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RETT SYNDROME RESEARCH TRUST WEBSITE
by Kelly Rae Chi
Rett Syndrome doesn’t usually run in the family. Researchers led by Alessandra Renieri at the University of Siena in Italy encountered two exceptional cases: one pair of sisters with the same mutation in the Rett-causing gene MECP2, and a second pair with identical deletions within the gene.
Prof. Alessandra Renieri
University of Siena, Italy
Despite having the same mutations in MECP2, the sisters represented the clinical spectrum of the disorder. For each pair, one sister had classical Rett Syndrome—she was unable to speak or walk or use her hands—while the other had a milder form of the disorder (called Zapella) and could talk using short phrases, walk and retained some hand function. Researchers describe these four women and possible genetic reasons why the severities of their symptoms were so different, in a PLOS ONE paper published a few months ago.
It’s not surprising that girls with Rett Syndrome generally show a wide range of symptoms. That’s partly because a mutated copy of the MECP2 gene is located on only one of two X chromosomes in a female cell; the other copy is healthy. One X chromosome becomes inactive in each cell early in development. In rare cases when many cells express the healthy copy of MECP2, women show a milder form of Rett. In the new study, however, both pairs of women were similar in how many of their maternally or paternally derived X chromosomes were inactivated, suggesting that something else might explain the differing severities of their disease.
Hypothesizing that other genes could contribute to these differences, the scientists sequenced a small proportion of the women’s genomes (about 1%) that is thought to code for proteins. (This strategy, called exome sequencing, is a less costly and less burdensome in terms of data analysis compared with whole-genome sequencing, and in recent years it has been shown to identify previously unknown genes for rare, inherited disorders, such as Freeman-Sheldon syndrome.)
The team located 112 genetic variants on 108 genes that were exclusive to the women with classical Rett. A subset of these variations, about 10 to 20, is believed to be relevant to impaired protein function in Rett, based on what’s already known about them.
These genes are involved in a range of functions. Interestingly, both women with classic Rett have variants on at least six genes that have been previously linked to oxidative stress. (The two people with Zappella had variants on three.) In a follow-up study, Renieri’s group found that the women with classical Rett, but not the two with Zappella, showed molecular signs of oxidative stress compared with healthy controls. But the link between MECP2 mutations and oxidative stress is still unknown, the authors note.
The women with Zappella had exclusive variants in 80 genes, but none of these were shared by both. Some genes are linked to immune function, and the variants may be involved in protection from a more severe phenotype, says Renieri, a professor of medical genetics.
Although exome sequencing will continue to bear genetic clues on the variability of Rett Syndrome, the meaning of these variants will need further study, Renieri says. “I’m not sure now that all the variants we describe in the paper are relevant,” she admits. “In the next few years we will learn better how to interpret these results.”
Renieri’s group hopes to sequence the exomes of more people with severe and mild Rett Syndrome, to understand their genetic similarities and differences. It is easier to compare the genes of sisters because their genomes are 50% identical. But because sisters with Rett are so rare, they will need to compare unrelated patients, she says.
RETT SYNDROME RESEARCH TRUST WEBSITE
The following piece comes to us from the blog of a UK newspaper, The Independent. This powerful and poignant piece was written by Beth Whitley mother to Hannah who has Rett Syndrome. (3/15/2013)
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Life with Rett Syndrome: ‘When my little girl was diagnosed, I had no concept how much things were going to change’
by Beth Whitley
I lost an old friend this week. Not in the idiomatic sense that he passed away, nor in the literal sense that I misplaced him in a crowded supermarket and never found my way back to him. Although, metaphorically, perhaps that’s exactly what happened: we lost each other in the crowded supermarket of life and by the time we realised we’d gone astray, there were just too many aisles and trolleys and shelves of tinned goods to find our way back. Of course, if I hadn’t been pushing a wheelchair maybe I’d have been able to keep up a bit better.
RETT SYNDROME RESEARCH TRUST WEBSITE
by Kelly Rae Chi
Rett Syndrome is caused by a variety of mutations in the MeCP2 protein, but in some instances, MeCP2’s end is missing. A graduate student in Developmental Biology at the Baylor College of Medicine in Houston, Steven Baker, who is also in the medical scientist training program, was sifting through the clinical literature on boys with such mutations when he noticed that a tiny difference in how much of the protein’s tail is shortened—by just three amino acids—seemed to make the difference between decades of life (albeit with Rett-like deficits) and death in infancy.
Steven Baker
Baker asked his adviser, Huda Zoghbi, whether she thought those extra few amino acids could so dramatically change the clinical progression of Rett.
Huda Zoghbi
“I don’t know,” Zoghbi, Howard Hughes Medical Institute Investigator and director of the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, recalls telling him, because other genetic factors could be contributing the dramatic difference in the progression of Rett. “The only way to know is to make mice that have the two different mutations.”
