David SegalProfessor David Segal heads a research laboratory at The Genome Center of the University of California Davis. A main focus of the Segal Lab is designing proteins that can bind to DNA and “turn on” or “turn off” the expression of specific genes. Such DNA binding proteins have the potential to be used in applications such as targeted gene expression therapy for conditions with a known genetic basis. For example this approach might allow people with Angelman Syndrome to make up for the loss or inactivation of the UBE3A gene on the chromosome inherited from the mother, by “turning on” or expressing the UBE3A gene inherited from the father.

Professor Segal recently co-authored a review article in BMC Neuroscience* entitled The prospect of molecular therapy for Angelman syndrome and other monogenic neurologic disorders. The article discusses the use of DNA-binding proteins called artificial transcription factors as potential treatments for Angelman Syndrome.  We asked Professor Segal about the technologies described in the review and the implications they could have for people with Angelman Syndrome.

What is targeted gene expression therapy?

In some types of genetic disease, there are genes that we would either like to turn on or turn off in order to treat the disease. Unfortunately, drugs don’t do this very well. Drugs are good at inhibiting enzymes that carry out chemical reactions in the cell, or binding to receptors on the cell’s surface to block signaling pathways. They usually cannot turn on specific genes. There are also some drugs that act on the general machinery that regulates gene expression. That would be “untargeted” gene expression therapy, because these drugs affect the expression of many genes. My lab believes that if you want to turn specific genes on or off, you should try to understand how nature does this. Nature uses proteins called transcription factors that bind to specific sequences of DNA. In this way, the transcription factors activate or repress just their target gene. We are trying to adopt this same approach to create a “targeted” gene expression therapy for Angelman Syndrome.

Why would Angelman Syndrome be a suitable condition to be treated using targeted gene expression therapy?

Angelman Syndrome is caused by loss of expression of the UBE3A gene in the brain which means that the UBE3A protein it codes for is not made. Regulation of UBE3A expression in the brain is unusually complex and the loss of expression can be due to several different types of mutations or errors that occur where UBE3A is located on the maternal chromosome, the chromosome a child inherits from the mother. However, in all cases there is a perfectly good copy of the UBE3A gene on the paternal chromosome, which is inherited from the father. In the brain this copy is normally “silenced” by the expression of yet another gene, called the UBE3A-antisense transcript. UBE3A and the UBE3A-antisense transcript lie in the path of each other, like two trains heading towards each other on the same track. Usually the UBE3A gene is the loser in this contest, so it doesn’t get to make its protein because its path is blocked by expression of UBE3A-antisense transcript. If we can either turn the UBE3A gene on stronger, so that it is more likely the winner, or if we could turn the UBE3A-antisense transcript down, so that it is more likely the loser, we may be able to restore UBE3A expression in the brain. It is worth mentioning that most other genetic diseases have both copies (maternal and paternal) of the gene mutated, and thus it doesn’t help to turn those genes up or down. In this case, the unusual gene regulation in the region of the UBE3A gene gives us a special opportunity for targeted gene therapy.

The use of artificial transcription factors for targeted gene expression therapy is discussed in your article. What are artificial transcription factors?

Transcription factors are one of the main tools that nature uses to turn genes on or off. They are proteins that are typically composed of two parts. One part is called an effector domain, which interacts with other proteins in the cell to cause the gene to be turned on or off. The other part is a DNA-binding domain, which can seek out and bind to a specific sequence of DNA and thus bring the effector domain to a specific gene. To make an artificial transcription factor, we steal an effector domain of a natural transcription factor and attach it to a programmable DNA-binding domain. A lot of scientific research has gone into how to make high quality programmable DNA-binding domains, and they are improving all the time. They go by names such as zinc fingers, TALEs and CRISPRs. The result is an artificial transcription factor that can turn on or off whatever gene to which we program it, in the case of Angelman Syndrome this is either UBE3A or the UBE3A-antisense transcript.

What advantages do you think they will have over other molecules being used or developed for gene therapy?

This approach should regulate the gene where it naturally occurs on the chromosome, and there may be some advantages to that. There is a lot about gene regulation that we don’t fully understand. For example, the UBE3A gene actually produces at least three slightly different forms of the UBE3A protein depending on variations in the process that converts the gene sequence into the protein it codes for. Although we think they are all doing pretty much the same thing, some forms of UBE3A protein end up in the cell nucleus and others in different parts of the cell. A traditional gene therapy would introduce a new healthy copy of the gene in a viral vector (a sequence of modified virus DNA) that expresses the gene using its own machinery. Using this method only one form of the protein would be made. Our method uses the cells normal machinery and would allow all the natural forms to be made.

Another important lesson we have learned about gene therapy is that dosage matters. Too little gene expression can be bad, but too much can be bad too. For example too much UBE3A expression has been associated with autism. Our approach may offer the ability to tailor the dose of gene expression to the individual, kind of like drug therapies do.

