Although the pathology of Huntington’s disease (HD) is still not completely understood, we know that HD is a genetic disorder where the root cause of every HD case is a longer-than-normal series of three repeated DNA base pairs, CAG, in the HD gene. A DNA sequence provides the instructions for the cell to make mRNA (messenger RNA), which in turn contains the instructions for making a protein – the building blocks and machines of cells. The process of using the information contained in genetic material (DNA and RNA) to form protein is called gene expression. Genetic changes in the HD gene sequence are thus propagated into the mRNA sequence and result in production of a mutated version of the huntingtin protein that ultimately results in degeneration of brain cells.
In order to develop a therapy that prevents the production of the mutant huntingtin protein, many scientists are currently using a technology known as gene silencing. This approach creates molecules that directly target and bind to the mRNA copies that contain the instructions for producing huntingtin protein. These molecules then use the cell’s own molecular machinery to destroy the problematic mRNA instructions (for more information about gene silencing, click here).
Gene silencing is a very promising approach, and many scientists are focusing their efforts on conducting studies and clinical trials to assess the feasibility and efficacy of gene silencing treatments for HD and other diseases. However, it has recently become possible to go one step further in manipulating gene expression. Instead of targeting the intermediate mRNA copies as in gene silencing, some scientists are pursuing strategies that will directly modify the DNA blueprint in cells and ultimately living organisms. This article will discuss this nascent technology known as genome editing and its potential as an HD therapeutic.
What is Genome Editing?^
Just as editing text involves adding, removing, or replacing words, genome editing is an approach in which the genome sequence is directly changed by adding, replacing, or removing DNA bases. However, the genome is relatively resistant to change. DNA in the body is not only responsible for encoding all of the necessary functions within a cell, but it is also crucial to determining differences between individuals. If DNA could be easily altered, many essential cell functions would be disrupted in undesirable ways. To deter any changes from being inadvertently made to DNA, cells have inherent mechanisms to proofread and repair their genetic code. These repair mechanisms and the overall stability of DNA is what makes genome editing such a novel approach and so difficult to achieve.
Remarkably, researchers have been able to take advantage of the cell’s DNA repair mechanisms to achieve genome editing. To accomplish this, scientists can use artificially engineered enzymes called nucleases to cleave DNA strands. In effect, these nucleases act as molecular scissors that form a break in the DNA double-stranded helix. Once a break is introduced in the DNA, the cell will detect a problem in its genetic code and quickly activate its repair machinery.
There are two major methods by which a cell can repair a break in its DNA. First, the cell can employ various enzymes to directly join the two ends of the DNA break back together. This process, known as nonhomologous end-joining, is very error-prone and often results in mutations – such as small insertions or deletions of nucleotides – in the resulting DNA strand. These small mutations can be neutral, but they can also render the entire gene in that location nonfunctional, achieving the disruption or knockout of the gene.
Second, the cell can also repair a DNA break by using another DNA sequence as a template. In genome editing applications, a DNA sequence can be designed to be inserted along with a nuclease, such that when a cut is made in the DNA, the cell’s own repair mechanisms can use the DNA sequence supplied to replace an existing DNA sequence as it repairs the break. This method allows scientists to directly change genetic information in cells by introducing a correct version of a DNA sequence to replace an unwanted mutation.
How can a specific gene be edited?^
By using a cell’s own repair mechanisms, scientists can disrupt or correct a mutation by genome editing, and both approaches could prove useful in the context of HD treatment. However, there must be a way to direct a nuclease to the desired location where a DNA break is to be introduced. To address this issue, many different types of nucleases have been developed. All nucleases consist of 2 components – the nuclease itself that is responsible for DNA cleavage and a secondary component responsible for recognizing a specific DNA sequence. There are three main classes of nucleases engineered for genomic editing purposes:
Zinc finger nucleases (ZFNs)^
ZFNs consist of a nuclease component linked to a DNA-binding component derived from an array of zinc finger proteins. Each zinc finger protein can bind three nucleotides, so combinations of zinc fingers linked together can be designed to recognize specific genomic sequences.