So they did. One mouse had the end of its MeCP2 cut off at the 270 mark (‘R270X’ mice) while another’s protein was shortened at the 273 mark (‘G273X’ mice). The stories of these mice were reminiscent of the boys Baker had noticed. The R270X mice died prematurely, around the same time as mice with no MeCP2. In contrast, the G273X mice, with their extra three amino acids, survived longer and showed symptoms later, although these features became more severe and the mice died before healthy mice did.
What do those amino acids do? In trying to find out, the scientists have refined their understanding of how MeCP2 works. Their results are published this week in Cell.
A first look at DNA binding:
Researchers know that MeCP2 binds throughout the genome, coating the DNA in neurons more heavily than in other cells, but what exactly it does after that is less clear. It’s thought to turn genes on or off, or alter the overall structure of DNA.
Zoghbi’s team thought that the two truncated forms of MeCP2 might bind to DNA differently — in a way that would help explain the different clinical progressions of the boys — but when they initially looked at several spots within the genome they saw that both forms bound to those spots similarly.
In fact, looking more broadly across the genome the group found that overall binding of the MeCP2 was pretty much the same, and it looked normal. (The latter wasn’t too surprising, though, because the front end of MeCP2 was already known to bind to DNA.) Both mutations also interfered with the normal ability of MeCP2 to repress genes.
Looking more closely at gene expression at various time points in brain development, however, the group found a key difference in the two mutants: at 4 weeks of age, a small group of genes was improperly expressed in the R270X mutant but not in the G273X mutant. Interestingly, most of those genes eventually became misregulated in the G273X group by 9 weeks.
The hook from the animal kingdom:
Meanwhile, the scientists asked evolutionary biologist, Olivier Lichtarge, a Professor at Baylor College of Medicine who uses computational tools to study the evolution of protein sequences, to help them compare the sequences of the protein’s tail across different species (the idea being that any regions of the protein that were highly similar across species might be important for the protein’s function). “We worked with Angela Wilkins in the Lichtarge lab and asked, ‘Is there something in this domain that’s really unique?” says Zoghbi, who is also a Professor at Baylor College of Medicine.
They found that three clusters of MeCP2’s tail were highly conserved across fish, frog, rat, mouse, and human. Shortening MeCP2 at the 273 mark removed the third of those conserved clusters, whereas cutting the tail at the 270 mark deleted the second and third clusters.
What are these clusters in MeCP2’s tail and what do they do? It turns out that they’re called AT-hooks. In 2005, a study by Adrian Bird’s group described the first of those three AT-hooks (which is the one still present in both of Zoghbi’s new models), though its function was unclear.
AT-hooks are regions of a protein that are already well known to bind DNA, however, so the team went back to the idea that the two truncated proteins might differ from one another in how they attached to the genome, even though their initial results had shown that binding was similar. Using a different assay, they found that missing the second AT-hook domain impaired the ability of the R270X to steadily interact with certain sequences in the genome.
The interaction between MeCP2 and DNA:
Our genomes are wound tightly around spool-like proteins called histones; the DNA and histones (together, called chromatin, which looks like beads on a string) are then packed in even more so that it can all fit inside cells. Using an experimental model of compaction in vitro, the team found results suggesting that R270X mice (which, remember, are missing an two AT-hooks instead of one) don’t pack up chromatin as well as 273X mice do.
The initial finding that both mutated forms of MeCP2 bind to DNA are still important though, Zoghbi says. “It tells us that the major binding to DNA happens. That’s the first step.” The researchers think that once the front end of MeCP2 sits on DNA, the AT-hook clusters on its tail come in manipulate the DNA further, likely bending or altering the structure to help it pack further into the cell.
“There were hints previously that MeCP2 might cause a change in the overall conformation of DNA. The new study is probably the most direct evidence,” says Howard Hughes Medical Institute Investigator Gail Mandel, at the Oregon Health and Science University in Portland, who was not involved with the study.
The protein partner ATRX:
When the MeCP2 sits on DNA, and likely alters the way it packs into a cell, there are other molecular partners that come and join it. One of those is the protein ATRX—whose mutations have been linked to Alpha-thalassemia mental retardation syndrome—and Adrian Bird’s lab has previously shown that its interaction with DNA is disrupted in mice missing MeCP2. Zoghbi’s team decided to look at this protein in their new mutant mice.
Compared with the healthy mice and the G273X mice, ATRX goes missing from the tightly packed DNA of neurons earlier in life for the R270X mouse, and this loss mirrors the quicker onset of Rett symptoms. “To us, that was really interesting,” Zoghbi says, “because this change is not because the neurons are sick, it’s because you don’t have MeCP2 functioning properly.”
