Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene / allele-specific silencing of mutant huntingtin

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion in the huntingtin gene (HTT) that results in a toxic gain of function in the mutant huntingtin protein (mHTT). Reducing the expression of mHTT is therefore an attractive therapy for HD. However, wild-type HTT protein is essential for development and has critical roles in maintaining neuronal health. Therapies for HD that reduce wild-type HTT may therefore generate unintended negative consequences. We have identified single-nucleotide polymorphism (SNP) targets in the human HD population for the disease-specific targeting of the HTT gene. Using primary cells from patients with HD and the transgenic YAC18 and BACHD mouse lines, we developed antisense oligonucleotide (ASO) molecules that potently and selectively silence mHTT at both exonic and intronic SNP sites. Modification of these ASOs with S-constrained-ethyl (cET) motifs significantly improves potency while maintaining allele selectively in vitro. The developed ASO is potent and selective for mHTT in vivo after delivery to the mouse brain. We demonstrate that potent and selective allele-specific knockdown of the mHTT protein can be achieved at therapeutically relevant SNP sites using ASOs in vitro and in vivo

Lenalidomide reduces microglial activation and behavioral deficits in a transgenic model of Parkinson’s disease

BACKGROUND: Parkinson’s disease (PD) is one of the most common causes of dementia and motor deficits in the elderly. PD is characterized by the abnormal accumulation of the synaptic protein alpha-synuclein (α-syn) and degeneration of dopaminergic neurons in substantia nigra, which leads to neurodegeneration and neuroinflammation. Currently, there are no disease modifying alternatives for PD; however, targeting neuroinflammation might be a viable option for reducing motor deficits and neurodegeneration. Lenalidomide is a thalidomide derivative designed for reduced toxicity and increased immunomodulatory properties. Lenalidomide has shown protective effects in an animal model of amyotrophic lateral sclerosis, and its mechanism of action involves modulation of cytokine production and inhibition of NF-κB signaling. METHODS: In order to assess the effect of lenalidomide in an animal model of PD, mThy1-α-syn transgenic mice were treated with lenalidomide or the parent molecule thalidomide at 100 mg/kg for 4 weeks. RESULTS: Lenalidomide reduced motor behavioral deficits and ameliorated dopaminergic fiber loss in the striatum. This protective action was accompanied by a reduction in microgliosis both in striatum and hippocampus. Central expression of pro-inflammatory cytokines was diminished in lenalidomide-treated transgenic animals, together with reduction in NF-κB activation. CONCLUSION: These results support the therapeutic potential of lenalidomide for reducing maladaptive neuroinflammation in PD and related neuropathologies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-015-0320-x) contains supplementary material, which is available to authorized users

Allele-Specific Suppression of Mutant Huntingtin Using Antisense Oligonucleotides: Providing a Therapeutic Option for All Huntington Disease Patients

<div><p>Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.</p></div

ASO screening pipeline.

<p>(A) HD-SNPs in the <i>HTT</i> gene: blue = HD-SNPs, pink = previous human fibroblasts screen, grey = Hu97/18 screen; green Rs numbers = SNPs identified as the most RNase-H-active sites (B) ASO development pipeline: The number of targeted SNPs and ASOs tested are shown above and below the column bars, respectively. 50 SNPs are enriched on HD alleles and ASOs targeting 24 of these were previously screened for mHTT mRNA silencing. ASOs targeting 10 SNPs were screened in primary Hu97/18 neurons for HTT protein suppression and tolerability. Then, ASOs with modifications to the wings targeting 4 of these SNPs were screened. Microwalk SAR and 7-base gap SAR was done for oligos targeting SNP Rs7685686. Lastly, higher ASO concentrations and longer treatment durations were tested.</p

ASO screen at 4 SNPs using two different cEt motifs.

