One bad gene: Harnessing genomic pioneering to save Caroline Fletcher
Caroline Fletcher lay on a plush memory foam pad near the television in her grandparents’ home in Houston’s West University neighborhood, legs folded underneath her tiny torso. Her face was illuminated by the laptop propped open in front of her as she used the tip of her fingers to navigate the screen. At eight years old, she is small for her age, but what she lacks in physical volume she makes up for in spirit.
A few feet away, her twin brother, Henry, jumped from cushion to cushion on the couch until his grandmother told him to stop. Caroline could not join him even if she’d wanted to—the young girl, who’s “brighter than all get out” and “never complains about anything,” according to her grandfather, was unable to walk or effectively use any of her limbs because of a single mistake in her DNA.
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Her condition, a rare form of Charcot-Marie-Tooth disease known as type 2D (CMT-2D), is characterized by peripheral neuropathy—muscle weakness and atrophy in her extremities. (The disease is named for the three physicians who identified it: Jean-Martin Charcot, Pierre Marie and Howard Henry Tooth.) It is caused by a mutation on a gene that is ultimately responsible for sending electrochemical signals to her muscles; as a result, she is unable to stand or walk and has difficulty breathing and swallowing. She is the only known person in the world to have this specific form of the disease, but that hasn’t stopped her grandfather, Stephen Fletcher, D.O., a pediatric neurosurgeon at Children’s Memorial Hermann Hospital, from doing everything in his power to find a cure. After seeking out top experts and pouring hundreds of thousands of dollars into research, he’s getting close. What’s more, the discoveries he’s helped forge may provide relief for hundreds of thousands of patients with rare genetic diseases.
At birth, no one would have suspected that anything was wrong with Caroline’s DNA. She developed alongside her twin brother, but at around eight months old, when she suddenly lost the ability to crawl, Fletcher took her to see one of his colleagues at Memorial Hermann. After an extensive workup, she was initially diagnosed with spinal muscular atrophy, but then her genetic tests came back.
“It turned out she had one abnormality in all of her DNA—one amino acid substitution that nobody knew much about,” said Fletcher, who is also an associate professor of neurosurgery at McGovern Medical School at UTHealth. “Hers was unique because this was the first amino acid substitution on this particular chromosome.”
Because Caroline’s condition was virtually unknown, many of the clinicians Fletcher spoke to questioned whether the abnormality was responsible for her deteriorating muscles. Caroline was sent for a battery of additional tests, but Fletcher grew increasingly impatient. He sifted through academic journals and combed the internet, finally stumbling upon Anthony Antonellis, Ph.D., chair of the department of human genetics at the University of Michigan Medical School and an expert in Charcot-Marie-Tooth genetics. Antonellis, Fletcher believed, could help answer questions about Caroline’s condition.
At first, though, Antonellis didn’t have the time or the personnel to pursue Caroline’s case, so Fletcher asked how much money he would need. Shortly thereafter, an unofficial Caroline Fletcher Research Fund was set up at the University of Michigan.
“We sent her DNA sequence up to him and his specialty is taking yeast and modifying them,” Fletcher said. “What he did was he grew some yeast that had this particular DNA sequence, he put a bunch of neurons and nerve tissue in there, and it killed all of it.”
Antonellis called Fletcher to deliver the news: Caroline’s mutation was toxic.
Armed with an answer—that the amino acid substitution was likely responsible for Caroline’s atrophying muscles—Fletcher forged ahead. He contacted a researcher at The Jackson Laboratory for Mammalian Genetics in Bar Harbor, Maine named Robert Burgess, Ph.D., who had created mouse models with Charcot-Marie-Tooth in an effort to better understand the disease and its process. Like Antonellis, Burgess politely told Fletcher he wouldn’t be much help. After all, what good would it do to create a mouse with Caroline’s Fletcher’s genetic mutation when there was no known treatment?
But Fletcher remained determined.
“I kept hounding the hell out of him,” Fletcher said. “And during that time, I also found a guy in Columbus, Ohio, that had done some stuff with Duchenne muscular dystrophy, where, if you’ve got a gene abnormality, maybe they can either reverse that or block it.”
The Columbus researcher was Scott Harper, Ph.D., a principal investigator at the Center for Gene Therapy at Nationwide Children’s Hospital. Harper was prepared to apologize to Fletcher as well since he didn’t even work with Charcot-Marie-Tooth disease, but he decided that he might as well look into what he was turning down.
That’s when Harper first connected with Burgess, the researcher in Maine. It turned out, the two had grown up about 20 miles apart. They didn’t know each other, even professionally, but after Harper read about Burgess’ work, he realized they might each hold a missing piece to Caroline’s genetic puzzle.
“Scott knew how to do what needed to be done therapeutically, and I had the mouse models and the expertise to do all the pre-clinical studies,” Burgess said. “Individually, I think we were both thinking we only had half the solution there, but … all of a sudden it instantly made sense.”
The abnormality in Caroline Fletcher’s DNA is a dominant mutation on a gene that directs the production of GARS (glycyl-tRNA synthetase), an enzyme essential for the basic function of the nerve cells that stimulate her muscles. Burgess’ lab in Bar Harbor had found that when they overexpressed the healthy GARS gene in their mice, the neuropathy did not improve. Therefore, to address the mutation, they would have to employ a “knockdown” strategy, blocking the negative effects of the mutant GARS and keeping the healthy gene, often referred to as the “wild type,” intact. Notably, GARS is considered an essential gene, so it was critical to protect the one healthy copy.