Studying female mutant mice that are missing a copy of MeCP2, the researchers found those brain cells with no MeCP2 also had less ATRX bound to tightly packed DNA compared to controls. In female mutants with too much MeCP2, an excess of ATRX latched on to DNA. MeCP2’s absence from liver and non-brain organs didn’t affect ATRX binding in those organs, suggesting that MeCP2 has a mechanism that’s specific for the brain.
Future directions:
Taken together, these results suggest that in the brain, the AT-hook clusters on MeCP2’s tail are manipulating DNA in a way that’s crucial for the other protein partners to bind and do their jobs, Zoghbi says.
“This new paper is beginning to shed light on the complexity of this interaction between MeCP2 and ATRX,” says Mandel. In addition, “we don’t know all the other proteins that bind to MeCP2, but the guess would be that there are likely more partners affecting whether genes are on or off.”
Zoghbi’s team hopes to understand how shortening MeCP2’s tail changed chromatin structure without dramatically changing gene expression — as well as the mutation’s affect on brain activity. They also plan to do biochemical and molecular experiments to figure out where ATRX is going and what it’s doing when its distribution is altered in the brain cells of the MeCP2 mutants.
For Zoghbi, the new findings underscore the importance of going back to patients to look for clues about MeCP2’s function. In 1999, Zoghbi first showed that various mutations in MeCP2 caused Rett. “Here we are 14 years later, some of these human mutations are teaching us lessons,” she says. “The variety you get and the breadth of human features you can dissect and go back and study in the mouse are really very humbling.”
RETT SYNDROME RESEARCH TRUST WEBSITE
by Monica Coenraads
This past November in a peaceful New York City suburb, twenty-eight scientists gathered for a three-day meeting organized and sponsored by RSRT.
In the age of email and Skype and webinars and GoToMeeting and a plethora of ways to connect people from across the world with a click of a mouse why does RSRT spend hard-earned money to bring scientists together for face-to-face meetings?
Science Magazine Editor-in-Chief, Bruce Alberts, addresses this question beautifully in a recent editorial. “Part of the answer is that science works best when there is a deep mutual trust and understanding between the collaborators, which is hard to develop from a distance. But most important is the critical role that face-to-face scientific meetings play in stimulating a random collision of ideas and approaches. The best new science occurs when someone combines the knowledge gained by other scientists in non-obvious ways to create a new understanding of how the world works. A successful scientist needs to deeply believe, whatever the problem being tackled, that there is always a better way to approach that problem than the path currently being taken. The scientist is then constantly on the alert for new paths to take in his or her work, which is essential for making breakthroughs. Thus, as much as possible, scientific meetings should be designed to expose the attendees to ways of thinking and techniques that are different from the ones that they already know.”
I’ve organized dozens of scientific meetings since 1999. In recent years I’ve come to favor small, invitation-only meetings on clearly defined topics, hosted in quiet locations far away from distractions. I find that more intimate and focused meetings catalyze deeper discussions and are better equipped to ensure participants of confidentiality, allowing them to share data long before publication, a process that can unfortunately take many months and sometimes years.
The success of a meeting is measured in part by how effectively the exchange of ideas, scientific tools and ensuing projects and collaborations move the field forward. It may take considerable time for the impact of a meeting to be known. Sometimes, however, success is instantaneous, with collaborations initiated before the meeting has even concluded. The concept for the modifier screen currently underway in the lab of Monica Justice, in which we have invested $1.5 MM, was born at a meeting I organized a number of years ago. The MECP2 Consortium evolved from interactions between Gail Mandel, Mike Greenberg and Adrian Bird at our science meetings over the last decade.
As the Rett/MECP2 field has matured, so has the nature of the science meetings. This year we heard a large number of presentations with clinical relevance; that certainly was not the case even a few short years ago. Where will the research take us in the next few years? I can’t wait to find out.
View our Facebook album to see all the pictures from the meeting and to put names to the faces.
Photo credit: Kevin Coloton
by Monica Coenraads
RETT SYNDROME RESEARCH TRUST WEBSITE
For almost 15 years now, I’ve been immersed in the science behind Rett Syndrome. As Executive Director of RSRT I understand that the work is methodical, that good research takes time, that breakthroughs often come after many tiny, incremental steps. And yet, as a mother witnessing my 16-year-old daughter deteriorate a little more each year, I feel a great urgency to push the research harder and faster. All families with intimate, daily experiences of Rett Syndrome’s harsh rule know the longing for their children to be free and well. RSRT is one-hundred-per-cent focused on that ultimate goal – and that’s what guides our choices about where to invest not just our hard-won funds but our hopes and dreams.