<p>(A) ASOs with two different cEt-modified wing motifs (ekek-9-keke and ekk-9-kke) were compared to the parent MOE oligos (5e-9-5e). Primary Hu97/18 neurons were treated with ASO at 1–1000 nM for 6 days. (B) HTT Western blot and quantitations. HTT levels are normalized to the internal loading control calnexin and then to the untreated sample for each allele. (C) Western blots showing full length and cleaved spectrin. Spectrin fragment is normalized to calnexin and then to the untreated sample. Membranes were probed for HTT and reprobed for spectrin. Representative images are shown. n = 6–8 per data point. Data are presented as mean ± SD. Two way ANOVA with Bonferroni post hoc test have been performed and p values are illustrated with *, **, ***, **** for p = 0.05, 0.01, 0.001, and 0.0001. The PS backbone is black, MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is underlined. The red dashed line represents the toxicity threshold.</p

Shortening the gap to 7 nucleotides and evaluation at higher doses.

<p>(A) Replacing PS-nucleotides with RNase H resistant chemical modifications and shortening the gap from 9 to 7 nucleotides. The top 4 candidates are shown. Primary Hu97/18 neurons were treated with ASO at 1–10000 nM for 6 days. (B) Western blot and quantitation of HTT protein levels. HTT levels are normalized to the internal loading control calnexin and then to the untreated sample for each allele. (C) Western blots showing full length and cleaved spectrin. Spectrin fragment is normalized to calnexin and then to the untreated sample. Membranes were probed for HTT and reprobed for spectrin. Representative images are shown. n = 8–14 per data point at 0–1000 nM and n = 4–6 at 1250–10,000 nM. Data are presented as mean ± SD. Two way ANOVA with Bonferroni post hoc test have been performed and p values are illustrated with *, **, ***, **** for p = 0.05, 0.01, 0.001, and 0.0001. The PS backbone is black, MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is underlined. The red dashed line represents the toxicity threshold.</p

Microwalk of the SNP position within the gap.

<p>(A, B) Diagram of microwalk ASOs and HTT mRNA silencing in primary human HD fibroblasts. (A) Starting from A3, we moved one cEt modification to the 5′ wing (ekkk-9-ke) and moved the SNP site from position 4 to 14 (B) Similarly, we moved one cEt modification to the 3′ wing (ek-9-kkke) and moved the SNP site from position 2 to 12. mHTT and wtHTT mRNA were normalized to total RNA and then to the untreated sample. n = 2 per data point. A subset of ASOs from preliminary fibroblast screen marked by #, were evaluated in primary Hu97/18 neurons at 4–1000 nM for 6 days. (C) Western blots of HTT protein and quantitations. HTT levels are normalized to the internal loading control calnexin and then to the untreated sample for each allele. (D) Western blots showing full length and cleaved spectrin. Spectrin fragment is normalized to calnexin and then to the untreated sample. Membranes were probed for HTT and reprobed for spectrin. Representative images are shown. n = 6–10 per data point. Data are presented as mean ± SD. Two way ANOVA with Bonferroni post hoc test have been performed and p values are illustrated with *, **, ***, **** for p = 0.05, 0.01, 0.001, and 0.0001. The PS backbone is black, MOE and cEt modifications are illustrated by orange and blue, respectively. The SNP is underlined. The red dashed line represents the toxicity threshold.</p

Selection of the best SNP targets.

<p>Primary Hu97/18 neurons were treated with 5e-9-5e ASOs targeted to 10 HD-SNPs at 6–1000 nM for 6 days. (A) HTT Western blots and quantitation for the 4 SNPs with the greatest activity. HTT levels are normalized to the internal loading control calnexin and then to the untreated sample for each allele. (B) Western blots showing full length and cleaved spectrin for the 4 ASOs. Spectrin fragment is normalized to calnexin and then to the untreated sample. Membranes were probed for HTT and reprobed for spectrin. Representative images are shown. n = 4–8 per data point. Data are presented as mean ± SD. Two way ANOVA with Bonferroni post hoc test have been performed and p values are illustrated with *, **, ***, **** for p = 0.05, 0.01, 0.001, and 0.0001. The PS backbone is black and MOE modifications are illustrated by orange. The SNP is underlined. The red dashed line represents the toxicity threshold.</p