Nacc1 Mutation in Mice Models Rare Neurodevelopmental Disorder with Underlying Synaptic Dysfunction.

A missense mutation in the transcription repressor Nucleus accumbens-associated 1 (NACC1) gene at c.892C>T (p.Arg298Trp) on chromosome 19 causes severe neurodevelopmental delay ( Schoch et al., 2017). To model this disorder, we engineered the first mouse model with the homologous mutation (Nacc1+/R284W ) and examined mice from E17.5 to 8 months. Both genders had delayed weight gain, epileptiform discharges and altered power spectral distribution in cortical electroencephalogram, behavioral seizures, and marked hindlimb clasping; females displayed thigmotaxis in an open field. In the cortex, NACC1 long isoform, which harbors the mutation, increased from 3 to 6 months, whereas the short isoform, which is not present in humans and lacks aaR284 in mice, rose steadily from postnatal day (P) 7. Nuclear NACC1 immunoreactivity increased in cortical pyramidal neurons and parvalbumin containing interneurons but not in nuclei of astrocytes or oligodendroglia. Glial fibrillary acidic protein staining in astrocytic processes was diminished. RNA-seq of P14 mutant mice cortex revealed over 1,000 differentially expressed genes (DEGs). Glial transcripts were downregulated and synaptic genes upregulated. Top gene ontology terms from upregulated DEGs relate to postsynapse and ion channel function, while downregulated DEGs enriched for terms relating to metabolic function, mitochondria, and ribosomes. Levels of synaptic proteins were changed, but number and length of synaptic contacts were unaltered at 3 months. Homozygosity worsened some phenotypes including postnatal survival, weight gain delay, and increase in nuclear NACC1. This mouse model simulates a rare form of autism and will be indispensable for assessing pathophysiology and targets for therapeutic intervention.

Toward a comprehensive catalog of regulatory elements.

Regulatory elements are the genomic regions that interact with transcription factors to control cell-type-specific gene expression in different cellular environments. A precise and complete catalog of functional elements encoded by the human genome is key to understanding mammalian gene regulation. Here, we review the current state of regulatory element annotation. We first provide an overview of assays for characterizing functional elements, including genome, epigenome, transcriptome, three-dimensional chromatin interaction, and functional validation assays. We then discuss computational methods for defining regulatory elements, including peak-calling and other statistical modeling methods. Finally, we introduce several high-quality lists of regulatory element annotations and suggest potential future directions.

Chemical engineering of therapeutic siRNAs for allele-specific gene silencing in Huntington's disease models.

Small interfering RNAs 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, enabling development of CNS-targeted therapeutics. Many genetically-defined neurodegenerative disorders are dominant, favoring selective silencing of the mutant allele. In some cases, successfully targeting the mutant allele requires targeting single nucleotide polymorphism (SNP) heterozygosities. Here, we use Huntington's disease (HD) as a model. The optimized compound exhibits selective silencing of mutant huntingtin protein in patient-derived cells and throughout the HD mouse brain, demonstrating SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. Targeting a disease-causing allele using RNAi-based therapies could be helpful in a range of dominant CNS disorders where maintaining wild-type expression is essential.

Allele-Specific Knockdown of Mutant Huntingtin Protein via Editing at Coding Region Single Nucleotide Polymorphism Heterozygosities.

Huntington's disease (HD) is a devastating, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Inactivation of the mutant allele by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mHTT protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.

Structurally constrained phosphonate internucleotide linkage impacts oligonucleotide-enzyme interaction, and modulates siRNA activity and allele specificity.

Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.

Huntington's Disease: Les Jeux Sont Faits?

In a meticulous study, Humbert, Durr, and colleagues showed evidence for aberrant neurodevelopment in both Huntington's disease (HD) and mouse models of HD. We consider the implications of prenatal pathological changes for the onset and progression of HD and their relatedness to current therapeutic plans.

Selective Neuronal Uptake and Distribution of AAVrh8, AAV9, and AAVrh10 in Sheep After Intra-Striatal Administration.