Transcription activator-like effector nucleases (TALENs)^
TALENs are very similar to ZFNs in that they also have a nuclease domain linked to a DNA recognition domain. The difference lies in the fact that in TALENs, the DNA recognition domain is a series of amino acid repeats. Each repeat corresponds to a single nucleotide base (A, G, C, or T), and TALENs can be designed to have different combinations of repeats to recognize specific genomic sequences.
The CRISPR/Cas system employs a nuclease called Cas9 to introduce a DNA double strand break. Unlike ZFNs or TALENs, this approach does not use a protein-based DNA recognition domain. In order to guide the Cas9 nuclease to a specific DNA binding site, an RNA sequence is designed to precisely bind to a complementary DNA sequence, allowing for the Cas9 nuclease to make a cut.
Unlike ZFNs or TALENs, each meganuclease has a long recognition sequence that allows them to make DNA breaks at specific sites. However, these long recognition sequences are naturally defined and cannot be engineered, thus meganucleases can only be used for some target genetic sequences.
Can genome editing be used therapeutically?^
Just like gene silencing, genome editing is already being used by scientists as one of their many tools to develop cell and animal models for studying different diseases. Is there a possibility that genome editing can be used in humans to cure genetic diseases like HD? Preliminary research suggests that genome editing may be a promising therapeutic approach, but more work is needed prior to clinical testing in humans.
Research has shown that simply delivering engineered zinc finger proteins (ZFPs) that do not have any nuclease activity is able to reduce the levels of mutant huntingtin. A study published in 2012 by a research group in Spain found that ZFPs can be designed to bind longer CAG repeats more strongly than shorter repeats, which means that these ZFPs could specifically recognize the mutant huntingtin gene with the CAG expansion. They further demonstrated that ZFPs reduced the levels of mutant huntingtin by 95% without affecting the levels of the wild-type huntingtin protein in an in vitro model of HD using mouse cells expressing a human version of the mutant HD gene. Moreover, they were able to demonstrate similar results in an HD mouse model, where ZFP treatment reduced the level of mutant huntingtin up to 60% and motor performance as measured on a rotarod was significantly improved. This study not only demonstrated that ZFPs can specifically bind to the mutant huntingtin gene, but also suggested that ZFPs can accomplish gene silencing by simply binding to the DNA and preventing the gene from being transcribed. These findings support the use of zinc finger nucleases (ZFNs), which could add to the repressive effect of ZFPs by actually disrupting or correcting the mutant gene.
ZFNs have been tested as a therapeutic approach in other diseases. For example, Sangamo Biosciences, a biopharmaceutical company, has explored the potential of ZFNs in treating hemophilia, a genetic disorder in which the ability of the blood to clot is impaired. The researchers used ZFNs to replace the mutated gene responsible for causing hemophilia with a correct gene that allows for normal function in a mouse model of hemophilia, and found that clotting times of the mice returned to normal after treatment. This study suggests that ZFNs are a viable strategy for correcting the genome in genetic diseases such as HD.
Many factors will still need to be considered before genome editing can be used as a viable therapeutic option for HD. Designing nucleases to be specific for one genetic sequence is a difficult and often expensive process, as it requires linking together the right combination of zinc fingers in ZFNs or the right combination of amino acid repeats in TALENs. Moreover, just like in gene silencing, DNA recognition by ZFNs or TALENs is not perfect and can result in off-target effects (binding to the wrong genetic sequence). Genome editing is still in its early stages, and it will be awhile before we will know if it can be used as a gene therapy for HD patients. But, many scientists are pursuing this new avenue of research with promising results.
de Souza, Natalie. (2012) Primer: genome editing with engineered nucleases. Nature Methods. 9: 27.
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotech. 31: 827-832.
Sangamo Biosciences. ZFP nucleases: http://www.sangamo.com/technology/zf-nucleases. Huntington’s disease: http://www.sangamo.com/pipeline/huntingtons-disease.html.
Garriga-Canut¬¬¬ M, Agustin-Pavón C, Hermann F, Sánchez A, Dierssen M, Fillat C, Isalan M. (2012) Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice. Proc Natl Acad Sci USA. 109(45): E3136-E3145.
-J. Choi, 10-24-13