While there is currently no cure for retinitis pigmentosa, that could change within the next 10 years due to revolutions in CRISPR technology that may allow scientists to fix “broken” genes.
By Todd Farley
Retinitis pigmentosa, an inherited eye disorder that results from genetic anomalies, leads to reduced night vision, loss of peripheral vision, and often blindness. While there is currently no cure, that could change due to revolutions in clustered regularly interspaced short palindromic repeats (CRISPR) technology that may allow scientists to fix “broken” genes.
“Genome surgery is coming,” says Dr. Stephen Tsang, MD, PhD, Laszlo T. Bito Associate Professor of Ophthalmology and associate professor of pathology and cell biology at Columbia University in New York, NY. “And ophthalmology will be the first to see genome surgery, before the rest of medicine.”
Dr. Tsang is confident about CRISPR’s role in the ground-breaking field of genome surgery—as well as ophthalmology’s pivotal role in that endeavor—in large part because of his own study, recently published in the American Academy of Ophthalmology’s journal Ophthalmology.1 In it, Dr. Tsang and his colleagues showed that the gene-editing tool CRISPR allowed them to restore retinal function in mice suffering the effects of retinitis pigmentosa. Equally noteworthy, Dr. Tsang’s study indicated the first instance of successfully using CRISPR technology to treat the more complicated dominant type of the disorder, not the recessive one.
From Farming to Medicine
CRISPR has been around since 2012. The gene-editing tool contains a family of DNA sequences with bits of DNA from various viruses, which bacteria then use to find and eliminate other viruses in a cell. Dr. Tsang describes the technology as “genetic scissors” or a “genetic scalpel.” For example, CRISPR/Cas9 is used primarily to edit a genome by delivering CRISPR-associated protein 9 (Cas9) into a cell along with “guide RNA.” When the RNA arrives at the desired location, the cell can be cut, with some existing genes removed and others added.
“Before CRISPR,” Dr. Tsang explains, “gene-editing consisted of adding a gene to a gene. We were essentially using a gene as a drug in the hopes that the drug would be effective indefinitely. In contrast, CRISPR is truly ‘genome surgery’ in that there’s some cutting involved. We’re doing actual surgery on the patient’s DNA: we can cut out a bad part and paste in a good one.”
It’s in the areas of agriculture and farming where the use of CRISPR is most prevalent, primarily in the production of genetically modified organisms (GMOs), according to Dr. Tsang. “Before CRISPR we could do some gene surgery, but it always left a scar,” he explains. “But this new CRISPR technique is so clean that the USDA doesn’t regulate vegetables that have undergone genome surgery. For example, if the USDA were to perform sequencing on genetically modified tomatoes, they couldn’t tell if the tomatoes had been bred naturally or altered with CRISPR.”
With this advanced technology at his disposal, Dr. Tsang turned his attention to finding a cure for retinitis pigmentosa, a relatively rare condition (affecting approximately 1 in 4000 people) that he calls “one of the most cruel diseases.” Inherited from one’s parents, retinitis pigmentosa is caused by one of 70 genes and results in the destruction or loss of cells in the retina, which are the tissues in the back of the eye that control light sensitivity. Symptoms include difficulty with night vision, loss of peripheral vision (leading to what is called “tunnel vision”), and frequently, total blindness. The symptoms of retinitis pigmentosa normally first appear in childhood and develop over time, progressing inexorably.
Retinitis Pigmentosa
“Retinitis pigmentosa is one of the most feared conditions for patients,” Dr. Tsang says. “There are two kinds of cells in the retina, night-seeing and day-seeing. The initial symptoms of retinitis pigmentosa are caused mostly by genes in the night-seeing cells. By the time people are about 50 years old, the day-seeing cells also die, and center vision can be completely lost. We have patients who say they can’t sleep because they are afraid if they wake up the next morning, either the tunnel will be smaller or their central vision will be gone. Typically, by the time patients are 50, the tunnel will be so small, like a tiny keyhole view, that it’s not useful anymore for vision.”
There are two main forms of retinitis pigmentosa: dominant and recessive. In the autosomal dominant form, a person inherits one copy of a mutated gene and one normal gene; in the autosomal recessive form, on the other hand, two copies of the mutant gene are inherited.
“The dominant form is even more fearsome,” Dr. Tsang explains, “because it is inherited by every generation in the family. Patients may go through college and have careers and children, but they know they’ll end up with vision like their parents or grandparents.”
