Hagemann TL, Powers B, Mazur C, Kim A, Wheeler S, Hung G, Swayze E, Messing A. Antisense suppression of glial fibrillary acidic protein as a treatment for Alexander disease. Ann Neurol. 2017 Dec 11. doi: 10.1002/ana.25118. [Epub ahead of print]
Alexander disease is a fatal leukodystrophy caused by autosomal dominant gain-of-function mutations in the gene for glial fibrillary acidic protein (GFAP), an intermediate filament protein primarily expressed in astrocytes of the central nervous system. A key feature of pathogenesis is overexpression and accumulation of GFAP, with formation of characteristic cytoplasmic aggregates known as Rosenthal fibers. Here we investigate whether suppressing GFAP with antisense oligonucleotides could provide a therapeutic strategy for treating Alexander disease.
In this study, we use GFAP mutant mouse models of Alexander disease to test the efficacy of antisense suppression and evaluate the effects on molecular and cellular phenotypes and non-cell-autonomous toxicity. Antisense oligonucleotides were designed to target the murine Gfap transcript, and screened using primary mouse cortical cultures. Lead oligonucleotides were then tested for their ability to reduce GFAP transcripts and protein, first in wild-type mice with normal levels of GFAP, and then in adult mutant mice with established pathology and elevated levels of GFAP.
Nearly complete and long-lasting elimination of GFAP occurred in brain and spinal cord following single bolus intracerebroventricular injections, with a striking reversal of Rosenthal fibers and downstream markers of microglial and other stress-related responses. GFAP protein was also cleared from cerebrospinal fluid, demonstrating its potential utility as a biomarker in future clinical applications. Finally, treatment led to improved body condition and rescue of hippocampal neurogenesis.
These results demonstrate the efficacy of antisense suppression for an astrocyte target, and provide a compelling therapeutic approach for 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.”…
“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….
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…
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….
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.