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Gene Silencing

Huntington’s disease (HD) is a genetic disease due to the abnormal CAG expansion of the mutated huntingtin gene.  The mutated gene instructs cells in the body to produce a version of the huntingtin protein that ultimately leads to neuronal damage in the brain and creates the symptoms of HD.  Currently, available HD therapies can only provide relief of symptoms, but what if there were a way to eliminate the root of the problem by correcting the mutated gene or preventing the production of the “bad” huntingtin protein?  We are still years away from being able to directly edit the genome in human patients, but much progress has been made in the field of gene silencing, a scientific technique which can hinder or stop the production of a protein.  This article discusses the mechanism of gene silencing, its potential as an HD therapeutic, and challenges this possible therapy faces on the path to clinical implementation.


What is Gene Silencing?^

As the name implies, gene silencing is a technique that aims to reduce or eliminate the production of a protein from its corresponding gene.  Genes are sections of DNA that contain the instructions for making proteins.  Proteins are essential molecules that perform an array of functions including signaling between cells, speeding up biochemical reactions, and providing structural support for the cell.  Each gene is responsible for producing a corresponding protein in a two-step process.  First, a copy of the information encoded in a gene is made in the form of messenger RNA (mRNA), a process known as transcription.  This occurs in the nucleus of the cell, the cellular structure where all of the cell’s genetic material is contained.  The mRNA subsequently travels out of the nucleus, and the genetic information it carries is used to produce a specific protein, a process known as translation.  (For more information about proteins and how they are made, click here.)

Instead of directly editing DNA or inhibiting the transcription process, the key idea behind gene silencing is intervening in gene expression prior to translation.  By designing a molecule that can specifically identify and breakdown the mRNA carrying instructions for making a certain protein, scientists have been able to effectively decrease levels of that protein.  Imagine the gene silencing molecule as a censor and mRNA as messages from genes that are broadcast into proteins:  the molecule will censor out a specified mRNA message, preventing the corresponding protein from being broadcast into the cell, and thus silencing the gene that is providing these instructions.  The ability to significantly lower the levels of a specific protein opens up many possibilities in scientific research and drug development, since proteins are critically involved in the proper function and structure of cells.


Types of Gene Silencing Techniques^

There are various gene silencing methods currently employed in research and being developed as potential disease therapeutics.  Nearly all of them involve disabling the function of mRNA by preventing it from being translated into a protein.  However, they differ in the design of the molecule used to disrupt mRNA and the manner of mRNA breakdown.  As a result, different silencing methods have specific advantages and drawbacks.  Two of the leading and most understood methods of gene silencing are RNA interference (RNAi) and antisense oligonucleotides (ASOs).

RNA Interference

In RNAi, the molecules that identify the target mRNA are called small-interfering RNAs (siRNAs). Unlike normal single-stranded RNA found in cells – such as mRNA – siRNAs are short, synthetically made double-stranded RNA molecules designed to pair with a specific mRNA strand.  This association of the siRNAs with a particular target mRNA causes the breakdown of the target mRNA by recruiting other proteins that degrade the mRNA target.  Because siRNAs are double-stranded, they are more stable and less susceptible to degradation than ASOs, allowing them to continue to perform their silencing function for a longer period of time in the cell.  For a more detailed description of how RNAi works, click here.

Antisense Oligonucleotides

Similar to siRNAs, ASOs are engineered by scientists to associate with a target mRNA strand.  The binding of the ASO to mRNA directs a protein to breakdown the mRNA.  However, unlike siRNAs, ASOs are smaller, single-stranded RNA molecules.  As mentioned above, single-stranded RNAs are not as stable as double-stranded ones; thus, ASOs are often chemically modified to increase their durability in a biological environment.  However, their smaller size and chemical structure allow ASOs to be transported in cells and living tissues much more effectively than siRNAs.  For a more detailed description of how ASOs work, click here.

Is one gene silencing method better than the other?

In terms of developing a drug therapy based on gene silencing, how do RNAi and ASOs compare to each other in effectiveness?  In cell culture experiments, gene silencing is often used to intentionally decrease levels of a certain protein for research purposes.  In such applications, siRNAs have sometimes been shown to produce stronger and longer lasting gene silencing than ASOs.  However, when developing silencing therapeutics, the strength and duration of gene silencing needed for treatment may vary; sometimes a shorter-acting or less complete gene silencing may be required.  Furthermore, when considering the efficacy of each method in live animal models, the results are not as clear-cut.  For example, as mentioned earlier, ASOs can often be distributed more easily than siRNAs throughout the target tissue because of their size and structure.  This observation would be expected to simplify delivery and lower costs of a therapeutic application.  The fact that there is no definitive answer to which gene silencing method is more effective has resulted in continued active research and development of both areas.

