Longtime MDA grantee Michio Hirano, M.D., talks about the science of treating genetic disorders of energy production
On a Thursday afternoon in July 2012, Michio Hirano, a professor of neurology, was in his office at Columbia University Medical Center, high above Manhattan’s upper west side.
|Longtime MDA grantee Michio Hirano, M.D.|
Hirano, a current MDA research grantee who’s had several MDA grants since 1989, co-directs the MDA neuromuscular disease clinic at Columbia and serves on the Association’s Medical Advisory Committee. He specializes in disorders of the mitochondria (singular, mitochondrion), popularly known as the “powerhouses” of cells.
“They’re more dynamic than people think,” he says, becoming animated as he warms to his subject. The same might be said of Hirano himself, whose restrained demeanor conceals an engine of scientific productivity matched by a profound concern for his patients, whose histories he seems to have memorized in detail.
Over the next two hours, Hirano described the mysteries of mitochondria and the cutting-edge strategies being used to fix defects in these vital energy producers.
Mitochondria, Hirano explains, take in fats and carbohydrates from the cells in which they dwell and convert them into ATP (adenosine triphosphate) molecules, the “currency” of cellular energy.
It’s a fairly well-accepted hypothesis that mitochondria were once independent organisms, resembling modern bacteria. Billions of years ago, they merged with more complex cells — the kind animals and humans have now — to form a symbiotic relationship. They produce most of the cell’s energy, and the cell provides critical fuel and specialized proteins to keep them going.
Although mitochondria are ancient microscopic organelles (little organs), human knowledge of them doesn’t even span a century.
“Mitochondrial structure has been recognized since the 1800s,” Hirano says. “’Mito’ means thread, and ‘chondria’ means granules. Under the first microscopes, they looked like thready, granular structures when stained with a dye.”
But it wasn’t until the 1950s that the biochemistry of energy production in mitochondria — a process known as oxidative phosphorylation — was first described. In the early 1960s, another crucial fact about mitochondria became known: They contained DNA. Until that time, all cellular DNA was thought to reside in the cell nucleus.
It was well-accepted that mutations in DNA in the cell nucleus — nuclear DNA — caused genetic disorders. But it would be more than 20 years before people were ready to believe that mutations in mitochondrial DNA could cause a human disorder.
“People used to think it was impossible for mitochondrial DNA to be pathogenic [harmful],” Hirano says. “Until the 1980s, it was believed that mutations in mitochondrial DNA would be so disastrous that they would inevitably be fatal, rather than disease-causing.”
Until the late 1980s, mitochondrial DNA was largely ignored, Hirano says. But then, clinical investigators began discovering disease-related mutations in mitochondrial DNA, resulting in an “explosion” of interest.
“The first point mutation [a DNA error in which one DNA component is substituted for another] in mitochondria was identified in 1988,” Hirano notes, referring to the discovery that such a mutation causes Leber’s hereditary optic neuropathy.
“Now we know about more than 250 point mutations in mitochondrial DNA, as well as deletions [missing pieces of DNA],” he says.
In 1988, Hirano was in a neurology training program (residency) at Columbia University.
He remembers the day that year when he and other neurology residents were introduced to two new patients, a brother and sister, both in their 40s and neither weighing more than 90 pounds — a result of abnormalities of the gastrointestinal tract. They both had peripheral nerve abnormalities, droopy eyelids and unusual features in their muscle biopsy samples that suggested malfunctioning mitochondria.
Their disorder seemed like a mitochondrial defect that had been described the previous year called MNGIE syndrome (for mitochondrial neurogastrointestinal encephalomyopathy, denoting abnormalities in the nervous system, gastrointestinal tract, brain and muscles).
But something didn’t fit. Disorders resulting from mutations in mitochondrial DNA are inherited through the maternal line. (Mitochondria from the egg, but not the sperm, contribute to a developing embryo at conception.) Mothers who transmit mitochondrial disorders often have symptoms of these disorders themselves, although they rarely recognize them as such.
