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Treatment Extends Survival in SMA Mouse — Researchers are Optimistic About HDAC Inhibitors

Spinal muscular atrophy (SMA) — a juvenile form of motor neuron disease leading to weakness and wasting of voluntary muscles — is caused by a loss of the SMN1 gene, but even the most severely affected patients retain a functional copy of an almost identical gene, SMN2. In mice, increasing SMN2 expression can compensate for absent SMN1, and investigators have therefore tried to increase expression of existing SMN2. (See “Understanding SMA” for more information about the disorder.)

Hopes have been highest for a class of compounds called histone deacetylase (HDAC) inhibitors, which turn off proteins that shut down gene expression.


Dr. Charlotte Sumner: “It is often the case that the earlier one can intervene, the better. In SMA, we dont test routinely for the gene mutation before the disease manifests, because there is no treatment. But if we demonstrate that earlier intervention would be helpful, then earlier identification would be important.”

In a study reported this month in the Journal of Clinical Investigation, researchers show that a powerful HDAC inhibitor can increase SMN2 expression, ameliorate neuromuscular abnormalities, and improve the clinical phenotype of an SMA mouse model (117:659–671).

“It is well established that there is a correlation between the level of SMN protein and disease severity,” said Charlotte Sumner, MD, assistant professor of neurology at Johns Hopkins University, who conducted the research while working in the NINDS lab of Kenneth Fischbeck, PhD. “That's why there has been such an active effort to increase the level of protein.”

One barrier to increased expression is the cell's own gene regulatory system. In the chromosome, DNA is wound around a core of histone proteins, whose interactions with other histones and non-histone proteins determine how easily the transcription machinery can reach the DNA.

Adding acetyl groups (small two-carbon structures) to histones tends to open the local structure, promoting gene expression, while removing them makes it more compact, and less likely to be transcribed to mRNA. Histones are removed by a group of at least a dozen enzymes, all known as histone deacetylases. When these enzymes are inhibited, SMN2 (and presumably other genes) remains accessible, and is therefore transcribed more often.


The first hint that HDAC inhibitors might affect SMA came in a 2001 study showing that the weak inhibitor phenylbutyrate increased protein levels in patient cell lines and SMA mice (Proc Natl Acad Sci 2001;98:9808–9813).

Because both phenylbutyrate and another weak HDAC inhibitor, valproate, are FDA-approved, clinical trials of these agents started rapidly. In the first trial (Neurology 2007;68:51–55), 12 weeks of phenylbutyrate treatment produced no beneficial effects, although the small number and relatively large age range of the patients may have hidden a modest effect. Other trials are still underway.

Even while these experiments were proceeding, Dr. Sumner wanted to study the phenomenon of HDAC inhibition in the SMA mouse. Mice with a homozygous SMN1 deletion are the offspring of heterozygous carriers, mimicking the SMA genetics in humans. Unlike humans, mice do not normally have an SMN2 gene, and die shortly after birth. Mice with a transgenic copy of the human SMN2 gene lose approximately 20 percent of their anterior horn cells, are extremely weak and underweight, and display other manifestations of disease, as well as die after two weeks.


“We wanted to take a step back to do a proof of concept, with a stronger HDAC inhibitor, to see whether this class of drugs could ameliorate symptoms after disease onset,” Dr. Sumner said. Previous animal studies had shown benefit from HDAC inhibition only before the onset of symptoms.

She chose trichostatin A, one of the oldest and most potent members of the class, though not one currently in clinical use. She found that even a single dose injected into the SMA mouse boosted the level of SMN2 expression, though not enough for an effect on symptoms. But daily doses of trichostatin A, given for eight days after symptom onset, increased SMN2 messenger RNA levels, increased protein levels of both normal and defective SMN, and increased assembly of the small nuclear ribonucleoprotein complexes that SMN forms with RNA.

Treatment also restored a more normal size to anterior horn cells, and increased both total muscle area and myofiber diameter. “It has long been thought that a characteristic of SMA is improper development of the motor unit,” Dr. Sumner said. “We think the drug is promoting better maturation, possibly through increased SMN expression, though perhaps another mechanism may be involved.”

Finally, treated mice lived longer and had better motor function. The median increase in survival was three days, approximately 20 percent of the lifespan in these mice. They walked more normally, were better able to right themselves, and had stronger forearm grip. “We know SMA is a devastating disorder, and to have found a drug that improves function and survival is the most important finding,” said Dr. Sumner.

Dr. Sumner is now planning experiments to test even earlier treatment with trichostatin A. “It is often the case that the earlier one can intervene, the better. In SMA, we don't test routinely for the gene mutation before the disease manifests, because there is no treatment. But if we demonstrate that earlier intervention would be helpful, then earlier identification would be important.”


Other HDAC inhibitors are in development for cancer treatment, and these, rather than trichostatin A, are more likely candidates for human SMA trials in the future. “There is a huge push to develop lots of these compounds,” Dr. Sumner said. “SMA may be a rare disorder, but we may be able to take advantage of that progress in other fields.”