Gene Silencing and HD^

HD is characterized by a mutation causing excess CAG repeats in the Huntington gene and the consequent production of the mutant huntingtin protein results in disease. As such, silencing of the mutant version of the huntingtin gene is a potential therapeutic strategy for HD treatment.  Indeed, HD and other related neurodegenerative diseases involving mutant CAG repeats, such as spinobulbar muscular atrophy and some types of spinocerebellar ataxias, have been at the frontier of the therapeutic development of gene silencing (for more information about trinucleotide repeat disorders, click here).

Approaches to Huntingtin Gene Silencing

Recall that everyone’s DNA is composed of two copies of a gene, called alleles, one from each parent.  In the majority of individuals with HD, one copy of the gene is mutated with excess CAG repeats, while the other copy is an allele with a number of CAG repeats within the normal range.  As a result, the body not only produces the mutant version of the huntingtin protein, but also makes the normal protein.  When considering gene silencing as a therapeutic approach to HD, it is crucial to think about the difference between silencing huntingtin mRNA in general and selectively disrupting mRNA that encodes for the mutant, and not the normal, huntingtin protein.

The huntingtin protein has many roles in proper development. Studies in mouse models have shown that completely eliminating huntingtin protein results in mice that do not survive past the embryonic stages of development, while mice that were induced to lose huntingtin after birth experienced severe neuronal degeneration (for more information about the function of wild-type huntingtin protein, click here). Thus, it is important for scientists to develop gene silencing drugs that specifically target mutant huntingtin mRNA.  This type of approach is known as allele-specific gene silencing.

While it may seem straightforward to target the excess CAG repeats to specifically decrease the levels of mutant huntingtin, it is important to remember that the molecules used for silencing are short RNA sequences, about 25 nucleotides in length.  Hence, they cannot effectively distinguish between the size of the normal and the expanded CAG repeats of the huntingtin gene. This is particularly true when trying to differentiate between 30 CAGs and 40 CAGs, CAG repeat ranges that are near normal.  To get around this obstacle, scientists are developing an approach that identifies single nucleotide polymorphisms (SNPs) – changes in a single nucleotide in the DNA sequence –closely linked with the mutant gene and not the normal allele.  SNPs are mutations that differ by a single nucleotide (e.g. ‘A’ à ‘C’) and result in the genetic variation between individuals.  What researchers have found is that many HD patients have common SNPs that are associated with the mutated huntingtin allele. Using silencing molecules to identify these SNPs provides a potential approach to allele-specific silencing of the mutant gene.

A recent study explored allele-specific silencing of the mutant huntingtin protein by targeting associated SNPs.  Since not all HD patients have the same SNPs, they sought to develop a panel of ASOs to maximize the coverage of the HD population.  They found that 85% of HD patients can be covered by targeting as few as three SNPs.  Moreover, injecting ASOs targeting an HD-associated SNP into HD mice showed a greater than 50% decrease in mutant huntingtin protein, while normal mice receiving the same treatment showed only a 3% drop in huntingtin levels.  These results indicate a relatively strong and selective silencing effect on mutant huntingtin.  Although further work must be done to expand coverage of the HD population by this approach and to assess its therapeutic efficacy outside of animal models, this study represents an initial step forward toward using allele-specific gene silencing as an HD therapeutic.

Although the above results are encouraging, some researchers have suggested using nonallele-specific silencing of huntingtin protein because  of the lack of a single SNP that will specifically target mutant huntingtin in all HD patients.  In this approach, instead of trying to decrease levels of the mutant huntingtin only, both the normal and mutated versions of the huntingtin protein are targeted and decreased.  This method is also advantageous because instead of employing different silencing molecules for different SNPs in different individuals, a single therapy can be developed for all HD patients, thus minimizing costs.  Although huntingtin has important functions in the body, interestingly, a study of nonallele-specific silencing using siRNA in HD mouse models found that when both mutant huntingtin and normal huntingtin levels were decreased by 75%, the treated mice demonstrated improved motor control and increased survival compared to controls.  This result suggests that nonallele-specific silencing may be a beneficial therapeutic for HD.  An important caveat to consider is that many therapies that show an effect in HD mouse models may not directly translate to humans.  For example, the mouse brain may be better able to tolerate a decrease in normal huntingtin than the human brain.  In any case, since both allele-specific and nonallele-specific silencing methods have their pros and cons, both continue to be under investigation as potential therapeutic options.

Challenges to Gene Silencing Therapeutics^

Even though gene silencing is a promising strategy for treating HD, there are still many hurdles to overcome before it can be applied in the clinic.  First and foremost, gene-silencing molecules have to be effectively delivered to the relevant parts of the body, which, in the case of HD, are the afflicted areas of the brain.  The blood-brain barrier prevents passage into the brain of most molecules that are injected or absorbed into the blood, making drug delivery difficult.  Some methods that scientists have used in animal models include direct injection of the silencing drug or implanting pumps that infuse the molecules into the brain.