But neither parent of the siblings with suspected MNGIE syndrome had symptoms, suggesting that the disease might be inherited in an autosomal recessive pattern, in which both parents are carriers and children who inherit a mutation from each parent develop the disorder. Autosomal recessive disorders originate in nuclear DNA, not mitochondrial DNA.
MNGIE, it appeared, might be a type of disorder that in 1988 had not yet been recognized — a disease in which mitochondrial malfunction is due to a mutation in a gene in the nucleus of the cell.
Hirano and a small group of colleagues sought out families with MNGIE. “We identified a family in Brooklyn in which four of the eight children were affected,” he recalls. “And then through that family and three others, we were able to map the gene to chromosome 22 [in the nucleus] in 1998.”
It was one of the first mitochondrial diseases to be linked to a nuclear chromosome. (A nuclear gene mutation for Leigh syndrome, identified in 1995, was the first.)
In 1999, in a study funded in part by MDA, Hirano and two colleagues pinpointed mutations in the gene for the thymidine phosphorylase enzyme as the root cause of MNGIE.
“A postdoctoral fellow from Japan found the gene,” Hirano says, graciously giving credit for the discovery to Ichizo Nishino, who was working in his Columbia University lab at the time.
Thymidine phosphorylase is present in most types of cells, where it breaks down thymidine and deoxyuridine. Without this essential enzyme, these two substances build up to toxic levels, poisoning mitochondria.
To make matters worse, thymidine phosphorylase deficiency also results in a shortage of the building blocks of mitochondrial DNA, resulting in mitochondrial DNA deletions (missing pieces of genetic material).
“Patients with MNGIE have less than 10 percent of normal thymidine phosphorylase activity,” Hirano says. The disease usually starts in the late teens, progresses for about two decades and results in death at an average age of 38.
We knew the thymidine phosphorylase enzyme was abundant in platelets [a type of blood cell],” Hirano says. They also knew that platelet transfusions had been used for years to treat other conditions, such as platelet depletion caused by cancer drugs. Why not, Hirano thought, see what platelet transfusions could do for MNGIE patients?
“We gave platelets to two patients,” Hirano says, “and it restored enzyme activity transiently, for two to three days after each infusion.”
The small study, which had MDA support, gave the scientists the needed “proof of principle” that infused platelets could take in thymidine and deoxyuridine from the bloodstream and break them down into harmless byproducts. But clearly a longer-lasting treatment was necessary. Hirano and his colleagues decided to try transplanting stem cells to see if they could provide the needed thymidine phosphorylase for a much longer period of time.
In 2005, Hirano infused stem cells from umbilical cord blood into a young man with MNGIE, hoping they would engraft into his bone marrow and continuously give rise to circulating blood cells, supplying a longer-lasting treatment than infused platelets could provide. Unfortunately, however, the donated cells failed to engraft.
The second individual was a young woman with MNGIE whose brother donated bone marrow stem cells, which, once again, the researchers hoped would incorporate into the bone marrow and supply long-term thymidine phosphorylase.
“She was 5-feet-2 inches tall and weighed 55 pounds,” Hirano says of the first woman to receive stem cells for MNGIE. “We did the transplant in October 2005, and a month later, 60 percent of the blood cells in her peripheral [circulating] blood were from the donor. A year later, it was 100 percent, and the thymidine phoshorylase activity was good.”
Slowly, the young woman began to show improvement. “She got stronger, was able to stop her intravenous feedings and resume eating. She gained five or 10 pounds and even returned to work,” Hirano says, smiling.
“Six-and-a-half years later, she’s doing OK,” he says proudly “She comes to see us every May. She can run and jump now.” So far, she has not needed any further infusions of stem cells.
Since those first two stem cell transplants, 24 more people have undergone the procedure at Columbia University — 10 remain alive, which is encouraging, considering that MNGIE is a fatal disease.
“All had biochemical improvement, and some showed clinical improvements,” Hirano says. “We’ve since held two international meetings at which we developed a protocol that we hope will maximize the safety and efficacy of bone marrow stem cell transplants in MNGIE,” Hirano says. “We’re planning to conduct a study soon to test the new protocol.”
