HD Insights™

SEQUOIA CLUB The Use of Antisense Oligonucleotides for Gene Silencing By: Emily Mitchell Sontag, PhD Huntington disease (HD) is caused by a single gene mutation and is therefore a good candidate for therapeutic gene silencing. While many potential HD therapeutic agents focus on ameliorating toxic effects following intracellular production of the mutant huntingtin protein (mHtt), gene silencing would disrupt the production of mHtt and could be the ultimate disease-modifying therapy for HD by preventing the toxic effects of mHtt. This article highlights some promising data from gene silencing and potential translational hurdles. Gene silencing offers many potential benefits. First, gene silencing does not require investigators to determine the exact mechanism by which mHtt causes disease. In addition, with gene silencing, the toxic effects of mHtt would be countered by the disruption of intracellular mHtt production. Finally, gene silencing therapies would remove any toxicity associated with mutant mRNA. The use of antisense oligonucleotides (ASOs) is a promising approach to gene silencing. ASOs are small single- stranded pieces of DNA that bind via complementary base-pair binding to the intracellular mRNA transcript (Figure). In HD, ASOs prevent the transcription of mHtt. ASOs have been found to reduce a number of different mHtt-associated abnormalities in animal models of HD1. A recent study showed that infusion of ASOs targeting mHtt into the brains of mouse models of HD could alleviate motor symptoms, prevent brain loss and increase survival rates1. The benefits of the ASO treatment persisted after the production of mHtt had returned to pre-treatment levels. 6 This effect, termed a "huntingtin holiday" by Carl Johnson of the Hereditary Disease Foundation, suggests that it may be possible for relatively less- frequent ASO treatments to give lasting benefit for patients. The study also found that infusion of ASOs into the cerebrospinal fluid delivered the ASOs to the brain and lowered mHtt mRNA levels in most brain regions in non-human primates. This method of delivery could be safer for human patients than direct intracerebral injection and may affect a wider range of brain tissues. This second point is significant because many research groups have shown that HD neuronal pathology is not limited to the striatum. Figure: Simplified mechanistic view of antisense oligonucleotide drugs. Despite the promise of gene silencing, challenges remain. First, the effect of reducing the levels of normal Htt, along with mHtt, is unclear. The majority of people with HD have one copy of the normal Htt gene and one copy of the mHtt gene. Htt is known to be essential in early development2, 3 and may be necessary for the survival of particular adult neurons4. Even though reducing Htt levels appears to be well tolerated in rodents and non- human primates5, 6, 7, it is possible that the human brain is more sensitive than animal brains to reduced Htt levels. One solution may be to target only the mutant HD gene for gene silencing, using ASOs or RNAi approaches. Several groups have utilized different approaches, including taking advantage of slight structural differences between mHtt mRNA and Htt mRNA8, or targeting mutations (polymorphisms) other than the expanded CAG repeat in the HD gene9. Additionally, other genes in the human genome also contain CAG repeats, but specifically targeting the mutant gene also appears to reduce "off-target" effects. Delivery is another potential roadblock for gene silencing techniques. It is not known whether spinal infusions will achieve the same widespread distribution of ASO in the much larger human brain, as that achieved in the brain of rodent and non-human primates. Convection enhanced delivery (CED)10 is a potential delivery approach that uses high pressure to deliver molecules deep into the brain. CED requires the insertion of tubes through the skull and into the brain, after which a pump is attached. This approach is obviously challenging from the patient's perspective. It may also prove difficult for surgeons to accurately place tubes into the brains of symptomatic HD patients, who typically suffer significant loss of brain volume. Copyright © Huntington Study Group 2012. All rights reserved. HD Insights, Vol. 3

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