Gene Silencing

Game-changing gene-silencing therapies for HD


NAME: Amber Southwell, PhD

TITLE: Assistant Professor of Neuroscience, Burnett School of Biomedical Sciences, University of Central Florida

EDUCATION: PhD, California Institute of Technology; post-doctoral research fellowship, University of British Columbia

HOBBIES: Scuba diving, sailing, partnered dancing, and cooking

The landscape of therapeutic development for genetic disease is rapidly evolving, and scientists are developing promising therapies to treat HD through gene-silencing therapies. HD Insights spoke with Dr. Amber Southwell about the status of  gene-silencing therapies for HD, as well as her own work on a selective antisense oligonucleotide (ASO) therapy.

What are gene-silencing therapies for HD?

Gene-silencing therapies reduce or prevent the expression of mHTT. They usually interfere with transcription or translation of mutant RNA, or cause the degradation of mHTT RNA.

What are the primary gene-silencing therapies under investigation?

The majority of gene-silencing therapies under current investigation are antisense oligonucleotides (ASOs), RNAi (RNA interference)-based reagents, and CRISPR/Cas9 gene-editing systems.

ASOs are short, synthetic DNA fragments that bind RNA through base pairing, and modulate its function. The majority of ASO drugs in development work through the degradative mechanism in which Ribonuclease H (RNase H) is recruited to recognize and cleave huntingtin transcripts. This process frees the ASO, enabling it to catalyze the degradation of multiple RNA molecules, effectively suppressing the gene product. With their diverse functionality, high target specificity, and suitability for direct CNS delivery through lumbar puncture ASOs are an excellent treatment option for HD. Currently, Ionis Pharmaceuticals has a nonselective ASO in a clinical trial. A nonselective ASO silences both wild-type and mHTT genes.

RNAi-based reagents—especially microRNA-based reagents—are also being developed. These reagents use small RNAs to bind to HTT mRNA molecules in order to silence their activity. RNAi-based reagents are not delivered naked—in other words, without a carrier—as are ASOs. Instead, they are typically delivered by a viral carrier. Two RNAi reagents that have shown a lot of promise are very close to translation to the clinic, one with Sanofi Genzyme and one with uniQure B.V. (see HD Insights, Vol. 16). One of the groups working on RNAi is doing its preclinical studies in the sheep model of HD, because sheep have a very long spinal cord, and a larger brain than nonhuman primates. That group hopes to maximize the therapeutics distribution in a larger brain and spinal cord.

In addition, a number of groups are developing gene-editing approaches to produce gene silencing, using the CRISPR/Cas 9 system. These approaches aim to completely inactivate the mutant copy of the gene. Currently, gene-editing strategies have low efficiency. Basically, you gene-edit some cells in culture, and then select for the cells in which the genes were actually edited. Next, you test the edited genes to make sure they did not receive an unwanted edit. Finally, you find that the cells have been changed in the way you want, and you expand those up to create your population. These studies are in very early preclinical stages, and there is a long way to go before efficiency and specificity will be high enough for use in the human brain.

What does your most recent research focus on?

We have been investigating a selective ASO that suppresses only mHTT and leaves wild type HTT expression intact. We ran a preclinical trial that will be published very soon and had excellent results. Basically, our ASO prevents the onset of HD in presymptomatic mice, and in symptomatic mice we saw recovery of motor and psychiatric phenotypes. Most importantly, we saw a complete rescue of cognitive phenotypes. We have shown that our ASO can not only prevent cognitive decline, but can also restore normal cognition when used post-symptom onset, meaning, after the mice have developed a cognitive deficit. We are very excited about this.

Is your selective ASO better than Ionis’s nonselective ASO?

Our selective ASO was co-developed with Ionis. I think the reason Ionis started with a nonselective ASO for its clinical trial was that it was the fastest way to get a therapy to all HD patients, with the idea that going back and developing selective therapies later would yield drugs that were safer for patients in the long term. I think that nonselective silencing is going to be preferable to not treating HD, but I also think that selective silencing is going to be better for the patient than nonselective silencing. In an effort to preserve wild-type HTT function, the nonselective ASO is being dosed to induce about 50 percent suppression in the cortex, and to have minimal activity in the basal ganglia.

With a selective ASO, it may be possible to use a higher dose, to induce almost total mHTT suppression in the cortex, and have more activity in the basal ganglia.

Is there competition among scientists to be the first to discover the best therapy?

