Saluti da Milano: Highlights from the 1st European STXBP1Summit and Researcher Roundtable

Back in mid-May, the first European STXBP1 Researcher meeting was held in Milan, Italy. The research-focused part of the Summit took up two days while a third day was devoted to family community development programs throughout Europe and family roundtable meetings.

This year’s meeting brought over 100 researchers from around the world together to share and discuss both basic and clinical research about STXBP1.

Day 1 - Morning Talks, Session 1

The first day of the research meeting was focused on ‘STXBP1 In the lab’ with the morning talks addressing mechanisms and models of STXBP1-related disorders (STXBP1-RD) and the afternoon talks tackling the preclinical development of potential therapies. Dr. Matthias Verhage from VU Amsterdam started off the meeting with an overview of the last 30 years of research into the STXBP1 protein, its gene, and its function, and how much we’ve learned over all this time. He introduced a disease concept that helped tie together many of the subsequent talks: Mutations in the STXBP1 gene lead to decreased STXBP1 protein, which in turn causes a decrease in synaptic function, or the ability of neurons to talk to one another. This loss of synaptic function leads to deficits in ‘neuronal circuits’, i.e., groups of neurons that are involved in specific functions in the brain. Dysfunction in multiple neuronal circuits leads to system deficits throughout the entire brain, which we experience as the various symptoms associated with STXBP1-RD.

The next couple of talks built on the disease concept that Dr. Verhage introduced. Dr. Amparo Roig Adam, who works with Dr. Verhage, looked at how cells in the mouse brain cortex change due to Stxbp1 haploinsufficiency (Stxbp1+/-) and found that the numbers and types of cells don’t change but the genes that are turned on or off do, i.e. the genes that they express. Neurons that express the neurotransmitters GABA (GABAergic neurons) or glutamate (glutamatergic neurons) show changes in gene expression and the extent of these changes are different depending on where in the neuronal circuitry of the cortex the neurons are located.

Dr. Jean-Francois Perrier, from the University of Copenhagen, performed electrical recordings in the brains of wild type and Stxbp1+/- mice from neuronal circuits where excitatory neurons (glutamatergic) ‘turn on’ inhibitory neurons (GABAergic), via an ‘AMPA’ receptor on the inhibitory neuron. He found that the excitatory neurons were deficient in turning on the inhibitory neurons in the Stxbp1 mutant mice and this leads to an excitatory/inhibitory imbalance, and that a drug, known as an ‘ampakine’, which enhances the AMPA receptor could partially fix this, thus ampakine drugs could be a potential therapy for STXBP1 related disorder.

Dr Noah Guiberson, from Weill Cornell, discussed his studies in Stxbp1 mutant mice demonstrating that mutant Stxbp1 protein alters the amount and potentially the function of other synapse-associated proteins.

Day 1 - Morning Talks, Session 2

The next set of talks focused on how model systems can help us better understand STXBP1 biology and function, starting with looking at individual neurons. Dr. Jakob Sorensen, from the University of Copenhagen, made neurons with STXBP1 mutations associated with either a severe or mild form of the disease. The neurons with the more severe form mutation had more electrical problems than the neurons with the mild form mutation. Mirriam Öttel, a graduate student in Dr. Verhage’s lab, also looked at glutamatergic neurons generated from patient stem cells. She generated neurons from 6 different patients, with missense, nonsense, or splicing mutations. Unlike Dr. Sorensen, she did not observe any difference in electrical activity in single neurons but did see a change in synaptic activity when looking at a network of neurons connected together. This may indicate that single neurons can compensate for a decrease in STXBP1 but not a network of neurons that work together. Dr. Faye McLeod, of Newcastle University, described how slices of brain tissue can be used to examine neurons and other brain cells in an intact network. When she reduced the amount of STXBP1 in the brain slices she saw a decrease in glutamatergic synapses and a decrease in activity in neurons, but an increase in GABAergic synapses, which also suggests some form of compensation is occurring.

Drs Pierre-Pascal Lenck-Santini, from INMED Marseille, and Klaus Linkenkaer-Hansen, from VU Amsterdam, finished off the morning session discussing a couple of EEG studies. Dr. Lenck-Santini examined Stxbp1+/- mice at ages 4 days to 19 days after birth, which roughly corresponds to a human child at birth to early childhood. He saw intrinsic hyperexcitability (abnormal increased activity) in a region of the brain called the hippocampus in the early day time points, which gradually decreased and recovered by 19 days. In the cortex he looked at evoked EEGs, where he measured what happens to the EEG after touching the mouse’s whiskers, and saw a decreased response at early time points and hyperexcitable responses at later time points. Dr. Linkenkaer-Hansen reviewed his work in understanding the spontaneous EEG activity in STXBP1 children. He found that EEGs from STXBP1 children have a pattern that indicates more inhibition than excitation compared to healthy control children. This type of EEG analysis may one day be able to be used as a biomarker for STXBP1 clinical studies.

