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An On/Off Switch for Genes: Meet Zinc Finger Transcription Factors

Scientists made a surprising discovery in 1985 while studying the African clawed frog. Along the frog’s DNA, they noticed elongated protrusions resembling fingers that tightly gripped tiny segments of genes. They soon learned that these “zinc fingers” (so called because they’re bound by a zinc ion) are like homing devices that recognize and bind to specific sites on DNA. Abundant in higher organisms, including humans, these sticky projectiles are part of a class of proteins known as “transcription factors,” molecules that are involved in determining which genes are turned on in each cell type in our bodies.

Now, more than three decades later, Pfizer has joined forces with Sangamo Therapeutics, a genomic medicine company, to deploy the zinc fingers to possibly develop a therapy targeting the most common inherited form of ALS and a related condition called frontotemporal dementia (FTD).

Both conditions are caused by a type of mutation in the C9orf72 gene known as a “repeat expansion” where a segment of nucleotides is repeated excessively—like a broken record. In the case of C9orf72, the repeat is a hexanucleotide, GGGGCC, in a non-coding region of the gene which can be expanded to thousands of times in patients. Deciphering how mutations in this mysterious gene cause disease is an active area of research, and it is clear that the expanded repeat produces a variety of abnormal RNA and protein molecules. However, currently there are no therapies to counteract the effect of these mutations.

The most common genetic cause of ALS/FTD is due to a “GGGGCC” repeat expansion on the C9orf72 gene. (Gitler, A.D. and Tsuiji, H.I., Brain Research, 2016. open access, Creative Commons.

The zinc finger technology has the potential to bind to the repeats and silence the harmful effects of the targeted gene, C9orf72. What’s more, this approach may potentially be used to treat a range of genetic conditions that are also caused by similar repeat expansions in other genes. “What’s interesting about this collaboration is that it’s exploring an approach to a class of mutations and by extension, a group of diseases,” says Christine Bulawa, a Senior Director in Pfizer’s Rare Disease Research Unit in Cambridge, Massachusetts. “We’re targeting a subset of ALS, but if successful, the approach might be applicable to other diseases that are caused by this type of molecular defect at the DNA level—a little string of nucleotides that tends to expand and get longer. The longer they get the worse the disease is.”

ALS

Among the most debilitating conditions is ALS, a degenerative disease caused by the gradual damage and death of the motor neuron cells around the brain and spinal cord. Individuals gradually lose their ability to speak, walk and eat until they’re fully paralyzed. More than 90 percent of ALS cases are sporadic and occur in patients with no prior family history of ALS, but about 10 percent are familial or genetic. Approximately 10% of sporadic and 30% of familial cases are linked to a “repeat expansion” mutation on the C9orf72 gene that produces a protein that is abundant in the brain and nerve cells.

Sticking to the Right DNA

Gene therapies that use viral vectors to deliver a healthy copy of a gene to replace a mutated one won’t work for ALS treatments. That’s because the inheritance is dominant, meaning individuals need to inherit only one copy of the mutated gene to develop the disease. Patients already have a healthy copy of the gene. Instead, scientists are looking to develop therapies that will selectively “knock down”, or debilitate, the mutated gene that is causing the condition. “Our strategy is to mitigate the deleterious effects of mutant form of C9orf72, to reduce the abnormal RNA and protein molecules that are produced from the expanded repeat,” Bulawa says.

With autosomal dominant conditions, offspring only need to inherit one copy of the mutated gene in order to express the disorder.  Source: U.S. National Library of Medicine.

The zinc finger transcription factor uses an engineered protein that has two domains with different functions. The zinc finger domain is specially engineered to recognize GGGGCC and binds preferentially to the expanded repeat. The repressor domain then shuts off the repeat, tamping down synthesis of the abnormal RNA and protein molecules.

“It’s a totally different approach to gene editing. We’re not making any cuts to or removing any of the repeats,” Bulawa says. “We’re basically making a potential therapy that can bind to the mutated nucleotides and then prevent them from being expressed.”

“We are very excited about the potential of our technology in addressing these challenging neurodegenerative diseases. In this case, we designed zinc finger protein transcription factors to selectively and potently repress expression of a mutant allele while allowing the expression of the healthy allele,” said Amy Pooler, PhD, Vice President of Neuroscience at Sangamo.

These “sticky” helping proteins discovered on frogs decades ago may someday help turn off the malfunctioning parts of our genes.

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BY BREAKTHROUGHS STAFF