I think most people would agree with me that we are all on the same team. HD is the adversary. It is an incredibly complicated disease, and one that we need to come at from every angle we can. There is still so much work to do—after all, the gene was identified in 1993, and today, more than 20 years later, we are only just starting the very first huntingtin-lowering clinical trials. There is enough room for everyone’s work, and there is a lot more collaboration than competition in the field.

Are the patients and families hopeful?

Yes, they are really excited. They often contact me and ask, “When is this going to be available for me?” Sometimes I feel that a single email asks me to be a scientist, a clinician, and a genetic counselor. I have to tell people that it is a really long way between mice and men. A therapy must go through so many steps, so many points of potential failure, and once it gets to human trials, it can take years to prove its safety and efficacy. We are also hindered by our tools; for example, we do not have enough biomarkers to evaluate these therapies. The excitement is pushing the HD community to think that there is going to be something available for them in 5 to 10 years, but we have no idea if that is the case. Yes, we can at last successfully treat a mouse. Yes, we have really great preventative and restorative preclinical data. Yes, we finally have a huntingtin-lowering clinical trial that is targeting the root cause of HD—but unfortunately, none of this means there is an effective treatment just over the horizon. We simply do not know yet.

Selected gene-silencing therapies in the pipeline






Ionis Pharmaceuticals and Don Cleveland

Research group

ASO Study found ASO infused into cerebrospinal fluid of symptomatic HD mice delays progression of HD and reverses disease phenotypes. Phase I clinical trial underway Kordasiewicz HB, et al. 2012. 1
Genzyme miRNA Research suggests two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, successfully target neurons that degenerate in HD. Clinical trial planned

(IND estimated 2018)

Hadaczek P, et al. 2016.2
uniQure miRNA Research demonstrates strong in vitro and in vivo allele-selective silencing of mHTT by miSNP50 and total HTT silencing by miH12. Preclinical research

(IND estimated 2017)

Miniarikova J, et al. 2016. 3
Sangamo Biosciences Zinc finger Research revealed that allele-specific zinc fingers lowered production of mutant protein by more than 90 percent, while reducing normal protein by 10 percent or less. Preclinical research

(IND estimated 2017)

Zhang HS, et al. 2014.4
Neil Aronin Research Group siRNA siRNA infusion is shown to lower mHTT levels in the striatum of mice without producing a robust immune response. Preclinical research Johnson E, et al. 2015.5
Ionis Pharmaceuticals

Michael Hayden Research Group

ASO At a wide range of doses, four ASOs potently and selectively silence mHTT throughout the central nervous system for 36 weeks or more after a single intracerebroventricular injection. Preclinical research Southwell AL, et al. 2014.6
WAVE Life Sciences ASO WAVE’s lead compounds are stereopure, allele-specific ASOs that target two difference single nucleotide polymorphisms (SNPs) associated with mutated Huntingtin to enable maximal target engagement with minimal off-target effects. Preclinical research (INDs expected 2017) WAVE Life Sciences 2017 Pipeline Update [press release], 2017.7
David Corey Research Group ASO Several ASOs targeted to the CAG repeat of HTT and containing a variety of modifications, such as bridged nucleic acids and phosphorothioate internucleotide linkages, demonstrated allele-selective silencing in patient-derived fibroblasts. Preclinical research Gagnon KT, et al. 2010.8
Beverly Davidson Research Group CRISPR/Cas9 CRISPR/Cas9-based strategy for allele-specific genome editing reduces expression of mutant HTT alleles in human HD fibroblasts and mouse brain. Preclinical research Monteys AM, et al. 2017.9






Jong-Min Lee Research Group CRISPR/Cas9 Protospacer Adjacent Motif (PAM)-altering SNPs target patient-specific CRISPR/Cas9 sites, with a goal of allele-specifically inactivating the mutant HTT for a given diplotype. Preclinical research Shin JW, et al. 2016.10
Gary Dunbar

Research group

CRISPR/Cas9 A CRISPR/Cas9 plasmid reduces the production of mHTT in mesenchymal stem cells from YAC128 mice. Preclinical research Kolli N, et al. 2017.11
Mahmoud Pouladi

Research Group

CRISPR/Cas9 Use of a CRISPR-Cas9 and piggyBac transposon-based approach to correct HD hiPSCs and associated phenotypic abnormalities. Preclinical research Xu, et al. 2017.12

1Kordasiewicz HB, Stanek LM, Wancewicz EV, et al. Sustained Therapeutic Reversal of Huntington’s Disease by Transient Repression of Huntingtin Synthesis. Neuron. 2012;74(6):1031-1044.

