Scientists made a surprising discovery in 1985 while studying the African clawed frog. Along the frog’s DNA, they noticed elongated loops 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, these sticky projectiles are part of class of proteins known as “transcription factors”, molecules that are involved in transcribing our DNA into RNA, determining which genes are turned on in each cell type in our bodies.
Now, more than three decades later, scientists are developing zinc fingers as a potential gene therapy to treat amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease. Pfizer has recently joined forces with Sangamo Therapeutics, a California bio-tech company, to deploy their zinc finger technology to possibly develop a therapy targeting the most common inherited form of ALS and a related condition called frontotemporal lobar degeneration (FTLD). Both conditions are caused by a type of mutation on the C9ORF72 gene known as a “repeat expansion disorder,” where a segment of nucleotides repeats excessively—up to hundreds and thousands of times—like a broken record on repeat. Currently there are no therapies to counteract the effect of these mutations.
The discovery of C9orf72 mutations as the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and FTLD has awakened a surge of interest in deciphering how mutations in this mysterious gene cause disease and what can be done to stop it. C9orf72 harbors a hexanucleotide repeat, GGGGCC, in a non-coding region of the gene and a massive expansion of this repeat causes ALS, FTLD, or both (FTLD/ALS).
What’s more, this approach may potentially be used to treat a range of genetic conditions that are also caused by repeat expansions in other genes. “What’s interesting about this collaboration is that it’s exploring an approach to a class of mutations and not just one therapeutic area,” says Christine Bulawa, a Senior Director in Pfizer’s Rare Disease Research Unit, based at the Kendall Square research site 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 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.”
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 and locked in their bodies. More than 90 percent of ALS cases are due to an unknown cause, but about 10 percent are familial or genetic.
Of those familial ALS cases, about one-third are linked to a “repeat expansion” mutation on the C9ORF72 gene that produces a protein that is abundant in the brain and nerve cells. When a specific six-nucleotide segment on this gene is repeated more than 30 times, up to thousands, it causes ALS. Scientists are still uncovering how this mutation leads to ALS symptoms.
Sticking to the Right DNA
Standard 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 normal 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.
The zinc finger transcription factor therapy is an engineered protein that works in two steps. First, the specially engineered zinc fingers can distinguish the mutated from the non-mutated, or wild type nucleotides. The zinc fingers bind to the mutated nucleotides and then a separate protein acts as a repressor that inhibits the DNA from being transcribed into RNA, 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 therapy that can bind to the outside of the mutated nucleotides and then prevent them from being expressed.”
“We are creating zinc finger protein transcription factors to differentiate the mutant from the wild-type allele and then to ‘turn down’ expression of the mutant selectively,” said Michael Holmes, PhD, Vice President of Research at Sangamo.
These “sticky” helping proteins discovered on frogs decades ago may someday help turn off the malfunctioning parts of our genes.