Once past the blood-brain barrier, silencing molecules have to locate neurons and other affected cells and enter these cells to silence huntingtin expression.  As mentioned earlier, due to their structure, ASOs distribute and enter cells more effectively than siRNAs.  To effectively deliver siRNAs into cells, scientists currently use viral-based delivery systems, which essentially take advantage of the machinery viruses use to infect our cells.  One of the drawbacks of using a viral delivery mechanism is the potential for an immune response against the molecules.  As an alternative to this method, Dr. Jan Nolta’s group at the University of California Davis has begun studying the possibility of using mesenchymal stem cells (MSCs) as a delivery system for siRNA (for more information about MSCs, click here).  A possible advantage of using a viral or stem cell delivery system is that they might be able to become a production facility for siRNA molecules, allowing long-term therapeutic treatment of HD, a great benefit for a chronic illness. Ongoing research is currently investigating whether this theoretical possibility could become a reality.

There are other concerns associated with gene silencing therapeutics.  For example, researchers have observed that high dosages of silencing molecules could have a toxic effect, highlighting the importance of finding an optimal dosage that is safe and effective.  In addition, there is the possibility for ASOs and siRNAs to accidentally bind to an undesired mRNA (mRNA coding for a protein that is not huntingtin).  To deal with this so-called off-target gene silencing phenomenon, researchers are studying the selectivity of how siRNAs and ASOs bind to the huntingtin mRNA, in order to better develop specific, effective, and safe HD gene silencing therapeutics.


Gene silencing as a therapeutic strategy is a highly active area of research and may one day yield an effective treatment for HD, since it acts by directly reducing the production of the mutant huntingtin protein.  However, the technology is still in preclinical stages, and there remain many issues to address and resolve before it can be approved for clinical trials.  Delivery methods, dosages, and selectivity of gene silencing drugs must be optimized to ensure safety and efficacy of treatment.  Clinical trials have begun using gene silencing for therapeutic applications in other diseases. These studies will help to inform current efforts to develop gene silencing for HD treatment.

Further Reading^



1.  Bennett CF, Swayze EE.  RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform.  Annu. Rev. Pharmacol. Toxicol. 2010. 50:259–93.

This is a technical article published by Isis Pharmaceuticals that gives an in-depth review of the mechanisms and pharmacology involved in developing RNA-targeting gene silencing therapeutics.

2.  Boudreau RL, McBride JL, Martins I, Shen S, Xing Y, Carter BJ, Davidson BL.  Nonallele-specific silencing of mutant and wild-type Huntingtin demonstrates therapeutic efficacy in Huntington’s disease mice. Molecular Therapy (2009) 17 6, 1053–1063.

A technical article that explains the potential benefit of nonallele-specific silencing in HD mouse models.

3.  Boudreau RL, Rodriguez-Lebrón E, Davidson, BL.  RNAi medicine for the brain: progresses and challenges.  Hum Mol Genet. 2011 Apr 15;20(R1):R21-7.

A medium-difficulty article that discusses the development of RNAi as a therapeutic, current preclinical data, and the key challenges that remain for its clinical implementation.

4.  Carroll JB, Warby SC, Southwell AL, Doty CN, Greenlee S, Skotte N, Hung G, Bennett CF, Freier SM, Hayden MR.  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.

A technical article that explains the results of a study to develop a panel of ASOs for allele-specific gene silencing of mutant huntingtin in mouse models.

5.  Dessy A, Gorman JM.  The emerging therapeutic role of RNA interference in disorders of the central nervous system.  Clinical Pharmacology & Therapeutics (2011) 89 3, 450–454.

A medium-difficulty article that gives a broad overview of the current status of RNAi as a developing therapy for neurodegenerative diseases.

6.  Scholefield J, Wood MJ.  Therapeutic gene silencing strategies for polyglutamine disorders.  Trends Genet. 2010 Jan;26(1):29-38.

A technical article that reviews the mechanism of gene silencing and discusses therapeutic studies that have been done and challenges that remain to be addressed for allele-specific silencing of polyglutamine disorders.

7.  Sah DWY, Aronin N.  Oligonucleotide therapeutic approaches for Huntington disease.  J Clin Invest. 2011;121(2):500–507.

Another technical article that explains and compares the various approaches to gene silencing therapeutics for HD.

8.  Sass, Meghan; Aronin, Neil. “RNA- and DNA- Based Therapies for Huntington’s Disease.” Neurobiology of Huntington’s Disease: Applications to Drug Discovery. Ed. Donald C. Lo and Robert E. Hughes. Boca Raton: CRC Press, 2010.

A technical article that broadly covers the mechanisms, current studies, and challenges of both RNAi and ASO therapeutics for HD.

J. Choi 04.04.12