The dominant form of retinitis pigmentosa proved a greater challenge for Dr. Tsang’s team in terms of treating it with CRISPR technology. With the recessive form, both copies of the gene are mutated, which means it is easier for CRISPR to find and replace the defective DNA. In fact, various companies have been working on curing the recessive form of retinitis pigmentosa with CRISPR and have already produced some positive results.
“In the US, the first CRISPR trial is going to happen in ophthalmology, and it will be for the CEP290 gene, which causes recessive retinitis pigmentosa,” Dr. Tsang predicts. “CEP290 is the low-hanging fruit for the genome-therapy field. There have been six different pharmaceutical companies trying to treat it.”
Dr. Tsang’s study was the first attempt to treat the dominant form of retinitis pigmentosa, which proved particularly challenging because CRISPR had to be used to do more than simply replace an entire cell, as in the case of the recessive form of the disease. Rather, CRISPR needed to be used to edit only the mutant part of the gene while leaving the healthy part unaltered. That was a challenge that Tsang’s team met by designing a “more agile” CRISPR tool that could effectively alter mutations in the rhodopsin gene. Up to 150 mutations in the rhodopsin gene can result in retinitis pigmentosa; and those mutations lead to about 30% of the cases of dominant retinitis pigmentosa.
The Mouse Study
Dr. Tsang designed two guides RNAs (instead of the usual one guide RNA) to help his CRISPR tool find and replace the defective genes of mice.
“Two cuts are better than one: that was the novelty of our approach,” Dr. Tsang says. “The other novelty was that there are some safeties built in; for example, no CRISPR cutting takes place unless there’s a rescue template, or a rescue gene, ready. It’s called ‘ablate and replace’ because there’s no cutting unless they cells are going to be rescued.”
Dr. Tsang’s technique also ensured that what was being added to the genome would not be lost. “CRISPR is genome surgery: we cut out the bad parts and then replace them,” he says, “but we replace them with something that cannot be recognized by those same genetic scissors.”
In the end, using the CRISPR tool with two guide RNAs allowed Dr. Tsang and his team to delete more of the genetic code than with one guide RNA, increasing the likelihood of disrupting the “bad” gene from 30% to 90%. Ultimately, Dr. Tsang’s work helped the mice in his lab to improve their retinal function.
“As cardiologists use an EKG to see how healthy the heart is, ophthalmologists use an ERG, or electroretinogram, to gauge how healthy the retina is” Dr. Tsangs says. “With our CRISPR technique, we were able to show on the ERG that we could restore the mice’s retinal function. And not just the function: we also saw an increased number of healthy cells.”
An added benefit of this new CRISPR technique is that its relative agility allows it to pursue more than one rhodopsin gene mutation, meaning it is able to do “genome surgery” on many of the different variations of the disease that exist.
“We don’t need to design 150 CRISPRs for the rhodopsin gene,” Dr. Tsang says. “The path forward for FDA approval will be easier with a broad treatment for many mutations instead of having to go through the FDA 150 times.”
The flexibility of Dr. Tsang’s new CRISPR technique suggests it might also work on non-dividing adult cells (like those in the eyes, brain, or heart), not just dividing cells, as had previously been the case. That would mean Dr. Tsang’s technique could be used to do genome-therapy on hundreds of inherited diseases, including those such as Marfan syndrome, Huntington’s disease, and corneal dystrophy.
“It would be the same approach: ablate and replace,” Dr. Tsang explains. “If we can prove it works in the eye, we believe the people in neurology for Huntington’s and cardiology for Marfan can apply it as well.”
An Eye Toward Human Trials
With his successful study of CRISPR genome-therapy on mice already completed, Dr. Tsang is looking forward to his next step. “Now that we’ve conducted studies in small animals, we have to show that it’s safe in large animal models,” he says. “Then, the hope is to start human trials in about 3 years.”
Still, Dr. Tsang doesn’t see CRISPR as a panacea for the universe of inherited diseases. “When cells are already dead, you need more of a stem cell approach. Our approach with CRISPR surgery is not to replace dead cells but to improve sick ones.”
Which is not to say Dr. Tsang is anything other than a huge fan of CRISPR. “This is absolutely a revolution in medicine,” he concludes, “and ophthalmology is leading the way.”
REFERENCE
Tsai YT, Wu WH, Lee TT, et al. Clustered regularly interspaced short palindromic repeats-based genome surgery for the treatment of autosomal dominant retinitis pigmentosa. Ophthalmology. 125(9):1421-30.