2012 gave us reason to be hopeful. We are grateful for the active engagement of our trustees, the unwavering commitment of the families who fundraise for us and the generous contribution of a wide range of people who give their time and talents freely to help us achieve our goal. We wouldn’t be where we are without the unique global partnerships that we enjoy with Rett Syndrome Research Trust UK and the Rett Syndrome Research & Treatment Foundation (Israel), and with national organizations such as GP2C, Kate Foundation, RMRA. Together you have produced an investment in science that will create a better future for our children.
But that future won’t just happen. Before Rett entered my life, I had never given much thought to the drug development process. Like most people, I assumed that academic scientists, industry and government worked together seamlessly to discover effective therapies for the horrible ailments that afflict us. Nothing could be further from the truth.
There is no “Department of Cures.” Laboratory breakthroughs don’t naturally bubble up and become drugs. The reality is that progress must be relentlessly driven, managed, nurtured and prodded, not to mention funded. It’s a messy, difficult and expensive process that can be slowed and derailed by a multitude of hurdles.
Disease-specific organizations such as RSRT cannot afford to be spectators, passively reviewing proposals and granting money. It is incumbent on us to set the research agenda and to facilitate its execution while staying nimble and vigilant to new opportunities.
Two such opportunities would not currently exist without RSRT: reactivating the silent MECP2 on the inactive X chromosome, and gene modifiers. Following the 2007 reversal, RSRT carefully evaluated the state of Rett research and made the decision to champion these explorations before others had even realized they were, in fact, promising approaches.
Will they lead to a cure? Ongoing research and clinical trials will tell. But in the meantime, RSRT will continue to encourage and support the research that holds the greatest promise to truly change our daughter’s lives. For we have the most to win if we succeed, and the most to lose if we fail.
There is no mystery about why a girl suffers from Rett Syndrome. The cause is that mutated copy of the MECP2 gene inhabiting her every cell. But since MECP2 is on the X chromosome and all females have two X’s, beside each mutated gene rests a healthy but silenced twin. What if we could replace the flawed gene with its perfect counterpart?
That’s the question Ben Philpot of the University of North Carolina at Chapel Hill has asked. RSRT has awarded Philpot, Bryan Roth and Terry Magnuson $2.2 million to answer it.
Philpot’s recent paper in Nature describes successful reactivation of the silenced gene in Angelman Syndrome, demonstrating that replacement is possible.
Joining Philpot and Roth in this effort is Terry Magnusson, a world-renowned leader in X-inactivation. The award will fund a team of three full-time post-docs and two technicians.
The goals of the 3-year project include:
- Screening of 24,000 compounds
- Performing whole genome analyses to test for drug specificity to help predict potential side effects (e.g. what other genes might be affected by the drug)
- Identifying the mechanism of MECP2 unsilencing, which will allow the prediction and design of additional therapeutic targets
- Optimizing drug efficacy through medicinal chemistry (e.g. by designing drugs to maximize transit through the blood-brain-barrier while minimizing off-target effects)
- Advancing lead candidates into preclinical trials. The project will be milestone-driven, with a set of pre-established deliverables. This will allow us to monitor progress utilizing a team of advisors with relevant expertise.
Interview with Benjamin Philpot Ph.D. from RSRT on Vimeo.
Along with activating the silent MECP2, RSRT has championed a second exciting approach.
In her Baylor College of Medicine laboratory, Monica Justice set out to identify modifier genes – altered genes able to dampen the ill effects of an MECP2 mutation.
The common belief has been that these genes would be hard to find. The reality? With the screen just 15% complete, Justice has already found five. What she is seeing in mice implies that Rett-like symptoms are unstable, and consequently easier to revert to a normal state than anyone had suspected.
None of the modifier genes can suppress the disease entirely, but each reduces a subset of Rett-like symptoms. While we had originally thought that the modifiers were specific to the central nervous system, it turns out they may operate elsewhere in the body. At least one of the modifiers suggests an alternative therapeutic target, using drugs already FDA-approved. With RSRT funding Justice is now testing the drugs in mice and has a manuscript currently under review. A clinical trial is being explored.
At RSRT we’re excited about will happen once the screen is completed. Justice is likely to find many more modifiers, some of which may point to tractable pathways. In support of this goal, RSRT has committed an additional $800K to the Justice lab, bringing its total commitment to the modifier screen to $1.5 million. This funding should provide sufficient resources to allow Dr. Justice to reach the 50 percent mark in the screen within two years – at which point she will propose a plan to us for completing the project. Many more modifiers await discovery. Further surprises are likely in store.
We have also awarded funding of $720K to the lab of Jonathan Kipnis at the University of Virginia. Kipnis and colleagues hope to gain better understanding of the immune system’s involvement in Rett by analyzing patient blood. The hope is that immune-based therapies can be developed.