There are other kinds of therapies that target the chromosomal copy of UBE3A or the UBE3A-antisense transcript, such as the antisense oligonucleotide therapy being pioneered by Dr. Arthur Beaudet¹ at the Baylor College of Medicine. That method uses short, man made sequences of nucleic acids, the building blocks of DNA, to block the expression of specific genes by preventing their proteins being made. It may have similar advantages or different advantages to artificial transcription factors. Ultimately, several methods may be useful, or one method may prove to be much more effective than the others, we will just have to see. In any case, testing several strategies ensures that the Angelman community is always the winner, because they will know that whatever proves most useful will be the best that all of us can do, not just the best that one person can do.

¹ Editor’s note: Dr.Beaudet’s antisense oligo approach aims to block the UBE3A-antisense transcript and allow for expression of paternal UBE3A.

Will it be possible to deliver artificial transcription factor therapeutics to specific areas of the brain that are affected in Angelman Syndrome?

Our current understanding is that UBE3A expression is lost throughout the brain in Angelman Syndrome. Our preliminary data suggests our artificial transcription factors can become widely distributed in the brain, much more so than any viral vector in use today. However, delivery remains an active area of research for this project, and we will continue to make improvements and incorporate the improvements of others to do the best we can with this.

Do you think this type of therapy could offer a treatment for all of the symptoms of Angelman Syndrome?

I don’t think anybody really knows if restoring UBE3A to full activity will fix all the symptoms of Angelman Syndrome. About 70% of affected individuals have a large deletion that removes other genes besides UBE3A. Even though there is still one copy of these genes on the paternal chromosome that is active (unlike the paternal UBE3A, which is silenced), the loss of even one copy of these genes from the maternal chromosome may contribute to some of the neurologic symptoms of Angelman Syndrome. Experiments to understand this better are in progress, and this is just one more thing that we still have to learn.

Are there likely to be any side effects to this kind of therapy?

Since our artificial transcription factor does not normally exist in nature, we are always concerned about an immune response against it. This would probably not cause harmful side effects but could reduce its effectiveness. Also, like drugs, there could be “off-target” effects if the factor were to regulate other genes unintentionally. The issue of side effects is something that we really want to understand before we think about any trials in humans.

How long could it take before we know whether they might be suitable as a treatment for humans?

The researchers and the whole Angelman community are lucky to have two very well funded sources of support in the Angelman Syndrome Foundation and the Foundation for Angelman Syndrome Therapeutics. My work is mostly funded by the latter, and they are very serious about moving things quickly into something that can help people. But, to paraphrase Einstein, science should move as quick as possible, but not quicker. We need to better understand side effects and immune response. We need to examine different dosing regimens and delivery methods. Unfortunately these experiments take time to do correctly. So I can’t make any promises about when we will be ready for clinical trials, I can only promise that I will keep trying.

Can you tell us anything else about the research into Angelman Syndrome that your lab has been doing and how it is progressing?

We have been developing artificial transcription factors for targeted gene expression therapy of Ube3a in a mouse model of Angelman Syndrome. The factors are based on DNA binding proteins known as zinc finger proteins which recognize and target specific areas of DNA. The factor was designed to turn the Ube3a -antisense transcript down on the paternal chromosome, which we hoped would restore Ube3a expression in the brain. Our preliminary data suggests that we are able to inject this purified factor into mice and it is indeed able to activate expression of UBE3A in the mice brains. This is a very exciting result for us for several reasons. Most injected proteins as well as many drugs are not able to cross the blood-brain barrier, a membrane of tightly packed cells that protects the brain from foreign substances in the blood. However, our engineered protein seems able to get in. It also seems to activate the Ube3a gene, and by using a special antibody that binds to the area of interest we are fairly convinced that it is the silenced paternal copy that is getting turned on. I say “seems” and “suggests” because we have not published these findings yet, which means our work has not been peer reviewed by other scientists. We want, and the community should also want, that everyone can look at our work and agree that it is true. As a scientist I need to be the biggest skeptic of my own work, and part of that is carefully building proof that what we think is happening is actually happening. We hope to publish the first chapter of our story soon.

However, there will be more chapters because there are still many things we need to learn before we can think about trying to activate UBE3A in a person. We need to understand the potential side effects of our treatment, and if any genes besides UBE3A are affected. We need to know how long the activation lasts after treatment. And of course we need to know what affect this treatment has on the behavior of the Angelman mouse. All those studies are also in progress right now. One of the things that we and others have been learning is that the symptoms that our mouse model of Angelman Syndrome display is a lot milder than what we typically see in people. We have some ideas about why that might be, and with the help of the Foundation for Angelman Syndrome Therapeutics (FAST), we are building better rodent models of the disease. If successful, these new models will help us and the rest of the research community to test drugs and develop new targeted therapies. I think the advances that have been made in just the past few years, Dr. Ben Philpot’s discovery of a Ube3a-activating drug, Dr. Ed Weeber’s gene therapy experiments restoring Ube3a expression in mice, and Dr. Art Beaudet’s antisense termination experiments and oligonuclotide therapy, are all very exciting and certainly seem to offer hope for better treatments. I have certainly been inspired by their work and the work and support of the broader Angelman Syndrome community, and I am glad to think that I might be able to contribute to that.

* Bailus and Segal: The prospect of molecular therapy for Angelman syndrome and other monogenic neurologic disorders. BMC Neuroscience 2014 15:76.