Arches. Photo by Daniel Chia
HOPES: Huntington's Outreach Project for Education, at Stanford

RNA Interference (RNAi)

People with Huntington’s disease have two different copies, or alleles, of the Huntington gene. As we discussed here, genes are sections of DNA that provide the information for making proteins. The non-HD allele produces a normally functioning protein, but the HD allele produces a protein that is either the cause or result of many problems in nerve cells. The proteins produced by the HD allele form clumps, or protein aggregates that prevent normal functioning of nerve cells. (For more information on protein aggregation, click here). Although the topic is still up for debate, some researchers suggest that these aggregations may somehow contribute to the progression of the disease. The next step, therefore, is to figure out how to remove the huntingtin protein aggregations or, better still, prevent them from ever forming in the first place. This chapter discusses RNA interference, a gene therapy technique that may do just that.

What is RNAi? How does it work?^

RNA interference (RNAi) is a way to “silence” genes by preventing the formation of the proteins that they code for. A type of gene therapy, it takes advantage of an intermediate step between DNA and protein. DNA acts as a blueprint for the final protein but it uses a kind of “middleman,” called messenger RNA (mRNA), in order to get there. Going from gene to protein is a two step process. The first step in protein synthesis, transcription, takes place in a cell’s nucleus, where the DNA template is used to make a single strand of mRNA. The mRNA then exits the nucleus and enters the cytoplasm, where now it serves as the template for making the protein. With the help of several different molecules, a string of amino acids forms according to the order of the mRNA bases, which are very similar to DNA bases. This process is called translation because the mRNA code is translated into the language of amino acids, the building blocks of proteins.

RNAi comes into play between the steps of transcription and translation. RNA is introduced into the cell and binds to and destroys its mRNA target. Scientists can tailor make pieces of RNA that are complementary (matched up) to a specific strand of mRNA. In some organisms, the whole strand of complementary RNA can be introduced and an enzyme called dicer cuts it up into small fragments once it is inside the cell. Experiments have shown that introducing large strands of RNA into mammals does not work, so scientists were able to overcome this problem by making small interfering RNA (siRNA), also called short interference RNA. This is basically creating smaller chunks of double-stranded RNA (RNA is usually single stranded, except in some viruses) before injecting it into the cell. When these pieces of siRNA match up with the mRNA, they initiate a process that cuts up the mRNA into small fragments. The cell recognizes these fragments as waste and degrades them, and the proteins never form. (See figure below for a representation of the RNAi mechanism.)

Fig H-2: RNA Interference - Simplified Mechanism

How can RNAi be used to treat HD?^

DNA serves as the template for mRNA. This means that the mRNA from the HD allele will be different from the mRNA from the non-HD allele in the same way that their DNA differs. Since the non-HD allele makes a functional protein, it is important that we only silence the disruptive mRNA from the HD allele (mouse studies have shown that shutting down both Huntington genes could be fatal). In order to do this we must first find a difference between the HD and non-HD mRNA. Unfortunately, targeting the most obvious difference between these two molecules, the extra CAG repeats, has proven ineffective (For more information on CAG repeats and HD, click here.) However, there are other differences within the HD allele that are present in most of the people who have it. These differences can be as small as one base substitution (remember how the DNA “alphabet” consists of only four letters, A, C, G, and T? This would be like substituting an “A” for a “C” somewhere in the middle of the chain.). These small differences are called single nucleotide polymorphisms (SNPs). Scientists can create pieces of RNA that are only complementary to the HD mRNA containing a SNP so that only the disruptive HD protein is prevented from being formed. This would allow the non-HD allele to continue to make the normal protein and prevent the harmful protein aggregations that form from the HD protein.

What are some of the challenges of RNAi?^

RNAi is a very promising new tool for treating many kinds of genetic disorders, but much more research and testing need to be done before it can be put to use. One of the main challenges right now is finding a vector, or delivery system, to bring the therapeutic RNA into the nerve cells in the brain. Some researchers have successfully used certain modified viruses for this purpose. This is done by first removing the virus’ own genetic material, thus removing its potential to cause harm, and then replacing it with the therapeutic RNA. One of the first successful RNAi tests was done on mice with a disease similar to HD called spinocerebellar ataxia type 1. (For more information on spinocerebellar ataxia type 1, click here). After injecting the mice with a modified virus, their condition improved. Mice receiving RNAi treatment stopped producing the mutant protein. With the disruptive protein out of the way, the mice no longer experience physical symptoms and are able to move around more easily.

While these are very promising results, we must remember that the testing was done on mice with a similar but different disease than human HD. Much work still needs to be done before the lessons learned from mouse experiments can be safely adapted for use in humans with HD. For instance, further testing has shown that the virus used in the mouse experiment will not work on humans, so another virus must be used. So far, researchers have had success with treating human cells in the laboratory using a virus similar to HIV that normally infects cats, not humans. It is also very important that the siRNA only silence the mRNA from the HD allele. As mentioned before, this requires finding a difference between the two alleles other than the extra CAG repeats. One problem with this in developing an effective treatment is to find a difference that is present in most or all people with HD. In the testing done on mice, the difference was a SNP that was present in 70% of mice with spinocerebellar ataxia. This begs the question, what about the other 30%? Scientists will need to find SNPs present in all people with HD so that they can eventually treat 100% of the population.

Many other important questions about RNAi have yet to be answered. At what age should people start to receive treatment? Would this be before or after they start showing symptoms of HD? Researchers must also run trials to see how much and how often patients should receive treatment. Right now we have no idea if RNAi therapy is long-term or only temporary. In addition, researchers need to continue their work on finding a vector that is both safe and effective. RNAi holds great promise for future treatment of HD but several more years of research and clinical trials need to be done before it can be widely available to the HD community.

For further reading:^

  1. Ambion, Inc. The RNA interference resource. Online
    This is a website with great articles of medium difficulty explaining RNAi and links to new research. Recommended are the articles listed under “New to RNAi? Start Here!”
  2. Check, Erika. Cure hoped for Huntington’s sufferers. 2004. Nature. Online
    This is an easy to read article explaining the initial successful research using RNAi.
  3. Crittenden, Jill. RNA modalities in Huntington’s disease therapy (Workshop). 2002. Online
    This is a discussion of what went on at a workshop of gene therapy researchers. It is of medium difficulty and touches on several key issues that must be worked on before RNAi can be put to use.
  4. Davidson, B.L. and Paulson, H.L. Molecular medicine for the brain: silencing of disease genes with RNA interference. 2004. The Lancet Neurology 3(3): 145-149.
  5. Dykxhoorn, et al. Killing the messenger: short RNAs that silence gene expression. 2003. Nature Reviews Molecular Cell Biology 4: 457-467. Online.
    This is a technical article that explains how RNAi is used to silence genes.
  6. Miller, Marsha. A major breakthrough in gene therapy. 2004. HDAC News Commentary. Online.
    This is a short commentary on the news that RNAi may eventually be able to treat Huntington’s disease.
  7. Miller, et al. Allele-specific silencing of dominant disease genes. 2003. Proceedings of the National Academy of Sciences. Online.
    This is a technical scientific article that explains research using siRNA technology to only silence the dominant allele in diseases similar to HD.
  8. Wexler, Nancy. RNA to our rescue. 2003. Hereditary Disease Foundation 10: 2, 7-9. Online.
    This is a very non-technical explanation of how RNAi may be applied to treat HD and some of the problems with achieving this goal.

-K. Taub, 11/14/04