Previous work from the Kipnis lab suggested that bone marrow transplants could be beneficial. Before proceeding to clinical trials with a procedure that is extremely serious and risky, RSRT committed funding in 2012 for independent corroboration of these findings.
We are also supporting Huda Zoghbi’s work to explore whether symptoms of the MECP2 Duplication Syndrome can be reversed once the protein level is normalized. $236K was awarded to this project via the MECP2 Duplication Syndrome Fund through the fundraising efforts of the duplication/triplication families.
RSRT is supporting work at John Bissonnette’s lab at OHSU (Oregon Health & Science University) to explore serotonin 1a agonists for their ability to reduce apneas, and Andrew Pieper’s lab at UTSW (University of Texas – Southwestern) for ongoing drug screening.
Finally an award of $65K was made to Dr. Sasha Djukic for her work at the Tri-State Rett Syndrome Center in the Bronx, NY with the majority of the funds coming from the annual Reverse Rett NYC event.
Please join me in wishing all of our scientists Godspeed. I look forward to keeping you apprised of their progress. One last heartfelt thank you to everyone who raises research funds for RSRT. These projects are your money and your effort at work.
Photo credit: Kevin Coloton
RETT SYNDROME RESEARCH TRUST
Kelly Rae Chi
[links to podcasts are below]
That mutations in the MECP2 gene cause Rett Syndrome has been known for over a decade. But what exactly the protein does is not yet clear.
In the early 90s, Adrian Bird’s group purified MeCP2—which stands for methyl-CpG binding protein 2—and named the protein for its ability to bind parts of the DNA with a chemical tag called a methyl. Methyls tend to dampen the expression of genes, suggesting that MeCP2’s function is to silence genes.
Studies published since then suggest MeCP2 activates or represses the expression of many genes. Other results suggest that the protein binds throughout the genome, influencing the way DNA packs into a cell.
New evidence, published today (December 21) in Cell, shows that MeCP2 binds to spots throughout the genome that are tagged with the chemical, 5-hydroxymethylcytosine (5hmC) in mice, and that this interaction may be important for understanding Rett Syndrome.
“[The study is] a very interesting new development in studying the functional significance of MeCP2, which we have to understand if we’re going to understand Rett Syndrome,” says Bird, professor of genetics at the University of Edinburgh in Scotland who was not involved with work.
Abundant in the DNA of certain brain cells, 5hmC seems to be a signpost of sorts for active spots within the genome — that is, the regions that are churning out new protein — the study found. MeCP2’s latching onto these sites supports its potential role as a gene activator, though it’s clear that the case of what MeCP2 does is far from closed.
“Whether or not [MeCP2 is] directly involved in activation is still a matter of further investigation. But we know that it can localize to a region that contains 5hmC and active genes,” says Skirmantas Kriaucionis, group head in the University of Oxford Nuffield Department of Medicine, who co-led the study with Nathaniel Heintz of the Rockefeller University in New York.
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Podcast with Skirmantas Kriaucionis
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Figuring out 5hmC
As a postdoctoral researcher in Heintz’s lab, Kriaucionis found 5hmC in 2009 by accident when he was looking at how a closely related chemical, 5-methylcytosine (5mC), influences genome structure in brain cells. Anjana Rao’s group, then at Harvard Medical School, working independently from Heintz’s lab, confirmed the existence of 5hmC in the same issue of the journal.
Researchers consider 5mC a fifth base, and 5hmC a sixth base, of DNA, which is traditionally thought of as a string of four different chemicals called nucleotides. 5mC and 5hmC resemble the traditional base cytosine, but with a methyl group added on, making 5mC, and a hydroxy group added to the methyl, creating 5hmC. The new study confirmed that patterns of these chemical modifications to the genome are different in each cell and influence which genes are turned on or off and when.
The discovery of 5hmC opened up a new area of work—and hundreds of new papers—focused on where the nucleotide is in the genomes of different cell types, and what it’s doing.
In the new study, using an explorative molecular assay to fish for a binding partner for 5hmC, the group identified the molecule as MeCP2, and nothing else. “It was really a surprise,” Kriaucionis says.
“This paper is the second paper to suggest a candidate binding protein for 5hmC,” says Rao, now a professor of signaling and gene expression research at the La Jolla Institute for Allergy and Immunology, who was not involved with the new study. Other findings have proposed a different molecule, MBD3, as a candidate, but those and the new results need further investigation, she adds.
“So far, both candidates — MBD3 and MeCP2—also bind 5mC, so an exclusive binding protein for 5hmC has not yet emerged,” Rao says.
Indeed, contradicting the new evidence that MeCP2 binds 5hmC and 5mC equally, some previous studies show that MeCP2 much prefers binding to 5mC over 5hmC. For example, a study published earlier this year shows that says MeCP2 is nearly 20 times more likely to bind 5mC than 5hmC.
