ALGS liver phenotypes in several mouse models generated by removing one copy of Jag1 in the germline with or without reducing the gene dosage of sex-determining region Y-box 9 in the liver. APPROACH AND RESULTS: Using an ASO established in this study, we show that reducing Poglut1 levels in postnatal livers of ALGS mouse models with moderate to profound biliary abnormalities can significantly improve BD development and biliary tree formation. Importantly, ASO injections prevent liver damage in these models without adverse effects. Furthermore, ASO-mediated Poglut1 knockdown improves biliary tree formation in a different mouse model with no Jag1 mutations. Cell-based signaling assays indicate that reducing POGLUT1 levels or mutating POGLUT1 modification sites on JAG1 increases JAG1 protein level and JAG1-mediated signaling, suggesting a likely mechanism for the observed in vivo rescue. CONCLUSIONS: Our preclinical studies establish ASO-mediated POGLUT1 knockdown as a potential therapeutic strategy for ALGS liver disease and possibly other diseases associated with BD paucity.

Therapeutic reduction of ataxin-2 extends lifespan and reduces pathology in TDP-43 mice.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that is characterized by motor neuron loss and that leads to paralysis and death 2-5 years after disease onset. Nearly all patients with ALS have aggregates of the RNA-binding protein TDP-43 in their brains and spinal cords, and rare mutations in the gene encoding TDP-43 can cause ALS. There are no effective TDP-43-directed therapies for ALS or related TDP-43 proteinopathies, such as frontotemporal dementia. Antisense oligonucleotides (ASOs) and RNA-interference approaches are emerging as attractive therapeutic strategies in neurological diseases. Indeed, treatment of a rat model of inherited ALS (caused by a mutation in Sod1) with ASOs against Sod1 has been shown to substantially slow disease progression. However, as SOD1 mutations account for only around 2-5% of ALS cases, additional therapeutic strategies are needed. Silencing TDP-43 itself is probably not appropriate, given its critical cellular functions. Here we present a promising alternative therapeutic strategy for ALS that involves targeting ataxin-2. A decrease in ataxin-2 suppresses TDP-43 toxicity in yeast and flies, and intermediate-length polyglutamine expansions in the ataxin-2 gene increase risk of ALS. We used two independent approaches to test whether decreasing ataxin-2 levels could mitigate disease in a mouse model of TDP-43 proteinopathy. First, we crossed ataxin-2 knockout mice with TDP-43 (also known as TARDBP) transgenic mice. The decrease in ataxin-2 reduced aggregation of TDP-43, markedly increased survival and improved motor function. Second, in a more therapeutically applicable approach, we administered ASOs targeting ataxin-2 to the central nervous system of TDP-43 transgenic mice. This single treatment markedly extended survival. Because TDP-43 aggregation is a component of nearly all cases of ALS, targeting ataxin-2 could represent a broadly effective therapeutic strategy.

The atlas of RNase H antisense oligonucleotide distribution and activity in the CNS of rodents and non-human primates following central administration.

Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.

Reduction of matrix metalloproteinase 9 (MMP-9) protects motor neurons from TDP-43-triggered death in rNLS8 mice.

Therapeutic strategies are needed for the treatment of amyotrophic lateral sclerosis (ALS). One potential target is matrix metalloproteinase-9 (MMP-9), which is expressed only by fast motor neurons (MNs) that are selectively vulnerable to various ALS-relevant triggers. Previous studies have shown that reduction of MMP-9 function delayed motor dysfunction in a mouse model of familial ALS. However, given that the majority of ALS cases are sporadic, we propose preclinical testing in a mouse model which may be more clinically translatable: rNLS8 mice. In rNLS8 mice, neurodegeneration is triggered by the major pathological hallmark of ALS, TDP-43 mislocalization and aggregation. MMP-9 was targeted in 3 different ways in rNLS8 mice: by AAV9-mediated knockdown, using antisense oligonucleotide (ASO) technology, and by genetic modification. All 3 strategies preserved the motor unit during disease, as measured by MN counts, tibialis anterior (TA) muscle innervation, and physiological recordings from muscle. However, the strategies that reduced MMP-9 beyond the motor unit lead to premature deaths in a subset of rNLS8 mice. Therefore, selective targeting of MMP-9 in MNs could be beneficial in ALS, but side effects outside of the motor circuit may limit the most commonly used clinical targeting strategies.

Frontotemporal dementia causative CHMP2B impairs neuronal endolysosomal traffic-rescue by TMEM106B knockdown.

Mutations in the endosome-associated protein CHMP2B cause frontotemporal dementia and lead to lysosomal storage pathology in neurons. We here report that physiological levels of mutant CHMP2B causes reduced numbers and significantly impaired trafficking of endolysosomes within neuronal dendrites, accompanied by increased dendritic branching. Mechanistically, this is due to the stable incorporation of mutant CHMP2B onto neuronal endolysosomes, which we show renders them unable to traffic within dendrites. This defect is due to the inability of mutant CHMP2B to recruit the ATPase VPS4, which is required for release of CHMP2B from endosomal membranes. Strikingly, both impaired trafficking and the increased dendritic branching were rescued by treatment with antisense oligonucleotides targeting the well validated frontotemporal dementia risk factor TMEM106B, which encodes an endolysosomal protein. This indicates that reducing TMEM106B levels can restore endosomal health in frontotemporal dementia. As TMEM106B is a risk factor for frontotemporal dementia caused by both C9orf72 and progranulin mutations, and antisense oligonucleotides are showing promise as therapeutics for neurodegenerative diseases, our data suggests a potential new strategy for treating the wide range of frontotemporal dementias associated with endolysosomal dysfunction.

Antisense oligonucleotide therapy rescues disturbed brain rhythms and sleep in juvenile and adult mouse models of Angelman syndrome.