“With these knockdown strategies, you’re trying to get rid of a toxic gene product as opposed to just replacing a defective gene product,” Burgess said.
First, Burgess and his team created a Caroline Fletcher mouse model in his lab—born with her exact mutation. Then Harper used a technique called RNA interference (RNAi) to harness RNA molecules to inhibit gene expression in the abnormal GARS gene. Known as gene silencing, the approach eliminates the mutant copy and leaves the wild type alone.
As far as the researchers know, this technique—creating an accurate mouse model with a specific human gene mutation, characterizing the animal’s phenotypes, and then using it to test a very specific gene therapy strategy—is unprecedented. And although gene therapy approaches have been used to treat recessive genetic diseases by restoring gene expression, reducing the expression of a toxic dominant mutation with such an approach was largely uncharted territory.
“We’ve essentially created a new gene, a microRNA gene, that can only find her mutant GARS gene, and it cannot find her normal one,” Harper said. “So we’re basically able to eliminate the bad one and leave the good one alone.”
The results were almost unbelievable.
At three-to-four weeks old, mice modeled after Caroline’s DNA exhibited obvious neuropathy—Burgess said they lacked muscle tone and were only able to belly-crawl, tails flopping behind them. But soon after his graduate student treated a “mutant” litter at birth with Harper’s gene-silencing therapy, one of the lab’s technicians went into Burgess’ office to alert him that there must have been a mistake. There was no way, she said, that those mice were mutants.
“That’s when you knew it worked,” Burgess said. “When you can’t tell the treated mice from their healthy littermate controls anymore.”
A more universal approach
Results of the study were published last fall in The Journal of Clinical Investigation, but there are still challenges to translating these findings into a therapy for Caroline.
Members of the Burgess team found that the longer they waited to treat the mice, the more modest the results—which begs the question of whether a person would need to be treated in utero or at birth for this approach to be effective.
“If we do this genetically and just shut off the mutant gene, can you actually regenerate the peripheral nervous system or not—or do we really have to do this early?” Burgess asked. “Right now, we don’t know the answer to that question. … I think that is still the big unknown in what we’ve done so far, just how much promise we can offer to people who’ve had the disease for years.”
For Caroline, it’s no longer a question of functional recovery, but rather preventing her from getting worse, Fletcher said.
“We don’t know what ‘worse’ is, because we don’t know how the disease will progress because nobody knows this disease,” he added.
Still, before Caroline could be treated, Harper’s therapeutic technique would need to undergo extensive additional testing, including safety studies in primates. But that requires funding, and neither the National Institutes of Health (NIH) nor private industry partners are likely to pour millions into a therapy that will help just one person.
“As far as we know, she’s the only person in the world that carries that exact DNA change in GARS,” Burgess said. “So even if we try to move this forward—I think our data says it might work—but just from a funding point of view and an FDA point of view, it’s truly targeting one person. And it’s not that people want to ignore rare diseases. I’ve actually been quite impressed by the rare disease community and also NIH’s commitment to rare diseases, but it’s just a challenge when you’re literally talking about one person.”
Yet like Fletcher, neither Burgess nor Harper were willing to give up.
“We started thinking: Is there a way that we can expand this? Develop a treatment that can affect thousands of people instead of just one?” said Harper.
The two are now collaborating on a more universal approach, one they’re calling “knockdown and replace.”
“Instead of doing this allele-specific silencing, we’re going to try to take out both copies of the GARS gene—the mutant one and the wild type one—at the same time with this RNA interference strategy, and then add back a resistant form of the normal GARS gene. So basically, the gene therapy approach would then be a combination: it would have this knockdown approach, where we’re taking out normal and mutant GARS that’s already present in her cells, and then adding back a normal copy of GARS that’s resistant to the knockdown strategy that we’re using.”
It still makes the normal protein, Harper explained, but the RNA is resistant to the effects of the inhibitor RNA that they are expressing. In theory, the strategy could work for any dominant mutation.
“For any gene, you can start to design these RNAi sequences that are going to knock down the gene, and you can design wild type replacements that are resistant to those RNAi sequences, so conceptually, you can pipeline that,” Burgess said. “It could potentially apply to lots of other diseases where you want to get rid of the bad gene product, but you need some sort of gene product there.”
The two researchers have received funding from NIH to work on developing this more universal approach, which would apply not only to Caroline but also to anyone with any mutation on their GARS gene.
Notably, Burgess’ mouse models have also been integral in research that showed the negative effects of a chronically activated integrated stress response in patients with a GARS mutation.
“The exciting thing about that is that, although nothing is approved yet, there are actually drugs that are being developed to target the integrated stress response,” Burgess said. “So now, in addition to gene therapy, we might actually have a drug pathway.”
A juggling act
Back in Houston, Fletcher continues to help care for his granddaughter when he’s not in the operating room. He and his wife, Julie, bought a house across the street from where Caroline lives with her family (her father, who is media-shy, is Fletcher and Julie’s son), which they renovated to make wheelchair accessible. Often, Caroline will spend the night with her grandparents when she’s sick or having trouble breathing.
Her scoliosis is getting worse, Fletcher said, and her right diaphragm is now almost completely paralyzed. She has a feeding tube, but no matter how many calories she takes in, her frame remains small, her muscles atrophied. Still, she has a rich life. She attends her local elementary school and has a large group of friends—and an incredibly supportive family.
“It’s a juggling act to keep her alive,” Fletcher said. “As a grandparent, I don’t look at it as a burden … you’re going to do everything you can. I don’t get tired of it.”
Meanwhile, Burgess and Harper continue their quest for a cure.