Relating to Rett
In the new study, Kriaucionis and his colleagues observed that a certain Rett-causing mutation, called R133C—which is responsible for a relatively milder form of the disorder—disrupts MeCP2’s binding to 5hmC.
“[The R133C mutation] is really interesting because it allows us to speculate that MeCP2 binding to 5hmC is important as a part of the function which causes Rett Syndrome,” Kriaucionis says.
The evidence now “strongly suggests” the potential involvement of 5hmC in Rett, says Peng Jin, an associate professor of human genetics who was not involved with the new study. A study his group published last year in Nature Neuroscience found that patterns of 5hmC are altered in mouse models of Rett.
Interestingly, the R133C mutation only slightly dampens MeCP2’s interaction with 5mC, suggesting that MeCP2’s binding with 5mC serves a different purpose than that of MeCP2 and 5hmC.
Other Rett-causing mutations in MeCP2 examined by the group don’t seem to affect binding to 5hmC, meaning 5hmC binding does not fully explain the symptoms of Rett. “It will be important to test further the contribution of 5hmC to Rett Syndrome,” notes Jin, adding that there are mouse models available to do so. Those mutant mice lack the enzymes needed to convert 5mC to 5hmC.
In the new study, researchers studied only a few types of neurons, but there are hundreds of cell types in the brain. Kriaucionis thinks that MeCP2 binds to 5hmC in other cells.
The 5hmC patterns themselves are cell-specific, however, perhaps further complicating the story of MeCP2.
“We really need to get more data to understand whether or not, how different 5hmC and MeCP2 localization would be in different cell types,” Kriaucionis says. “It’s an important component to understand MeCP2 function,” and how scientists might think about future treatment.
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Cell podcast with Nat Heintz (click on Paperclick on right)
RETT SYNDROME RESEARCH TRUST WEBSITE
by Kelly Rae Chi
In September of 2011, RSRT met with the National Institute of Neurological Disorders (NINDS) and other public and private organizations that fund Rett Syndrome research to discuss crucial knowledge gaps in the field. The main findings of the workshop were published recently in Disease Models & Mechanisms.
In particular, the meeting focused on how the research community can improve its chances of success in clinical trials. Preclinical studies require a huge investment of time and effort studying disease in rodent models. Even then, for a variety of reasons, drugs that show promise in preclinical studies will often fail in the clinic.
Here are a few big hurdles in preclinical animal studies — some that are specific to Rett research — and how experts are meeting those challenges.
1. Studying female mouse models of Rett.
“It’s important that when we do a drug trial, that we really impact features that are clinically meaningful, features that are going to impact patients,” Huda Zoghbi of the Baylor College of Medicine in Houston, Texas, told RSRT in a recent interview.
Like girls with Rett, however, female mouse models of the disorder vary in the type and severity of their symptoms, which makes them harder to study than males.
That’s because the gene missing or mutated in Rett, MECP2, is located on the X chromosome. Female mice — which, like girls, have two X chromosomes, only one of which is active — will have either mutated protein or normal protein levels, depending on which copy is expressed in the cell. Rarely are they missing all of their MeCP2 protein.
In contrast, male mouse models missing the Rett gene have no protein at all. Although these mice have paved the way in understanding the protein’s role in the brain, when it comes to treating Rett, results from studies of male mouse models may not be the ideal model to work with.
More researchers are turning to female mouse models. Zoghbi and Rodney Samaco, also at Baylor, for example, published a study in October in Human Molecular Genetics, describing two different female mouse models of Rett in detail. Detailed characterization of these mice will help lay the groundwork for preclinical studies.
2. Unknowns about how an animal’s environment affects therapeutic efficacy.
No two research labs are alike. The ways in which they differ, including animals’ access to food, housing, lighting or other environmental factors, might well influence an animal’s response to a drug.
What’s more, an individual mouse’s genetic environment — meaning the genetic background on which Rett mutations are made — affects some of its symptoms, such as obesity and abnormalities in social behavior. These genetic differences may also affect how animals respond to treatment.
Variability in genetic and environmental conditions plague scientists studying many conditions, not just Rett syndrome. One way to help address this obstacle, according to Rett researchers in the Disease Models & Mechanisms workshop summary, is to study symptoms and potential therapies across a variety of models and in many lab settings. Those mouse models that show consistent results across different environments will be most useful for translational studies.
3. Recapitulating speech problems in mice.
Of the many symptoms seen in Rett, loss of speech is among the most challenging to study in a mouse model. Some groups have shown that Rett mouse pups produce unusual vocalizations when they’re separated from their mothers in early postnatal life. These sounds are either more or less frequent than in healthy controls, depending on the mouse model studied. Future work will need to sort out these conflicting results and identify a mouse model that best captures this hallmark symptom of Rett, researchers say.