BACKGROUND: Transgenic sheep are currently the only large animal model of Huntington's disease expressing full-length mutant human huntingtin. These transgenic sheep provide an opportunity to test adeno associated virus (AAV) therapies directly targeting the huntingtin gene. A recent study demonstrated that self-complementary (sc) AAV with artificial miRNA against human huntingtin reduced mutant human huntingtin in caudate and putamen after a single injection near the internal capsule. OBJECTIVE: To identify an AAV serotype among AAVrh8, AAV9 and AAVrh10 with the highest neuronal uptake and distribution, with no obvious cell loss in the neostriatum of the sheep. METHODS: We tested AAVrh8, AAV9 and AAVrh10 by stereotactic direct unilateral injection into the neostriatum of sheep, near the internal capsule. Four weeks after administration, we examined the viral spread and neuronal uptake of each serotype of AAV containing GFP. We compared single stranded (ss) and scAAVs. Further, we measured the distribution of AAVrh8 and AAV9 to a variety of tissues outside the brain. RESULTS: Sc AAV9 had the best combination of neuronal uptake and distribution throughout the neostriatum. scAAVrh10 demonstrated good spread, but was not taken up by neurons. scAAVrh8 demonstrated good spread, but had less neuronal uptake than AAV9. Six hours after convection-enhanced administration to the neostriatum, both AAVrh8 and AAV9 viral genomes were detected in blood, saliva, urine, feces and wool. By four weeks, viral genomes were detected in wool only. Administration of AAVrh8, AAV9 and AAVrh10 was not associated with loss of neostriatal, medium spiny neuron number as measured by DARPP32 immunohistochemistry. CONCLUSIONS: Altogether, we found scAAV9 had the best neuronal uptake and spread, showed no loss of neurons at one-month post-injection, and was not measurable in body fluids one month after injection. This information will guide future clinical experiments requiring brain injection of AAV for therapeutics for gene or miRNA deliveries in sheep transgenic for the human huntingtin gene.

Artificial miRNAs Reduce Human Mutant Huntingtin Throughout the Striatum in a Transgenic Sheep Model of Huntington's Disease.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CßA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.

Alterations in mRNA 3' UTR Isoform Abundance Accompany Gene Expression Changes in Human Huntington's Disease Brains.

The huntingtin gene has two mRNA isoforms that differ in their 3' UTR length. The relationship of these isoforms with Huntington's disease is not established. We provide evidence that the abundance of huntingtin 3' UTR isoforms differs between patient and control neural stem cells, fibroblasts, motor cortex, and cerebellum. Huntingtin 3' UTR isoforms, including a mid-3' UTR isoform, have different localizations, half-lives, polyA tail lengths, microRNA sites, and RNA-binding protein sites. Isoform shifts in Huntington's disease motor cortex are not limited to huntingtin; 11% of alternatively polyadenylated genes change the abundance of their 3' UTR isoforms. Altered expression of RNA-binding proteins may be associated with aberrant isoform abundance; knockdown of the RNA-binding protein CNOT6 in control fibroblasts leads to huntingtin isoform differences similar to those in disease fibroblasts. These findings demonstrate that mRNA 3' UTR isoform changes are a feature of molecular pathology in the Huntington's disease brain.

Safe and Efficient Silencing with a Pol II, but Not a Pol lII, Promoter Expressing an Artificial miRNA Targeting Human Huntingtin.

Huntington's disease is a devastating, incurable neurodegenerative disease affecting up to 12 per 100,000 patients worldwide. The disease is caused by a mutation in the Huntingtin (Htt) gene. There is interest in reducing mutant Huntingtin by targeting it at the mRNA level, but the maximum tolerable dose and long-term effects of such a treatment are unknown. Using a self-complementary AAV9 vector, we delivered a mir-155-based artificial miRNA under the control of the chicken ß-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CßA promoter, produced the artificial miRNA at supraphysiologic levels. Embedding the antisense strand in a U6-mir-30 scaffold reduced expression of the antisense strand but increased the sense strand. In mice treated with scAAV9-U6-mir-155-HTT or scAAV9-CßA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CßA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioral abnormalities and striatal damage. In conclusion, miRNA backbone and promoter can be used together to modulate expression levels and strand selection of artificial miRNAs, and in brain, the CßA promoter can provide an effective and safe dose of a human huntingtin miRNA.