UBE3A encodes ubiquitin protein ligase E3A, and in neurons its expression from the paternal allele is repressed by the UBE3A antisense transcript (UBE3A-ATS). This leaves neurons susceptible to loss-of-function of maternal UBE3A. Indeed, Angelman syndrome, a severe neurodevelopmental disorder, is caused by maternal UBE3A deficiency. A promising therapeutic approach to treating Angelman syndrome is to reactivate the intact paternal UBE3A by suppressing UBE3A-ATS. Prior studies show that many neurological phenotypes of maternal Ube3a knockout mice can only be rescued by reinstating Ube3a expression in early development, indicating a restricted therapeutic window for Angelman syndrome. Here, we report that reducing Ube3a-ATS by antisense oligonucleotides in juvenile or adult maternal Ube3a knockout mice rescues the abnormal electroencephalogram (EEG) rhythms and sleep disturbance, two prominent clinical features of Angelman syndrome. Importantly, the degree of phenotypic improvement correlates with the increase of Ube3a protein levels. These results indicate that the therapeutic window of genetic therapies for Angelman syndrome is broader than previously thought, and EEG power spectrum and sleep architecture should be used to evaluate the clinical efficacy of therapies.

Reduction of nemo-like kinase increases lysosome biogenesis and ameliorates TDP-43-related neurodegeneration.

Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although mutations in TARDBP, encoding transactive response DNA-binding protein 43 kDa (TDP-43), account for less than 1% of all ALS cases, TDP-43-positive aggregates are present in nearly all ALS patients, including patients with sporadic ALS (sALS) or carrying other familial ALS-causing (fALS-causing) mutations. Interestingly, TDP-43 inclusions are also present in subsets of patients with frontotemporal dementia, Alzheimer's disease, and Parkinson's disease; therefore, methods of activating intracellular protein quality control machinery capable of clearing toxic cytoplasmic TDP-43 species may alleviate disease-related phenotypes. Here, we identify a function of nemo-like kinase (Nlk) as a negative regulator of lysosome biogenesis. Genetic or pharmacological reduction of Nlk increased lysosome formation and improved clearance of aggregated TDP-43. Furthermore, Nlk reduction ameliorated pathological, behavioral, and life span deficits in 2 distinct mouse models of TDP-43 proteinopathy. Because many toxic proteins can be cleared through the autophagy/lysosome pathway, targeted reduction of Nlk represents a potential approach to therapy development for multiple neurodegenerative disorders.

Antisense oligonucleotide therapy for KCNT1 encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are characterized by pharmaco-resistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, which encodes for sodium activated potassium channel protein KNa1.1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene-silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared with mice treated with a control ASO (nonhybridizing sequence). ASO administration at neonatal age was also well tolerated and effective in controlling seizures and extending the life span of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.

Antisense oligonucleotide therapy reduces seizures and extends life span in an SCN2A gain-of-function epilepsy model.

De novo variation in SCN2A can give rise to severe childhood disorders. Biophysical gain of function in SCN2A is seen in some patients with early seizure onset developmental and epileptic encephalopathy (DEE). In these cases, targeted reduction in SCN2A expression could substantially improve clinical outcomes. We tested this theory by central administration of a gapmer antisense oligonucleotide (ASO) targeting Scn2a mRNA in a mouse model of Scn2a early seizure onset DEE (Q/+ mice). Untreated Q/+ mice presented with spontaneous seizures at P1 and did not survive beyond P30. Administration of the ASO to Q/+ mice reduced spontaneous seizures and significantly extended life span. Across a range of behavioral tests, Scn2a ASO-treated Q/+ mice were largely indistinguishable from WT mice, suggesting treatment is well tolerated. A human SCN2A gapmer ASO could likewise impact the lives of patients with SCN2A gain-of-function DEE.

The Distinct and Cooperative Roles of Toll-Like Receptor 9 and Receptor for Advanced Glycation End Products in Modulating In Vivo Inflammatory Responses to Select CpG and Non-CpG Oligonucleotides.

Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner. In addition, we found the receptor for advance glycation end product (RAGE) receptor to be involved in the initiation of inflammatory response to suboptimal concentrations of both CpG- and non-CpG-containing ODNs. In contrast, dosing RAGE KO mice with high doses of CpG or non-CpG ODNs lead to a stronger inflammatory response than observed in wild-type mice. Together, our data provide a previously uncharacterized in vivo mechanism contingent on ODN-administered dose, where TLR9 governs the primary response and RAGE plays a distinct and cooperative function in providing a pivotal role in balancing the immune response.

Comprehensive Structure-Activity Relationship of Triantennary N-Acetylgalactosamine Conjugated Antisense Oligonucleotides for Targeted Delivery to Hepatocytes.

The comprehensive structure-activity relationships of triantennary GalNAc conjugated ASOs for enhancing potency via ASGR mediated delivery to hepatocytes is reported. Seventeen GalNAc clusters were assembled from six distinct scaffolds and attached to ASOs. The resulting ASO conjugates were evaluated in ASGR binding assays, in primary hepatocytes, and in mice. Five structurally distinct GalNAc clusters were chosen for more extensive evaluation using ASOs targeting SRB-1, A1AT, FXI, TTR, and ApoC III mRNAs. GalNAc-ASO conjugates exhibited excellent potencies (ED50 0.5-2 mg/kg) for reducing the targeted mRNAs and proteins. This work culminated in the identification of a simplified tris-based GalNAc cluster (THA-GN3), which can be efficiently assembled using readily available starting materials and conjugated to ASOs using a solution phase conjugation strategy. GalNAc-ASO conjugates thus represent a viable approach for enhancing potency of ASO drugs in the clinic without adding significant complexity or cost to existing protocols for manufacturing oligonucleotide drugs.