4. Avoiding bias, which can prevent preclinical errors.
Unintended biases can creep into animal studies. This can lead researchers to conclude a treatment is effective when it isn’t, or it can cause overestimations of a drug’s efficacy.
In recent years, researchers across numerous fields have stepped up efforts to improve study rigor. In June of this year, NINDS convened a panel of scientists, funders and journal editors to talk about how researchers can do a better job reporting methods in preclinical animal studies; both in grant applications and journal publications. At the very least, the panel concluded in a perspective published October in Nature, researchers should report on the following practices:
- Randomization, where animals are randomly assigned to receive either treatment or placebo;
- Blinding, where researchers doing the experiments or analyzing the data are unaware of whether of which animals are receiving treatment or placebo;
- Sample-size estimation, a calculation of an appropriate sample size at the study’s outset;
- And how data is handled, for example, deciding on study’s primary endpoints, or how to handle missing data points or outliers, before starting the study.
Not reporting such details has, in the past, been linked to overestimations of therapeutic efficacy, according to the NINDS report.
Now Rett researchers have added their voices to the mix in the Disease Models & Mechanisms report, voicing their support of NINDS’s recommendations and emphasizing the need for rigorous experimental design.
by Monica Coenraads
In January of this year a gentleman who has a granddaughter with Rett Syndrome introduced me to his neighbor, David Scheer, a 31-year veteran of the life sciences industry. I was eager to meet David, whose entrepreneurial focus lies at the intersection of finance and science. Our planned hour of conversation turned into a three-hour discussion as we delved into David’s network, experiences, and the potential synergies we might explore. Over the past 9 months I’ve come to rely on David’s insights, perspective and advice. I’m delighted that he has agreed to serve on RSRT’s Professional Advisory Council and look forward to working closely with him on our drug development strategies.
MC: David, please start us off by telling us a bit about your background.
DS: I began with an undergraduate degree in biochemical sciences from Harvard and went on to study cell and molecular biology and pharmacology at Yale. While in graduate school during the early 80’s, I started doing consulting work in the emergent fields of molecular biology and biotechnology. This became a life sciences consulting practice that brought together venture capital, transactional advisory services, and corporate strategy. Scheer & Company is best known for having launched a series of companies spanning the fields of infectious disease, cardiology, oncology, and neurology, to name a few. We’ve had some pretty significant successes among the entrepreneurial enterprises we have launched and built. One of our companies launched a product that was ultimately acquired by Johnson & Johnson; another, a company called Esperion Therapeutics, developed a product in HDL (good cholesterol) therapeutics which was sold to Pfizer in 2004 for $1.3 billion. In the roles of founder and director of numerous companies, I have been involved in identifying technology, recruiting people and raising capital. In most of my more recent companies, I have served as Chairman of the Board.
MC: David, one of your companies, Aegerion Pharmaceutical, operates in the ultra-orphan disease world. Can you tell us a bit about that company?
DS: Aegerion has developed a drug for a very rare disease present in only one in a million people: Homozygous Familial Hypercholesterolemia (HoFH). The disease causes very high bad cholesterol that confers high risk for cardiac events like strokes or heart attacks, even in individuals as young as teenagers, for those who are untreated. We are pursuing approval with both FDA and European regulatory authorities, and we have put in place management and commercial infrastructures in the US and Europe to make the drug available to patients in need, when there is the proverbial regulatory green light. As Chairman of the Board of this company, I have become quite interested in the area of rare diseases.
MC: Lately, pharmaceutical companies seem to all be starting rare disease initiatives. This is in stark contrast to the traditional focus on blockbuster and “me-too” drugs. What is driving this change?
DS: The traditional view has been if there are not enough patients, it’s not really worth developing a drug. However, pioneering companies such as Genzyme, BioMarin, and more recently, Alexion, have successfully developed life-saving drugs to treat rare diseases while providing a return on investment for their shareholders. In so doing, they have opened the eyes of people in the pharmaceutical industry. This shift holds clear potential for organizations working toward the development of therapies for rare diseases, such as the Rett Syndrome Research Trust.
I suspect that rare diseases may also provide an easier path for drug approval. Much of the cost of drug development comes at the end, during the extremely large and expensive clinical trials that are needed for blockbuster drugs. In the rare-disease space, due to the small population sizes, trials will be much smaller, and therefore less expensive. Also, the regulatory process may be fast-tracked for a rare disease, as the FDA recognizes the enormous unmet need and cooperates with sponsors and patient advocates to provide new agents sooner.
So interest in innovation in the rare disease category has turned bullish, which makes this an exciting time for someone engaged in your work, Monica, and frankly also for people like me who are really interested in efficient and effective development of new drugs.