Day 1 - Afternoon Talks, Session 1

The afternoon talks on day 1 concentrated more on preclinical studies involving potential therapies or therapeutic approaches. Dr. Lukas Neukomm from the University of Lausanne presented his approach of using fruit flies to screen for drugs that could be used to treat STXBP1-RD. Knocking out the Rop gene, the fly equivalent of STXBP1, in specific neurons located in the fly wing causes the neurons to die, which can be observed (fly wings are mostly transparent). By exposing the fly to 2700 different drug compounds, Dr. Neukomm was able to identify about 40 drugs that stopped the neurons from dying.

Dr. Ben Prosser discussed how his lab, at the University of Pennsylvania, is using antisense oligonucleotides, or ASOs, to interact, not with DNA but with STXBP1 RNA to increase the amount of functional protein made from the RNA. He has also developed a ‘humanized mouse’ or a mouse with a human copy of the STXBP1 gene, that will be very valuable in testing gene therapies, like some ASO therapies, that require interaction with the specific human sequence.  Dr. Prosser also presented some work from Dr. Mike Boland at Columbia University (who was unable to attend the meeting). Dr. Boland has been developing a gene therapy approach called CRISPRa to increase STXBP1 expression in the brain and had had some encouraging results in testing this approach in cortical organoids. Organoids are large 3-dimensional groups of cells that have some common properties with organs, like the brain, and represent a model system between single cells and animals that can be used to test potential drugs and therapeutics.

Dr. Lara Janssen, from VU Amsterdam, finished the first set of afternoon talks with a discussion on ‘poison exons’. These are pieces of DNA that are mistakenly used to make the STXBP1 mRNA. This results in a damaged mRNA which causes the cell to destroy the bad mRNA, and even some normal mRNA as well. Dr. Janssen found 3 potential poison exons in the STXBP1 gene and proposed an ASO therapy that would stop these exons from being incorporated into the mRNA and thus result in more stable RNA and more STXBP1 protein.

Day 1 - Afternoon Talks, Session 2

The second set of afternoon talks began with Dr. Ruud Toonen, from VU Amsterdam, who discussed a strategy to test various therapies to treat STXBP1-RD by taking patient skin biopsies and turning those into neurons which are then used for a variety of different tests to see how drugs or gene therapies improve the functioning of the neurons. Since multiple tests can be performed during a relatively quick time period this approach should help to speed up new drug discovery.

Dr. Mingshan Xue, from Baylor College of Medicine, described his lab’s work on testing AAV-viral vectors expressing STXBP1 in a Stxbp1+/- mice. This gene replacement approach can stop seizure activity and restore several behavioral abnormalities, including motor and cognitive problems, in these mice. He is working with Capsida Biotherapeutics, a biopharma company, and he shared data that new AAV vectors being developed by the company show great promise for eventual use in people. The final talk of the day was provided by Dr. Manuel Alvarez Dolado, from Cabimer, Sevilla, who described how transplanting GABAergic neurons into the brains of STXBP1 mice can reduce seizures and improve locomotion and cognition.

Day 2 - Morning Talks, Session 1

The second day of talks centered on ‘STXBP1 in the clinic’ with the morning talks focused on the clinical spectrum and standard of care for STXBP1-RD and the afternoon talks looking at the future therapeutic landscape for treating and measuring STXBP1-RD. Dr. Hilgo Bruining, from Amsterdam University Medical Center, started us off. He discussed the importance of understanding the clinical symptoms and presentations of STXBP1-RD in each individual patient and optimizing clinical outcome measures. Coupled with this understanding, he also discussed the need to develop clinical trial designs that could be used in the STXBP1-RD population.

Dr. Anna Fetta from the Institute of Neurological Sciences in Bologna talked about sensory processing (i.e. how we process or deal with various senses such as touch, taste, smell, sight, and hearing) in STXBP1-RD kids. She found that many children are ‘passive’ in that they need others to present sensory stimulation to them in order to become engaged and that targeted ‘sensory integration’ therapies could be used to help these kids.

Dr. Jackie Gofshteyn from Encoded Therapeutics gave a summary of the STXBP1 ENGAGE study being conducted by the company. This study is designed to obtain an understanding of how patients feel, function, and survive with STXBP1-RD and what the family journey looks like though the use of individual caregiver interviews and online surveys. Dr. Gofshteyn presented some preliminary data which showed that communication issues, seizures, and movement problems had the greatest impact on children and families and that families face social isolation, financial challenges, and disrupted routines.

The first set of morning talks was concluded with presentations by Dr. Scott Demarest of Colorado Children’s Hospital (COCH) and Dr. Francesca Furia from the University of Southern Denmark. Dr. Demarest gave an overview of the multi-disciplinary clinic at COCH, which as so far seen 13 STXBP1-RD patients, and the development of a specific ‘Clinical Severity Assessment’ for STXBP1-RD. Dr. Furia discussed the development of a European-wide patient registry for STXBP1-RD that will be used to recruit patients into the European STXBP1 Consortium (ESCO) natural history study.

Day 2 - Morning Talks, Session 2

The second round of the morning session began with a talk from Dr. Steffen Syrbe from the University of Heidelberg who discussed their clinic. STXBP1-RD is the fifth most common genetic seizure disorder that they see and appears to be somewhat unique in its multi-seizure type phenotype. He also discussed the potential use of surgery to alleviate seizures in some patients.