In the chromosome, DNA is wound around a core of histone proteins referred to as chromatin. In areas where chromatin is tightly compacted and condensed, genes are inactive and less likely to be transcribed to mRNA. Where the chromatin is expanded and open, the genes are active. Histones are removed by a group of at least a dozen enzymes – histone deacetylases; when these enzymes are inhibited, SMN2 (and other genes) are accessible and are transcribed more often.

“The most fundamental and worrisome question with this class of drugs has always been its effects on non-target genes,” she continued. “Other genes are being activated. It has been surprising that this class of drugs is as well tolerated as it is, but it is a concern.” And it is likely to remain more of a concern in SMA than in cancer, according to Dr. Sumner, since treatment starts in childhood and is likely to be much longer.

Along with testing existing HDAC inhibitors, another promising avenue is to develop drugs that target one or a few of the dozen histone deacetylases. This will depend on discovering which enzymes are most important in increasing expression of SMN2, which is not yet known.

Finally, Dr. Sumner is careful to say that the mechanism at the heart of this study — HDAC inhibition and increased SMN2 expression — is not the only possible explanation for her results, although it seems the most likely.


Other investigators, who were not involved in the study, were impressed with the results. This study is “a great step forward,” said Chris Lorson, PhD, who studies the molecular basis of SMA. “It does a great job of tying up the molecular activation of HDAC inhibitors, and shows not only an in vitro effect, but a functional effect in an excellent model of the disease.” Dr. Lorson is associate professor in the department of veterinary pathology at the University of Missouri in Columbia.

Kathryn Swoboda, MD, praised Dr. Sumner for the quality of her research, as well as her caution. “She is such a careful investigator, and this is such a beautiful result,” said Dr. Swoboda, associate professor of neurology at the University of Utah School of Medicine. Dr. Swoboda led a trial of valproic acid in human SMA, whose results are due out soon.

“What is most fascinating about HDAC inhibitors, and not just for SMA, is that they are gene therapy without having to put the gene in,” Dr. Swoboda said. She also stressed the importance of early identification and treatment. “No drug is going to bring back neurons in a teenager who has had the disease for a decade. But we have the potential to do newborn screening in SMA, and really make a difference before these children begin to lose neurons.”

The excitement in the SMA community is palpable, she said. “The potential of the HDAC inhibitors has brought a lot of optimism to the community, which has really had nothing before this.” But that optimism, plus the availability of FDA-approved drugs with an HDAC inhibitory effect, has also prompted many families to find doctors who will prescribe these drugs, without knowing whether they work, or at what doses, or in which subset of patients. “The problem is we sometimes can't get enough kids into trials, because of prior use. Everybody is struggling with this issue.”

Nonetheless, SMA parents are solidly behind future trials. According to Dr. Swoboda, a recent trial of carnitine and valproic acid was funded entirely by donations, to the tune of $2.5 million. “Moving forward,” she said, “there is so much for all of us to do. This mechanism might really work, and we need to pursue it.”


Spinal muscular atrophy (SMA) is a juvenile autosomal recessive form of motor neuron disease caused by progressive degeneration of motor neurons in the spinal cord. There are several forms: type 1 in infants is lethal; type 2 starts later and children live longer and may sit, but do not walk; type 3 starts even later — between ages 2 and 17 — but affected children can live decades or more.

Children with SMA are missing the SMN1 gene, but might have a functional SMN2 gene. Both SMN1 and SMN2 encode the same protein, called SMN, for “survival of motor neuron.” The protein binds to RNA in the nucleus to form “small nuclear ribonucleoproteins,” which help splice the messenger RNA of many other genes, in preparation for nuclear export and transcription. It is unknown how loss of SMN protein causes the symptoms of spinal muscular atrophy. But researchers know that when SMN1 is missing or defective, SMN2 cannot fully compensate for its loss. While SMN1 encodes a full-length protein, SMN2 often does not. Instead, most SMN2 transcripts lack a critical exon, and the resulting protein is unstable and is degraded.

Some SMN2 transcripts are complete, and function properly however, and previous studies have shown that simply adding more SMN2 genes completely prevents development of SMA in mice that lack SMN1. But such gene therapy approaches in humans are notoriously difficult. Thus, the goal in humans is to increase output of the existing SMN2 gene, to increase the production of whole copies.


• Avila AM, Burnett BG, Sumner CJ, et al. Trichostatin A increases SMN expression and survival in a mouse model of spinal muscular atrophy. J Clin Invest 2007;117:659–671.
• Mercuri E, et al. Randomized, double-blind, placebo-controlled trial of phenylbutyrate in spinal muscular atrophy. Neurology 2007;68:51–55.
• Chung JG, et al. Treatment of spinal muscular atrophy by sodium butyrate. Proc Natl Acad Sci 2001;98:9808–9813