MC: Are large pharmaceutical companies set up to tackle drug development for rare diseases?
DS: I think the answer depends on the drug company. The bulk of the rare-disease experience in drug development has traditionally come from innovative smaller companies. For example, Genzyme, which started off as an idea on the back of an envelope, became a multimillion-dollar pharmaceutical company with a large number of products in its portfolio, and was recently acquired by Sanofi-Aventis. Sanofi now has a franchise that is very capable and active in the rare-disease field. Other companies have either built from scratch, made smaller acquisitions, or are making partnerships or deals with companies that have assets or programs in rare-disease drug development. Pfizer and GlaxoSmithKline have done this, and both now have rare-disease units.
I think it is too early to predict the success of these larger companies. Can the big companies be as effective as some of the smaller companies have been? Will the entrepreneurial spirit of a Genzyme in the early days be retained or lost as part of a larger company? We shall see.
MC: How do FDA drug reviews for a rare disease differ from those for a common one?
DS: The FDA has really had to modify its approaches to adapt to the needs of the rare-disease community. In fact, there is an orphan-disease unit within the FDA that is specifically tasked to review rare-disease drugs. These people are very familiar with how to evaluate a drug through a very different lens than is ordinarily used. It is important to keep in mind that regulatory decisions are always based on a favorable risk-benefit relationship.
MC: The Rett community, including families, clinicians and researchers, is highly concerned—-and properly so—with rigorous validation of pre-clinical advances and the complexities of developing solid protocols for outcome measures. Though Rett patients are a small population, the range of symptoms is staggering, so there are many issues to address.
DS: There are no perfect drugs – even Tylenol can have dreadful side effects if taken in excess, such as liver failure. The FDA must balance the solemn task of making available new and important therapies, while ensuring that such agents demonstrate safety and efficacy commensurate with the condition being treated.
MC: You were only recently introduced to Rett. What have you found most interesting thus far?
DS: That it has attracted some very talented individuals. The scientists, thought-leaders, and patient advocates involved in Rett Syndrome research represent an incredibly impressive group. Perhaps this is because Rett Syndrome is believed to be scientifically very tractable. It certainly helps that it is a single-gene disorder. The number of chronic conditions that can be attributed to a single gene is relatively low. So if I may say this, those in need of therapeutics for Rett Syndrome, may be fortunate in that there is an important foundation for discovery of potentially novel, disease-modifying therapeutics.
MC: I think the 2007 proof-of-concept reversal has also helped put Rett on the map; it had been a much more obscure disorder before that breakthrough.
DS: I agree. Each year in my not-for-profit work, I organize and chair a conference in New Haven, in conjunction with Yale and the Long Wharf Theater, called Global Health and the Arts,. For the past four years, this conference has promoted examination of important disease topics in public health and global health. This past May, we explored the neuropsychiatric disease arena. We had a major scientific symposium, with some of the most well known academic and industrial thought leaders from around the world, who were able to give us an update on relevant areas of science and technology, drug development, genetics, genomics, and translational medicine. In the middle of this event we actually had several individuals comment on the importance of the work being done in Rett and related disorders.
Monica, you need to continue doing what you do so well: ensure you have the best information from scientists at the cutting edge of this field, and then position that knowledge in a way it can be most effectively translatable. The more quickly drug development gurus can bring their expertise to the table, the better the chances of a successful outcome. I am very much looking forward to help you achieve that success.
MC: Thank you, David, and on behalf of every Rett family, welcome.
RETT SYNDROME RESEARCH TRUST
Ever wondered why most labs use male Rett mice for their experiments even though the females are the better model? What human symptoms are replicated in the Rett mice? What are some of the surprises these mice have in store for us? What are the complexities of doing well-designed and executed trials in mice? What are some of the pitfalls that the Rett field needs to avoid? What is the potential for the newly unveiled Rett rat? Listen and find out….
RSRT has an amazing opportunity over the next 2 weeks to show the internet community the importance of Rett Syndrome research. We have been nominated for a $250,000 grant, and need your support to win.
Any Facebook user, anywhere in the world, can vote.
Voting is open from 9/6 through 9/19. This is not a daily vote.
BONUS VOTE: Share a link to the Chase Community Giving app on your FB timeline and if a friend links back to the app and casts a vote of their own you will earn an extra vote.
SPREAD THE WORD: Ask your friends, family, neighbors and co-workers to vote, too!
CHASE CUSTOMERS: You have 2 extra votes. Just go to Chase.com/ChaseGiving, log in, search charities, and cast your ballots!
The charity that gets the most votes over this two-week period will win the money – funds that will be immediately invested in RSRT’s research projects.
It’ll take a few seconds of your time – and could make all the difference.
NOTE: In case of a tie, the winner will be the organization who got the highest number of votes the earliest in the voting period. So…
