Edith L. Pfister

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits

Huntingtons disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1–400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

Five siRNAs Targeting Three SNPs May Provide Therapy for Three-Quarters of Huntington's Disease Patients

Among dominant neurodegenerative disorders, Huntingtons 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.

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

Huntingtons disease is an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system contributes to Huntingtons 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 Huntingtons 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 Huntingtons 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 Huntingtons disease.

Huntington's disease: Silencing a brutal killer

Huntington’s disease (HD) is a progressive and invariably fatal autosomal dominant neurodegenerative disorder caused by expansion of the CAG repeat in exon 1 of the Huntingtin gene. CAG repeat expansion generates an extended, toxic polyglutamine tract in the mutant Huntingtin protein (The Huntington’s Disease Collaborative Research Group, 1993), which disrupts a stunningly large number of cellular pathways. Mutant Huntingtin is especially damaging to the medium spiny neurons of the striatum (Graveland et al., 1985), and degeneration in this region is thought to underlie the motor disturbances or —chorea that characterize the disease. In parallel, loss of striatal and cortical neurons disrupts patient cognition and alters mood and personality (Heinsen et al., 1994). The age of onset of HD varies with CAG repeat length (Brinkman et al., 1997; Duyao et al., 1993) 36 or more repeats is considered pathological, but some patients with 36 to 39 repeats remain asymptomatic well into old age. The vast majority of patients are heterozygous for the HD mutation; most have 39 to 50 repeats and develop symptoms between age 30 and 50. No current treatment can prevent or delay the progression of HD.

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

BACKGROUND Huntingtons 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. Huntingtons 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 Huntingtons 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.

Artificial miRNAs reduce human mutant Huntingtin throughout the striatum in a transgenic sheep model of Huntington’s disease

Huntingtons 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 CβA. 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.

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.

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 β-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CβA 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-CβA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CβA-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 CβA promoter can provide an effective and safe dose of a human huntingtin miRNA.

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 Huntingtons 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.

B23 Immune dysfunction in HD human myeloid cells is caused by NFκB pathway dysregulation and is reversed by lowering HTT levels

Background Altered innate immune responses are observed in HD, with hyper-reactive central and peripheral immune cells producing increased levels of pro-inflammatory cytokine in HD patients. Bone marrow transplantation studies and KMO inhibitor administration have both shown that the immune system is a modifier of HD progression. However, a mechanistic understanding of how this relates to mHTT expression has been lacking. Aim Characterise the abnormal function of peripheral myeloid cells and examine alterations in signalling pathways responsible for those changes. Results The production of pro-inflammatory cytokines in response to LPS was elevated in both monocytes and macrophages from HD patients. Induction of mHTT exon 1 expression in a myeloid cell line showed that the hyper-reactive cytokine production is caused by a cell intrinsic effect of exon 1 mHTT expression. Significant changes in the expression levels of several key molecules in the NFκB pathway, such as IRAK1 and AKT1, were identified using PCR signalling arrays. IκB, the endogenous inhibitor of NFκB activity, was more rapidly degraded following LPS stimulation in HD patients compared to controls, potentially leading to increased NFκB activity and altered gene expression. A direct interaction between HTT and IKKγ was demonstrated by co-immunoprecipitation, suggesting the possibility of a scaffolding function of HTT in the NFκB pathway that may be altered by an expanded polyQ-repeat. Knocking-down total HTT protein levels using GeRP-delivered siRNA in human HD macrophages led to a reduction of cytokine production by LPS-stimulated HD cells. Conclusions Taken together, these data suggests that the dysfunction of primary myeloid cells from HD patients is directly caused by the expression of mHTT and its effects on the NFκB pathway. Lowering HTT levels in human macrophages can at least partially restore normal cell function, underlining the importance of HTT-lowering as a therapeutic approach.