Potent and Selective Antisense Oligonucleotides Targeting Single-Nucleotide Polymorphisms in the Huntington Disease Gene / Allele-Specific Silencing of Mutant Huntingtin

Abstract

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.

Huntington disease in the South African population occurs on diverse and ethnically distinct genetic haplotypes

“…The study further supports the need to identify population-specific therapies, when envisaging gene-silencing methodology targeting SNPs present on the expanded HTT allele. [38][39][40] The subpopulations in this study emphasise the diversity that is present on the African continent. It is thus important to recognise that one population group cannot be used as a proxy for another, especially when these groups have different ethnic and geographical origins.…”

Palmitoylation of caspase-6 by HIP14 regulates its activation

“…The N-terminal 1212 amino acids of HTT with 128Q and the 4C mutations (D513A, D530A D552A, and D589A) were transiently transfected and overexpressed together with WT CASP6 or mutant CASP6 C264/277S in COS7 cells for 24 h. 47 Cell pellets from COS7 cells or Q111 and Q7 cells were lysed with SDP+ lysis buffer 65 and diluted in SDP+ lysis buffer to a final protein concentration of 2 μg/μl. 66,67 The detection of the 586 HTT fragment was performed using a combination of the monoclonal BKP1 antibody raised against the HTT N-terminus and an in-house 586 neo-epitope antibody raised against the C-terminus of 586 cleaved HTT.…”

Allele dependent silencing of collagen type I using small interfering RNAs targeting 3′UTR indels—A novel therapeutic approach in osteogenesis imperfecta

“…Allele specific silencing has been studied utilizing several heterozygous SNPs in Huntington's disease [36][37][38] and a mutation direct approach has been employed in e.g. epidermolysis bullosa simplex [39], Parkinson's disease [40] and vascular Ehlers-Danlos syndrome [20].…”