2Hadaczek P, Stanek L, Ciesielska A, et al. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington’s disease. Mol There Methods Clin Dev.. 2016;3:16037.

3Miniarikova J, Zanella I, Huseinovic A, et al. Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington’s Disease. Mol Ther Nucleic Acids. 2016;5:e297.

4Zhang HS, Zeitler B, Froelich S, et al. 769.12. Engineered zinc finger transcriptional repressors selectively inhibit mutant huntingtin expression and reverse disease phenotypes in Huntington’s disease patient-derived neurons and in rodent models. [abstract]. Neuroscience 2014; November, 2014; Washington, DC.

5Yu MS, Johnson E, Chase K, McGowan S, et al. Safety of Striatal Infusion of siRNA in a Transgenic Huntington’s Disease Mouse Model. J Huntington’s Dis. 2015;4(3):219-229.

6Southwell AL, Skotte NH, Kordasiewicz HB, et al. In Vivo Evaluation of Candidate Allele-specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides. Mol Ther. 2014;22(12):2093-2106.

7WAVE Life Sciences 2017 Pipeline Update [press release]. January 06, 2017; Accessed April 29, 2017.

8Gagnon KT, Pendergraff HM, Deleavey GF, et al. Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat. Biochemistry. 2010;49(47):10166-10178.

9Monteys AM, Ebanks SA, Keiser MS, Davidson BL. CRISPR/Cas9 Editing of the Mutant Huntingtin Allele In Vitro and In Vivo. Mol Ther. 2017;25(1):12-23.

10Shin JW, Kim K-H, Chao MJ, et al. Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9. Hum Mol Genet. 2016;25(20):4566-4576.

11Kolli N, Lu M, Maiti P, Rossignol J, Dunbar GL. CRISPR-Cas9 Mediated Gene-Silencing of the Mutant Huntingtin Gene in an In Vitro Model of Huntington’s Disease. Int J Mol Sci. 2017;18(4).

12Xu X, Tay Y, Sim B, et al. Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent Stem Cells. Stem Cell Reports. 2017;8(3):619-633.

HSG Evening Blog: I’ve been here before, and I need to know what’s new

At HSG 2015, it's all about the connections made that will help us build our future.

At HSG 2015, it’s all about the connections made that will help us build our future.

In conversations today, we overheard the experience of one person who has been a family caregiver to a loved one with HD, and who is now working in the field of HD research. This person, who was attending the HSG for their first time and who had been participating in the session on Practical Pointers and Perspectives on Huntington’s for Local Practitioners, said “I remember all this. We just had no idea that is what it was.” We asked this person (who requested to remain anonymous) to tell us more about this sentiment. What did she mean when she said that she had been “here” before?

This person had been a caregiver for a parent with HD, and has a sibling with HD. When they were in the session and talking about neuropsychiatry, it brought back a series of experiences and a life history of Huntington’s. We got to thinking, just how many other people involved in the field of HD research, and treatment and care were here because they too came from an HD family? How many generations of HD families have contributed to the knowledge base of how to care for someone with HD? HD is a multigenerational disease. It leaves a genetic legacy

This afternoon we had the chance to hear about Huntingtin lowering therapies. David Corey from UT Southwestern Medical Center discussed gene silencing mechanisms. We know that HD drugs are needed, but Huntingtin, or HTT, the mutant protein that causes HD, presents some challenges. We can trace the problems of HD back to this mutant protein. David described HTT as an “undruggable target.” This leaves gene silencing, essentially changing the way a person’s genetic makeup expresses HTT in body, as a possible treatment. Turn off that potential genetic legacy, and you can turn off the disease.

It’s this genetic legacy that brings us to where we are in the world of HD research. As we look to fix the mutant HTT protein, we have to get in to some sophisticated therapies like gene silencing. The science in to gene silencing is complex and time taking. Getting gene silencing therapies to work in the body takes a combination of delivery and safety. Gene silencing therapies have to get the best coverage in the regions of the brain affected by HD, but also have to make sure there are no effects on other proteins. Get the therapeutic intervention in to the brain, and keep it focused just on the mutant HTT.