(See a video from the Mayo Clinic called How a Stem Cell Transplant Works.)
MNGIE is an unusual mitochondrial disease, because the missing thymidine phosphorylase protein can be supplied by blood cells. In this situation, the blood cells act like miniature dialysis machines, cleansing the blood of substances that would otherwise build up and become toxic to mitochondria.
But for most mitochondrial disorders (as well as many other genetic conditions), supplying a protein via blood cells won’t help.
To treat most mitochondrial muscle diseases (mitochondrial myopathies) or mitochondrial muscle and brain diseases (mitochondrial encephalomyopathies), a therapeutic gene or protein would have to be delivered to muscle or nerve cells.
One way to get a cell to make a protein it hasn’t been producing is via gene therapy, an approximately 30-year-old science in which new DNA is inserted into cells.
That strategy is being investigated for many genetic diseases in which the defect is in nuclear DNA, including Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy. And, putting a functional gene into the cell nucleus is a viable strategy in mitochondrial diseases that are caused by mutations in nuclear DNA, such as MNGIE, some forms of Leigh syndrome and some forms of progressive external ophthalmoplegia (PEO).
But for some mitochondreal disorders, getting a gene into the nucleus is only part of the challenge. Further steps must be taken in disorders where the defect is in mitochondrial DNA, such as Kearns-Sayre syndrome (KSS), maternally inherited Leigh syndrome (MILS), mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS), myoclonus epilepsy with ragged red fibers (MERRF), neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome and some forms of PEO.
“So far, no one has figured out how to get DNA into human mitochondria,” Hirano says. But in 2002, researchers at Columbia and elsewhere, with partial support from MDA, showed they could use current nuclear gene therapy methods to correct a mitochondrial gene defect.
Knowing that a mutation in a mitochondrial gene called ATPase 6 causes two devastating mitochondrial disorders — MILS and NARP — these investigators reasoned that they might be able to “convert” a functioning mitochondrial gene into a nuclear gene that would produce a protein that could be delivered to mitochondria.
The genetic code in mitochondrial genes is slightly different from that of nuclear genes, so the researchers first had to do the mitochondrial-to-nuclear code conversion. Then, they had to attach an additional code to notify the cell to insert the protein into the mitochondria.
They succeeded in doing both, finding that human cells with a mutation in the mitochondrial gene for ATPase 6 were restored to health by the therapy.
The cells were in a laboratory dish — not in animals or people — but the strategy is sound and could be further developed and applied to other disorders of mitochondrial DNA, Hirano says.
Thus, it may not be necessary to put DNA into mitochondria if mitochondrial genes can be modified and inserted into the nucleus and generate proteins that can be imported into the mitochondria.
For now, Hirano says, most people with mitochondrial diseases can’t be treated with genes or proteins that correct fundamental defects in energy production. However, there are a number of supportive therapies — from cardiac pacemakers and even cardiac transplants, to seizure-controlling medications, to surgeries to lift droopy eyelids, to nutritional supplements like coenzyme Q10 — that help some people.
But Hirano knows that isn’t really enough, and therefore, he’s joined with his colleagues in mitochondrial disease research to create the North American Mitochondrial Disease Consortium (NAMDC). A member of the Rare Diseases Clinical Research Network sponsored by U.S. National Institutes of Health, the network’s ultimate goal is to conduct clinical trials in mitochondrial disease.
But crucial first steps are establishment of a patient registry database, in which information about these disorders will be recorded; and the collection of tissue and blood samples from people with mitochondrial diseases for research purposes.
The registry, which protects personal data from unauthorized access, allows people with mitochondrial disorders to be notified about clinical trials for which they may be eligible and provides periodic updates about research in the field.
To learn more about the NAMDC and the privacy-protected registry, visit the Rare Diseases Clinical Research Network NAMDC registry.
“We need more data,” Hirano says. “We want to revolutionize how we approach these diseases.”