CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression

Abstract

Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion of the CAG trinucleotide repeat region in exon 1 of the Huntingtin gene (HTT), leading to the formation of mutant HTT transcripts (muHTT). The toxic gain-of-function of muHTT protein is a major cause of the disease. In addition, it has been suggested that the muHTT transcript contributes to the toxicity. Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT knock-down of both mRNA and protein was successfully achieved. Diminished phosphorylation of HTT gene-associated RNA-polymerase II is demonstrated, suggestive of reduced transcription downstream the ON-targeted repeat. Different backbone chemistries were found to have a strong impact on the ON efficiency. We also successfully use different delivery vehicles as well as naked uptake of the ONs, demonstrating versatility and possibly providing insights for in vivo applications.

Concomitant attenuation of HMG-CoA reductase expression potentiates the cancer cell growth-inhibitory effect of statins and expands their efficacy in tumor cells with epithelial characteristics

“…In theory, dual inhibition of the same target protein can ease the consequences of these limits, as has been shown for the attenuation of the oncogenic kinase, Bcr-Abl's activity [ 47 ]. Alternatively, downregulation of target protein expression is emerging as a viable therapy in neurodegenerative diseases, such as Huntington's Disease [ 48 ]. For HMGCR, this could also be achieved by targeting proteins that regulate the expression level of mevalonate pathway enzymes, such as the SREBP family- and Myc transcription factors, mTORC1, constituents of the PI3K-AKT pathway or AMPK (reviewed in Ref.…”

Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats

“…The presence of 3 complementary CAG-CTG triplets in the duplex allows, in principle, four possible Watson-Crick positional isomers to exist, with identical numbers of base pairs; we refer to these as mobile 3WJs. We reveal that these secondary structures undergo two distinct types of dynamics, which has implications for understanding and treating trinucleotide REDs, and their development as drug targets 23,24 .…”

Targeted Oligonucleotides for Treating Neurodegenerative Tandem Repeat Diseases

“…In a similar way, ONs directed towards genomic DNA (anti-gene ONs) may target, or be similar in sequence, to the NAT depending on their composition. In the experiments recently conducted in our laboratory using anti-gene ONs directed against the HTT gene, the anti-gene ONs target the template strand and are hence identical in sequence to a stretch in the HTT mRNA [ 111 ]. An effect on the NAT is unlikely, because that presumably would result in the upregulation of the sense HTT mRNA, whereas we observed the opposite.…”

“…Following a related DNA targeting concept, we have recently reported that LNA/DNA mixmer ON binding of CAG repeats in the HTT gene in primary patient cell lines resulted in efficient downregulation of transcription [ 111 ]. The ON used was directed against the template strand and we found no evidence of hybridization with HTT transcripts.…”