ARTICLE IN BRIEF
Novel antisense oligonucleotides gradually eliminated levels of the mutant protein, glial fibrillary acidic protein, implicated in animal models similar to Alexander disease.
Researchers have successfully used antisense oligonucleotides (ASOs) to block a mutation in experimental mice that causes pathology similar to the genetic defect implicated in Alexander disease, a rare and often fatal leukodystrophy in infants and small children. The findings of the study were published online December 11, 2017 in Annals of Neurology.
ASOs are short, synthetic single-strand nucleic acids — DNA, RNA, or an analog — that bind to and highjack messenger RNA (mRNA) to prevent, reduce, or alter a gene's transcription and expression of a protein.
Several ASOs have shown the ability to target and prevent overproduction of a nerve protein that damages the brain's white matter in Alexander disease (AxD), a progressive disorder that results in imperfect growth or destruction of myelin.
While it can be inherited, AxD is primarily caused by sporadic new mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament protein in astrocytes. When overexpressed, GFAP results in aggregation of abnormal deposits known as Rosenthal fibers, which cripple or destroy astrocytes. About half of all cases occur in infants, who usually die before the age of six.
In the study, the novel ASOs demonstrated the ability to prevent translation of GFAP in wild-type and AxD-mutated mouse models, gradually eliminating downstream levels of the protein. Treatment almost eliminated GFAP in the brains and spinal cords of mice, and resulted in reversal of Rosenthal fiber aggregates as well as markers of microglial and other stress-related responses. Treatment also led to improved body condition and rescue of hippocampal neurogenesis, as well as GFAP clearance from cerebrospinal fluid.
“Our study demonstrates proof of concept,” said senior researcher Albee Messing, VMD, PhD, a professor of neuropathology and director of the Waisman Center at the University of Wisconsin-Madison.
“We believe that GFAP knockdown using antisense technology is a viable approach for treating Alexander disease, but much work remains to be done, including assessing the ability to produce improvements in motor and other behavioral phenotypes,” he told Neurology Today.
That treatment also reduced GFAP in the cerebrospinal fluid of treated animals suggests that GFAP itself could be used as a biomarker to assess ASO efficacy and duration of suppression in patients, Dr. Messing said.
“The results exceeded all of our expectations,” he said. “We are a long way from human application, and I cannot guarantee that this treatment will not cause side effects, but this really is the first positive sign that ASOs might work.”
STUDY METHODS, RESULTS
The researchers evaluated several ASOs for their ability to reduce GFAP mRNA on molecular and cellular phenotypes in cell cultures. They identified 10 that showed superior GFAP mRNA suppression. These were then tested in vivo in two strains of wild-type mice and in mice with elevated GFAP and AxD pathology. A single intracerebroventricular injection of each ASO was delivered into the right lateral ventricle of the animals.
Three ASOs suppressed GFAP transcripts in the AxD mice to nearly undetectable levels in all central nervous system regions tested, with greater than 90 percent GFAP mRNA reduction at two weeks. The brains and spinal cords of the mice showed reduction in GFAP in all regions analyzed, including the hippocampus, olfactory bulb, cerebral cortex, and cervical spinal cord.
In wild-type mice, these ASOs elicited a dose-responsive 77 percent reduction of cortical GFAP mRNA at two weeks compared to untreated control animals. Cortical GFAP mRNA reduction was maximal at 93 percent at four weeks, and persisted to a lesser extent (60 percent) throughout the 16-week trial. GFAP mRNA in the spinal cord was reduced by more than 95 percent, a rate that was also sustained throughout the study.
Other astrocyte transcripts showed signs of recovery within two weeks after injection, and the hippocampus, olfactory bulb, and cortex in ASO-treated mice all showed significant reduction in GFAP promoter activity.
“The rapid clearance of GFAP and other proteins suggests that the protein degradation machinery is not permanently impaired,” the researchers noted.
“Experiments are underway to determine the precise timeline in which GFAP mRNA and protein decrease and return after ASO treatment. It will also be interesting to see if weight loss, a prominent feature of the untreated animals, occurs upon the return of mutant GFAP,” said Dr. Messing.
One limitation with the study is that while GFAP mutant strains of mice show pathological and biochemical changes that closely match AxD in humans, they do not develop changes in white matter signifying leukodystrophy, and do not get as sick as humans. However, Dr. Messing said the researchers are now working with a rat model they hope more closely matches the leukodystrophy in humans.
“I am very optimistic about this research. We have been working to find a way to reduce GFAP since 2003, trying other approaches using different drugs with limited success. We tested lithium, for example, but it was too toxic,” he said.
Another issue is that treatment results in elimination of GFAP, and whether this will be a problem in humans is unknown, he said. “The big question is how much suppression you actually need.”
Several of the authors receive salaries from and/or are shareholders in Ionis Pharmaceuticals, which develops ASOs for various diseases. Dr. Messing and the other authors from the University of Wisconsin-Madison reported no conflicts of interest. The work in Dr. Messing's laboratory was funded by the National Institute of Child Health and Human Development and by the Juanma Fund.
“This is the first targeted therapy for Alexander disease, and the idea that we soon might be able to address this gene defect is very exciting,” said Florian Eichler, MD, assistant professor of neurology at Harvard Medical School and director of the leukodystrophy service at Massachusetts General Hospital, who was not involved with the study.
“This is a devastating disorder, and in all of my years seeing children and adolescents with the disease, there has never been any prospect of therapy,” he told Neurology Today.
Although the mouse models do not develop leukodystrophy, he said, their pathology is similar to that of adult-onset chronic disease and the lack of white matter problems is secondary to the fact that, for the first time, ASOs were able to target and correct the GFAP mutation.
“Moving forward, clinical trials will be necessary to determine not only if ASOs can target the genetic defect in humans, and what dosages work best, but [also] whether changes in areas of the brain, after delivery, are adequate to prevent symptoms,” Dr. Eichler said.
Dr. Eichler is principal investigator for the Starbeam trial, an investigational gene therapy study to stop progression of cerebral adrenoleukodystrophy. Interim results, announced in early October, indicated that the approach could stabilize the disease. He receives grant support from bluebird bio for that ongoing study, and from REGENXBIO for his work on gene therapy for leukodystrophies.
Amy Waldman, MD, FAAN, assistant professor of neurology and medical director of the Leukodystrophy Center at Children's Hospital of Philadelphia, said the US Food and Drug Administration's recent approval of the ASO nusinersen for spinal muscular atrophy provides precedence for the use of ASOs in humans with neurologic diseases.
“The findings offer tremendous hope for individuals with Alexander disease and their families,” she told Neurology Today. “The medical and Alexander disease communities are anxiously awaiting development of human ASOs, and with the encouraging animal data from this study and others, a treatment may one day be available.”
However, she said several concerns with the approach still need to be addressed. “The route of administration for the mice was via an intracerebroventricular injection, and such delivery is not feasible for repeated injections of ASOs in humans. There are other methods, but they would add a layer of complexity. In spinal muscular atrophy, for example, ASOs are delivered via a lumbar puncture,” she explained.
“It remains to be seen whether the circulation of ASOs and penetration to various brain structures after a lumbar puncture will be sufficient to reduce GFAP in astrocytes in the frontal white mater, basal ganglia, and brainstem,” Dr. Waldman added.
Dr. Waldman is the principal investigator for an Alexander disease natural history and outcome measures study, funded by Elise's Corner and Ionis Pharmaceuticals. •
EXPERTS: ON THE POTENTIAL OF AN ASO THERAPY FOR ALEXANDER DISEASE