ARTICLE IN BRIEF
Investigators engineered zinc finger protein transcription factors that target the expanded CAG repeats on the mutant huntingtin (Htt) gene and suppress production from the mutant gene by 90 percent in cells derived from HD patients, while reducing the production of normal Htt protein by less than 10 percent.
Scientists at Sangamo BioSciences, Inc. of Richmond, CA, have developed a method of suppressing the production of mutant huntingtin protein (Htt) that causes Huntington's disease without interfering significantly with levels of normal Htt.
Huntington's disease (HD) results from a CAG-trinucleotide repeat expansion within the gene for Htt. Repeat lengths up to 28 CAGs are considered normal. People who carry one normal and one defective copy of the gene for Htt have more than 40 repeats, and produce a mutant form of the protein that devastates neurons, causing chorea, cognitive difficulties and psychiatric symptoms, leading invariably to death, usually within 10- to 20-years.
In rodent models of HD, reducing levels of huntingtin protein — both the normal and mutant protein — through RNA interference and antisense oligonucleotides has delayed or even prevented the onset of symptoms, but since huntingtin protein plays an important although poorly understood role in the functioning of nerve cells, excessive suppression of normal huntingtin could lead to problems. Mice genetically engineered to produce no Htt die before birth.
H. Steve Zhang, PhD, and colleagues at Sangamo have engineered zinc finger protein transcription factors (ZFP TFs) that target the expanded CAG repeats on the mutant huntingtin gene and suppress production of the mutant gene by 90 percent in cells derived from HD patients, while reducing the production of normal Htt protein by less than 10 percent.
METHODS BEHIND REDUCING HTT
At the Society for Neuroscience meeting in New Orleans last month, Dr. Zhang described the method and pointed out the advantages of selectively reducing mutant Htt without significantly reducing normal Htt. “The method that selectively targets mutant transcription would be more desirable because that allows more complete elimination of the mutant protein without having to worry about levels of normal protein dropping below a certain threshold that might be required for normal biological activity,” he said.
Zinc finger proteins, found in every living creature, contain one or more zinc ions that function like “knots” that hold the protein in a characteristic 3-dimensional folded shape. (The “finger” refers to a thumb-shaped loop that appears in drawings of some of the proteins.)
Because these proteins can be engineered to contain “gene activation” or “gene repression” domains, they can interact with DNA sequences to selectively switch genes on or off. This was done for the first time in 1994, when a three-finger protein was used to block expression of an oncogene in a mouse cell line. Since then ZFPs have been used to inhibit the human immunodeficiency virus, disrupt the herpes simplex virus, and activate the expression of vascular endothelial growth factor.
Dr. Zhang and colleagues, in a 2010 paper in The Journal of Neuroscience, described how they alleviated motor symptoms in a rat model of Parkinson's disease by administering engineered ZFP transcription factors to activate the expression of the endogenous glial cell line-derived neurotrophic factor gene.
Dr. Zhang and colleagues plan to deliver their ZFP repressor of Htt with an adeno-associated viral (AAV) vector through direct brain injection. “Safety and some efficacy of AAV-based therapy have been demonstrated in multiple clinical trials for Parkinson's disease,” Dr. Zhang told Neurology Today.
Researchers at the Centre for Genomic Regulation (CRG) in Barcelona have also used an AAV vector to deliver ZFPs into the brain of a mouse model of Huntington's disease. In an article recently published in the Proceedings of the National Academy of Sciences, they reported up to 60 percent reduction of mutant Htt, resulting in delayed onset of symptoms. In a statement released by the CRG, first author Mireia Garriga-Canut, PhD, a researcher at the Gene Network Engineering group at the CRG, said the ZFPs the team designed “recognize and specifically bind to more than 35 repetitions of the CAG triplet, preventing the expression of the gene containing these repeats and reducing the production of the mutant Huntingtin protein.”
DELIVERY CAN BE A CHALLENGE
But at least one HD researcher expressed some concerns about the technique. Delivery remains a significant obstacle to the use of ZFPs to reduce mutant huntingtin protein in Huntington's disease, said Steve Finkbeiner, MD, PhD, professor of neurology and physiology at the Gladstone Institute of Neurological Disease at the University of California, San Francisco, and director of the Taube-Koret Center for Huntington's Disease Research.
“The delivery methods being suggested for antisense oligonucleotides or siRNAs [small interfering RNAs] may be a little more feasible and face fewer safety risks and FDA hurdles than those, such as viral vectors, used for zinc finger nucleases,” he said. “In addition, to be effective, the drug will also probably have to be delivered to both the striatum and cortex. That is a large volume of brain, and depending on the delivery method, the scale of the target tissue could be challenging to access.”
WILL IT BE SAFE IN HUMANS?
Nevertheless, Dr. Zhang said the technique that he and his colleagues at Sangamo have developed shows great promise. “We have, through a rigorous battery of tests, which I presented at the Society for Neuroscience conference, shown that our therapeutic ZFP candidates are not only potent repressors of mutant Htt, but are also highly selective,” said Dr. Zhang. “They repress the mutant Htt gene but not the normal gene. They also are highly specific. In the entire genome only 1 or 2 genes other than Htt were regulated.”
Although techniques that reduce both mutant and normal Htt by about 50 percent have been shown to be safe in rodent HD models, “it remains to be seen whether long-term suppression of normal Htt in humans is safe, and whether partial suppression of mutant Htt levels is sufficient to modify disease progression in humans,” Dr. Zhang added.
“However, because there is currently no disease-modifying therapy for this devastating disease, and the non-allele specific approaches are closest to human trials, having them tested in patients as soon as possible is certainly warranted,” he continued. “Given the well-established link between mutant Htt and HD, and the embryonic lethality caused by total loss of Htt expression, approaches that selectively reduce mutant Htt levels, such as the zinc finger protein transcription factors we are developing, are highly desirable.”
FOR FURTHER READING:
• Laganiere J, Kells AP, Zhang HS, et al. An engineered zinc finger protein activator of the endogenous glial cell line-derived neurotrophic factor gene provides functional neuroprotection in a rat model of Parkinson's disease. J Neurosci
• Stiles DK, Zhang Z, Sah DW, et al. Widespread suppression of huntingtin with convection-enhanced delivery of siRNA. Exp Neurol
• Garriga-Canuta M, Agustín-Pavóna C, Isalan M, et al. Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice. Proc Natl Acad Sci USA 2012;E-pub 2012 Oct. 10.