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells.

Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington's disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection.

Cellular Analysis of Silencing the Huntington's Disease Gene Using AAV9 Mediated Delivery of Artificial Micro RNA into the Striatum of Q140/Q140 Mice.

BACKGROUND: The genetic mutation in Huntington's disease (HD) is a CAG repeat expansion in the coding region of the huntingtin (Htt) gene. RNAi strategies have proven effective in substantially down-regulating Htt mRNA in the striatum through delivery of siRNAs or viral vectors based on whole tissue assays, but the extent of htt mRNA lowering in individual neurons is unknown. OBJECTIVE: Here we characterize the effect of an AAV9-GFP-miRHtt vector on Htt mRNA levels in striatal neurons of Q140/Q140 knock-in mice. METHODS: HD mice received bilateral striatal injections of AAV9-GFP-miRHtt or AAV9-GFP at 6 or 12 weeks and striata were evaluated at 6 months of age for levels of Htt mRNA and protein and for mRNA signal within striatal neurons using RNAscope multiplex fluorescence in situ hybridization. RESULTS: Compared to controls, the striatum of 6-month old mice treated at 6 or 12 weeks of age with AAV9-GFP-miRHtt showed a reduction of 40-50% in Htt mRNA and lowering of 25-40% in protein levels. The number of Htt mRNA foci in medium spiny neurons (MSNs) of untreated Q140/Q140 mice varied widely per cell (0 to 34 per cell), with ∼10% of MSNs devoid of foci. AAV9-GFP-miRHtt treatment shifted the distribution toward lower numbers and the percentage of cells without foci increased to 14-20%. The average number of Htt mRNA foci per MSN was reduced by 43%. CONCLUSIONS: The findings here show that intrastriatal infusion of an AAV9-GFP-miRHtt vector lowers mRNA expression of Htt in striatum by ∼50%, through a partial reduction in the number of copies of mutant Htt mRNAs per cell. These findings demonstrate at the neuronal level the variable levels of Htt mRNA expression in MSNs and the neuronal heterogeneity of RNAi dependent Htt mRNA knockdown.

Does the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon?

BACKGROUND: Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington's disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments. OBJECTIVES: We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA. METHODS: Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection. RESULTS: Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites. CONCLUSIONS: Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage.

Safety of Striatal Infusion of siRNA in a Transgenic Huntington's Disease Mouse Model.

BACKGROUND: The immune system In Huntington's disease (HD) is activated and may overreact to some therapies. RNA interference using siRNA lowers mutant huntingtin (mHTT) protein but could increase immune responses. OBJECTIVE: To examine the innate immune response following siRNA infusion into the striatum of wild-type (WT) and HD transgenic (YAC128) mice. METHODS: siRNAs (2'-O-methyl phosphorothioated) were infused unilaterally into striatum of four month-old WT and YAC128 mice for 28 days. Microglia number and morphology (resting (normal), activated, dystrophic), cytokine levels, and DARPP32-positive neurons were measured in striatum immediately or 14 days post-infusion. Controls included contralateral untreated striatum, and PBS and sham treated striata. RESULTS: The striata of untreated YAC128 mice had significantly fewer resting microglia and more dystrophic microglia than WT mice, but no difference from WT in the proportion of activated microglia or total number of microglia. siRNA infusion increased the total number of microglia in YAC128 mice compared to PBS treated and untreated striata and increased the proportion of activated microglia in WT and YAC128 mice compared to untreated striata and sham treated groups. Cytokine levels were low and siRNA infusion resulted in only modest changes in those levels. siRNA infusion did not change the number of DARPP32-positive neurons. CONCLUSION: Findings suggest that siRNA infusion may be a safe method for lowering mHTT levels in the striatum in young animals, since treatment does not produce a robust cytokine response or cause neurotoxicity. The potential long-term effects of a sustained increase in total and activated microglia after siRNA infusion in HD mice need to be explored.

HTT-lowering reverses Huntington's disease immune dysfunction caused by NFκB pathway dysregulation.