Dr. Zach Grinspan, from Weill Cornell, then gave an update on his phenylbutyrate trial. Of the 10 STXBP1-RD children enrolled, 5 are considered responders, 2 were non-responders, and 3 were indeterminant in their response. These kids are now being followed-up for an additional year and will be evaluated for any changes to sleep, communication, and quality of life.

Samuel Pierce, a physical therapist from Children’s Hospital of Philadelphia (CHOP), summarized how several different standardized clinical outcome assessments meant for healthy children can be used to evaluate children with STXBP1-RD; how they can be implemented, how they can be scored, and what potential problems might be expected when using them.

Dr. Megan Abbott, from COCH, gave a talk about cortical visual impairment (CVI) in STXBP1-RD. CVI is visual problems caused by damage or impaired function of the brain and not the eye or the eye nerve. It is characterized by kids who pay attention to bright lights or certain colors like red, who have problems keeping track of visual stimuli, who tend to look at objects using their peripheral or side view instead of head on, and have problems walking on certain surfaces especially changes in surfaces, like from carpet to hardwood floors or stairs. Dr. Abbott discussed how they test for CVI at the COCH clinic and found it appears to be much more common than originally thought, with maybe 50% of kids having some degree of CVI.

Dr. Andrea Miele, also from COCH, finished off the morning session with a discussion of how neuropsychological evaluations are conducted in kids with STXBP1-RD. These are approximately 3-hour sessions that include obtaining background information, testing with standardized assessments, caregiver interviews/questionnaires, and feedback to the caregiver about where their child is on various developmental scales.

Day 2 - Afternoon Talks, Session 1

I started off the afternoon session of day 2 with an overview of potential therapeutic approaches that could be used for treating STXBP1-RD and considerations that impact the use of these approaches. Dr. Elena Gardella from the University of Southern Denmark discussed EEG analysis in STXBP1-RD patients. She showed that EEGs from STXBP1-RD patients have an increase in a type of EEG wave called a delta wave compared to healthy controls or individuals with other epileptic encephalopathies. She also showed that the delta wave increase was greater in the front of the brain compared to the back of the brain. Further, Dr. Gardella found that STXBP1-RD patients could be clustered into two different categories based on their delta- EEG and these correlated with some clinical symptoms.

Dr. Anne Hallemans, from the University of Antwerp, gave a talk about analyzing how people with Dravet syndrome walk. Dravet syndrome is another neurodevelopmental disorder like STXBP1-RD. Walking involves coordination between sensory, motor, and cerebellar systems in the brain. She found that Dravet patients have a unique gate that includes crouching, slow walking speed and short steps and strides. Since changes in how a person walks can be measured, it might be something that could be used in the STXBP1-RD population to test if it changes over time or in response to a therapeutic drug.

The next talk was given by Dr. Emilio Russo from the University Magna Graecia of Catanzaro, Italy. He discussed the role of gut microbiota, i.e. gut bacteria, in autism and epilepsy. There is some form of communication between the brain and the gut/gut microbiota that is only now beginning to be understood. He described that if gut bacteria are transplanted from human autism patients into mice, the mice develop some behavior problems and that the gut microbiota are different in mice with genetic epilepsies and those without. Most intriguing is that there may be a correlation between types of gut bacteria and the effectiveness of a ketogenic diet in controlling epilepsy.

Day 2 - Afternoon Talks, Session 2

The last set of speakers in the afternoon started with three talks centered on natural history studies. Sarah Ruggiero from CHOP outlined the natural history study that the Foundation is sponsoring and that will be carried out at 4 clinical sites, CHOP, COCH, Weill-Cornell, and Baylor College of Medicine. Dr. Ganna Balagura, from the University of Genoa, discussed the ESCO natural history study that will involve clinical sites in 9 different countries. The U.S. study and the ESCO study are working together to make sure that most of the data collected will be comparable between the two countries by aligning the age groups of participants and many of the assessments that will be performed. The hope is that pharma companies will be able to use the data collected by these two studies to help support clinical drug trials. The third talk was given by Dr. Xavier Liogier from the Loulou Foundation, who discussed the CANDID study, a natural history study being conducted in patients with CDKL5 disorder, another monogenic neurodevelopmental disorder similar to STXBP1-RD. This study has just begun but he focused on the problems associated with getting the study funded and running, which is information very important for our own natural history study.

The final talk of the afternoon was given by Julie Xian from Ingo Helbig’s lab at CHOP. She talked about seizure variability in STXBP1-RD and if any predictable pattern could be found that would make it easier to test therapeutics. Using data from about 100 patients, she was able to predict seizure activity in about 2/3 of patients with over 80% accuracy and was able to also identify age ranges where a clinical trial would be most likely be able to find changes in seizures in response to a drug treatment.

This year’s Researcher Roundtable meeting really highlighted the excellent work being done by researchers and clinicians from throughout the US and Europe, how much we have learned about STXBP1 and STXBP1-RD, and what we still need to learn and discover. Next year’s meeting will be back in the U.S. where I’m sure we’ll learn some answers to the questions brought up during this meeting.