Even though the gene for HD was discovered in 1993, gene silencing is just the beginning. That genetic legacy, that for generations of people when they were diagnosed with HD meant the end of their life, may be just the beginning of new cures. That genetic legacy could be on its way to saving lives, and curing HD. Oh yeah, the families living with HD have been here before. But where they’ve gotten us? That’s what’s new.

-Jared Husketh, contributor and director of clinical services for HD Reach

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.

Figure: Simplified mechanistic view of antisense oligonucleotide drugs.

Figure: Simplified mechanistic view of antisense oligonucleotide drugs.

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.

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.

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 nonhuman primates, 5, 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.

The optimal timing and treatment regimen for HD patients is also not known. Studies suggest that relatively early treatments are more beneficial; however, tracking the benefits of a treatment administered before the appearance of symptoms remains difficult. Groups worldwide are working to establish more quantifiable indicators of early symptoms of HD. Further, even though ASO treatment in animal models reduced symptoms for longer than expected, human patients must deal with the disease for decades, and it is not known how often a “booster” might be needed for continued benefit.

A number of clinical trials utilizing ASOs have been completed, and more are currently underway. Previous trials have used ASOs for the treatment of various cancers, asthma, arthritis, Duchenne muscular dystrophy, Crohn’s disease, heart disease and familial amyotrophic lateral sclerosis (ALS). The ALS clinical trial is of particular interest because it uses spinal infusion for the delivery of ASOs. Phase I of this clinical trial was completed in early 2012, and its results may provide information on the safety of ASO spinal infusion.

Many different gene silencing techniques are being used to lower mHtt expression in animal models of HD. RNA-based strategies, including short-hairpin RNAs (shRNA)11; small interfering RNAs (siRNA)2; and microRNAs (miRNA)3, also show promise in alleviating mHtt-mediated phenotypes. Some of these strategies are moving toward human clinical trials. Despite the challenges of gene silencing, ASO therapy and other mHtt knockdown approaches for HD remain exciting avenues of treatment. Each new development brings us closer to the discovery of disease-modifying therapy for HD.


1 Kordasiewicz HB, Stanek LM, Wancewicz EV, et al. Sustained Therapeutic Reversal of Huntington’s Disease by Transient Repression of Huntingtin Synthesis. Neuron2012 Jun 21;74(6): 1031-44.

2 Nasir J, Floresco SB, O’Kusky JR, et al. Targeted disruption of the Huntington’s disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell. 1995 Jun;81(5):811-23.

3 Zeitlin S, Liu JP, Chapman DL, et al. Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington’s disease gene homologue. Nature Gen. 1995 Oct; 11(2):155-63.

4 Dragatsis I, Levine MS, Zeitlin S. Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice. Nature Gen. 2000 Nov;26(3):300-6.

5 Boudreau RL, McBride JL, Martins I, et al. Nonallele-specific silencing of mutant and wild-type huntingtin demonstrates therapeutic efficacy in Huntington’s disease mice. Molecula therapy. 2009 Jun;17(6):1053-63.

6 Grondin R, Kaytor MD, Ai Y,et al. Six-month partial suppression of Huntingtin is well tolerated in the adult rhesus striatum. Brain. 2012 April;135(4):1197-209.

7 McBride JL, Pitzer MR, Boudreau RL, et al. Preclinical safety of RNAi-mediated HTT suppression in the rhesus macaque as a potential therapy for Huntington’s disease. Molecular therapy. 2011 Dec;19(12):2152-62.

8 Gagnon KT, Pendergraff HM, Deleavey GF, et al. Alleleselective inhibition of mutant huntingtin expression with antisense oligonucleotides targeting the expanded CAG repeat. Biochemistry. 2010 Nov 30;49(47):10166-78.

9 Carroll JB, Warby SC, Southwell AL, et al. Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene/allele-specific silencing of mutant huntingtin. Mol Ther 2011 Dec;19(12): 2178-85.

10 Stiles DK, Zhang Z, Ge P, Nelson B, et al. Widespread suppression of huntingtin with convection-enhanced delivery of siRNA. Exper Neurol 2012; 233(1):463-71.

11 Harper SQ, Staber PD, He X, et al. RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proc Nat Acad Sc USA. 2005 Apr;102(16): 5820-5. 12 DiFiglia M, Sena-Esteves M, Chase K, et al. Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits. Proc Nat Acad Sc USA. 2007 Oct; 104(43):17204-9.

13 McBride JL, Boudreau RL, Harper SQ, et al. Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Nat Acad Sc USA. 2008 Apr;105(15):5868-73.