Huntington's disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntington's disease pathogenesis and has been targeted successfully to modulate disease progression, but mechanistic understanding relating this to mutant huntingtin expression in immune cells has been lacking. Here we demonstrate that human Huntington's disease myeloid cells produce excessive inflammatory cytokines as a result of the cell-intrinsic effects of mutant huntingtin expression. A direct effect of mutant huntingtin on the NFκB pathway, whereby it interacts with IKKγ, leads to increased degradation of IκB and subsequent nuclear translocation of RelA. Transcriptional alterations in intracellular immune signalling pathways are also observed. Using a novel method of small interfering RNA delivery to lower huntingtin expression, we show reversal of disease-associated alterations in cellular function-the first time this has been demonstrated in primary human cells. Glucan-encapsulated small interfering RNA particles were used to lower huntingtin levels in human Huntington's disease monocytes/macrophages, resulting in a reversal of huntingtin-induced elevated cytokine production and transcriptional changes. These findings improve our understanding of the role of innate immunity in neurodegeneration, introduce glucan-encapsulated small interfering RNA particles as tool for studying cellular pathogenesis ex vivo in human cells and raise the prospect of immune cell-directed HTT-lowering as a therapeutic in Huntington's disease.

Increased Steady-State Mutant Huntingtin mRNA in Huntington's Disease Brain.

BACKGROUND: Huntington's disease is caused by expansion of CAG trinucleotide repeats in the first exon of the huntingtin gene, which is essential for both development and neurogenesis. Huntington's disease is autosomal dominant. The normal allele contains 6 to 35 CAG triplets (average, 18) and the mutant, disease-causing allele contains >36 CAG triplets (average, 42). OBJECTIVE: We examined 279 postmortem brain samples, including 148 HD and 131 non-HD controls. A total of 108 samples from 87 HD patients that are heterozygous at SNP rs362307, with a normal allele (18 to 27 CAG repeats) and a mutant allele (39 to 73 CAG repeats) were used to measure relative abundance of mutant and wild-type huntingtin mRNA. METHODS: We used allele-specific, quantitative RT-PCR based on SNP heterozygosity to estimate the relative amount of mutant versus normal huntingtin mRNA in postmortem brain samples from patients with Huntington's disease. RESULTS: In the cortex and striatum, the amount of mRNA from the mutant allele exceeds that from the normal allele in 75% of patients. In the cerebellum, no significant difference between the two alleles was evident. Brain tissues from non-HD controls show no significant difference between two alleles of huntingtin mRNAs. Allelic differences were more pronounced at early neuropathological grades (grades 1 and 2) than at late grades (grades 3 and 4). CONCLUSION: More mutant HTT than normal could arise from increased transcription of mutant HTT allele, or decreased clearance of mutant HTT mRNA, or both. An implication is that equimolar silencing of both alleles would increase the mutant HTT to normal HTT ratio.

Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington's disease patients.

Among dominant neurodegenerative disorders, Huntington's disease (HD) is perhaps the best candidate for treatment with small interfering RNAs (siRNAs) [1-9]. Invariably fatal, HD is caused by expansion of a CAG repeat in the Huntingtin gene, creating an extended polyglutamine tract that makes the Huntingtin protein toxic [10]. Silencing mutant Huntingtin messenger RNA (mRNA) should provide therapeutic benefit, but normal Huntingtin likely contributes to neuronal function [11-13]. No siRNA strategy can yet distinguish among the normal and disease Huntingtin alleles and other mRNAs containing CAG repeats [14]. siRNAs targeting the disease isoform of a heterozygous single-nucleotide polymorphism (SNP) in Huntingtin provide an alternative [15-19]. We sequenced 22 predicted SNP sites in 225 human samples corresponding to HD and control subjects. We find that 48% of our patient population is heterozygous at a single SNP site; one isoform of this SNP is associated with HD. Several other SNP sites are frequently heterozygous. Consequently, five allele-specific siRNAs, corresponding to just three SNP sites, could be used to treat three-quarters of the United States and European HD patient populations. We have designed and validated selective siRNAs for the three SNP sites, laying the foundation for allele-specific RNA interference (RNAi) therapy for HD.