Gene Silencing

VITAL SIGNS

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

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; http://www.businesswire.com/news/home/20170106005730/en/ 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.

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.

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 development^{2, 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.