It’s an exciting time! There is a lot going on in the world of therapeutics for Angelman syndrome. Below, is a chart showing current progress in the work to find therapeutic treatments and a possible cure for Angelman Syndrome and who is involved.
Therapeutics for Angelman Syndrome
There are four broad strategies for therapies—gene therapy, reactivation, pathway intervention and symptomatic treatment. Below, you will find an explanation of each type, how the therapeutic is administered and some pros and cons.
Gene therapy is the process of delivering the missing gene — UBE3A, directly to the appropriate cell type using an engineered virus. This is most typically done using an adeno-associated virus (AAV), which is commonly used and very safe, but can also be done using lentivirus. All of the approaches being considered thus far are using AAV.
How it Works
When AAV is used to deliver a gene, the DNA that encodes UBE3A is put on a circular piece of DNA and packaged into a virus particle, called a capsid. The virus capsid contains very little virus information, but it carries the DNA with UBE3A. The virus capsid finds the cell and infects it (or transduces) and delivers the circular DNA to the cell. The cell can then use the circular UBE3A DNA to produce more RNA and more UBE3A protein. The protein is what is important.
The difficulties about this approach include how do we get an AAV virus capsid to each of the neurons in the brain? How do we get only one virus capsid per cell? Many individual capsids of the AAV can transduce the same cell, and each deliver one circular UBE3A DNA piece. Too much UBE3A is probably a bad thing. Furthermore, the UBE3A DNA is read (i.e. transcribed) using a signal called a promoter, which is also encoded in the DNA circle. This promoter signal determines how much UBE3A protein is ultimately made from each DNA circle. If the promoter is strong, too much UBE3A can be made, and too many circles in once cell can be quite bad. However, if the promoter is too weak, the cells that only got one copy of the circle might not have enough UBE3A. Getting the balance right—the number of circles per cell and the amount of UBE3A made from each circle is tricky. Getting circles to as many cells as possible in the brain is also hard.
This type of therapeutic is currently being pursued by PTC, UPenn, UNC and Bamboo/Pfizer.
Reactivation approaches are finding ways to turn on the father’s copy of UBE3A. Every individual with AS has a perfectly good copy of UBE3A inherited from their father. This copy is silent and does not make UBE3A protein. Paternal UBE3A is silenced because another gene, UBE3A antisense or UBE3A-ATS is made from the opposite direction. Think of two trains on the same track, and they can’t pass. Reactivation strategies try to stop the UBE3A-ATS train to let UBE3A be produced from dad’s chromosome 15. There are a few different approaches to reactivation.
Reactivation using Topoisomerase inhibitors
ASF Funded Research by Ben Philpot (UNC) first showed that it is possible to turn on the paternal copy of Ube3a in mice using topoisomerase inhibitors. These drugs bind to the UBE3A-ATS train tracks and stop the train at many different places. Topoisomerase inhibitors stop other “trains” in the genome as well, so they’re not specific for AS. Topoisomerase inhibitors are chemotherapy drugs, which means that they could cause some undesirable side effects. Some companies may be continuing to work on this approach. There are also potentially other types of small molecule drugs that may reduce UBE3A-ATS and activate UBE3A in ways we do not yet understand.
Reactivation using Antisense oligonucleotides (ASOs)
Antisense oligonucleotides (ASOs) and locked nucleic acids (LNAs) are being pursued currently by Ionis, Roche, and GeneTx/Ultragenyx. These two drugs bind to the RNA specifically using the UBE3A-ATS sequence. They’re very targeted to UBE3A-ATS. They cause machinery in the neuron to cut the RNA, which then leads to the derailing of the UBE3A-ATS train. Crash averted and UBE3A restored! These drugs can’t cross the blood-brain barrier, so they need to be injected into the cerebrospinal fluid (CSF). One dose can last a few months, but treatment will require repeated injections.
Other approaches to Reactivation
shRNAs, miRNAs, ribozymes, and siRNAs are also RNA cutters that may accomplish the same tasks as ASO/LNAs. They can create a cut at a very specific spot in the RNA, which is determined by their sequence. This would derail the UBE3A-ATS train. They make cuts using different machinery in the cell, though. shRNAs, miRNAs, and siRNAs use proteins that exist in the cell to make their cuts. shRNAs and miRNAs can be delivered by putting them onto a circular DNA and packaging them in an AAV capsid, like gene therapy. siRNAs are the final product that is made from shRNAs and miRNAs in the cell. They would need to be delivered by injection into the cerebrospinal fluid, like ASOs.
Ribozymes are RNAs that cut themselves—no extra machinery in the cell is required. Stormy Chamberlain’s lab (UConn Health) in collaboration with Steve Gray and Ryan Butler (UT-Southwestern) is exploring these approaches, but they are in an early stage of study. It’s hard to know whether any of these will work as well as ASOs/LNAs, but if they do, they could provide a one-and-done treatment for AS. Because they only activate paternal UBE3A, there is little chance of making too much UBE3A.
Reactivation using CRISPR/Cas9
CRISPR reactivation makes use of a bacterial protein, Cas9 and a small RNA targeted to the UBE3A-ATS DNA to physically block the UBE3A-ATS train and avert collision. This strategy is being pursued by Mark Zylka’s group. This requires the delivery of the Cas9 protein and RNA in an AAV virus capsid.
One challenge for this approach is that Cas9 is a foreign protein, and it is not known how the immune system will respond to it. There are MANY people globally trying to use CRISPR as a therapeutic approach, so this is being addressed. Another challenge is that the most widely used Cas9 is too big to fit in an AAV vector. This can be overcome with smaller versions of Cas9—there are some that exist. This also would represent a one-and-done, permanent solution to restoring UBE3A expression.
For all of these activation strategies, it must considered how other parts of UBE3A-ATS would be impacted. UBE3A-ATS is at the very end of a very long RNA that makes a lot of other RNAs important for other things, including Prader-Willi syndrome. Some of these RNAs have unknown function, and it’s not clear whether it is okay to reduce the amount of RNAs that are made as well.
There may be ways to address a specific deficit in an Angelman syndrome neuron without knowing exactly how it might be expected to help with AS symptoms. One example of this is OV101, which is being trialed by Ovid Therapeutics.
OV101 improves tonic inhibition in Angelman syndrome neurons. This is known to be a deficit based on ASF-funded studies from the mouse model of AS. OV101 was originally a drug developed to help with sleep issues that was found to be very safe. Because the drug was safe and because it was expected to correct a known deficit in AS neurons, it has been trialed in individuals with AS and is now in phase 3 clinical trials for AS. It is not exactly clear what symptoms are being improved in AS, but physicians are seeing an improvement in individuals on the drug. This is not a cure, but may be a helpful tool in managing multiple symptoms of AS. In time, we will find out whether younger patients treated with OV101 or long-term treatment with OV101 provides even more benefits than have been seen so far.
Future studies by academic labs will continue to look for pathways that are disrupted or specific deficits in mouse or human neurons and seek to find therapeutics that address those specific pathways/deficits.
This approach addresses drugs that address a specific symptom. The best example of this are seizure medicines. Until all seizures are well controlled with a drug with very little, if any, side effects, there will continue to be searches for better seizure drugs.
Currently, Sage Therapeutic’s compound, SAGE-324, is being investigated for seizures and myoclonus.