By Nicole Villas
DSF Board President
DSF hosted its 9th annual Research Roundtable on November 29, 2018, in conjunction with the American Epilepsy Society’s annual meeting in New Orleans, Louisiana. The roundtable has become the highlight of the meeting for many professionals, and we are pleased to see attendance grow each year.
Dr. Jack Parent, Dr. Lori Isom, and Dr. Scott Baraban organized the speakers and moderated the evening, which consisted of 6 presentations, question and answer time, and a brief discussion period. Links to the speakers’ most recent AES poster abstracts, or published literature where appropriate, are included after each description.
Evangelos Kiskinis, PhD, of Northwestern University, received a grant in 2016 from DSF to study patient-specific induced pluripotent stem cells (iPSCs). He presented his work on these cells, which involved creating an “isogenic” control population by editing the patient’s mutation out of their iPSC before differentiating them into different types of neurons and other cells. This allows the team to study a single patient’s mutation in the context of that patient’s particular genetic makeup, called the “genetic background,” a task that is difficult to do in a mouse model of Dravet syndrome. Distinct differences were observed between the healthy control populations and the mutated populations. Using a similar CRISPR/Cas9 editing procedure, they were able to study isolated neurons and how they compromised their ability to interact with control neurons. Dr. Kiskinis also presented the concept of extending his recently published “Optopatch” technique to SCN1A-mutated neurons. The optopatch technique involves stimulating neurons and imaging their responses with light, which could be useful in higher throughput screening of drugs.
Lori Isom, PhD, of the University of Michigan, presented her lab’s work with Stoke Therapeutics’ Targeted Augmentation of Nuclear Gene Output (TANGO) in a Dravet syndrome mouse model. TANGO is a potential disease-modifying approach that increases production of healthy sodium channels by increasing the cell’s editing efficiency of the Scn1a gene transcript. The cell normally destroys a percentage of transcripts after reading the Scn1a gene through nonsense-mediated decay (NMD). TANGO rescues a portion of those transcripts headed for NMD, instead sending them outside of the nucleus to be translated into healthy sodium channels. Because it uses antisense oligonucleotides (ASOs), which contain a specific sequence of nucleotides unique to Scn1a, it selectively upregulates Scn1a without affecting the other sodium channels. This approach has proven extremely successful in increasing Scn1a mRNA and Nav1.1 sodium channels in a Dravet syndrome mouse model to the levels seen in healthy mice. When injected directly into the mouse brain shortly after birth, TANGO rescued 99% of the animals from SUDEP, or sudden unexpected death in epilepsy, which usually occurs in approximately 60% of animals by 35 days after birth. Furthermore, when healthy mice were treated with TANGO, no obvious adverse events were recorded, suggesting TANGO does not over-upregulate Scn1a expression. In an attempt to mimic what might happen in humans, when the disease is not found until after the first spontaneous seizure, Dr. Isom’s team is injecting the mice around two weeks of age (when seizures normally begin in the mice), and preliminary results show that a single dose rescues a subset of the mice. Further studies on dosage, timing, and seizure effect are needed, as well as many other studies before this might be ready for a small test in human patients.
Joseph Sullivan, MD, of the University of California at San Francisco, gave an update on Zogenix’s formulation of fenfluramine, now named “Fintepla.” Two Phase 3 pivotal trials were conducted: one with patients who were not taking stiripentol, and one with patients taking stiripentol. The first study looked at both a low dose and a high dose of fenfluramine, while the second study included a single dose of fenfluramine. In the first study, patients taking the high dose of fenfluramine had a 64% reduction in mean number of monthly convulsive seizures. 75% of patients on high dose experienced at least a 50% reduction in seizures and 45% had more than a 75% reduction, including 25% that were nearly seizure free. Similarly, in the second study patients experienced a 55% reduction in mean monthly seizures, and 54% had at least a 50% reduction in seizures, and 34% had more than a 75% reduction, with 14% being nearly seizure free. Common side effects included reduced appetite, diarrhea, fatique, lethargy, and insomnolence. Because fenfluramine was pulled from the market because of cardiac concerns including valve thickening or valvulopathy, patients in the trials underwent frequent cardiac workup. Echocardiograms performed several times throughout both the blind portion of the trial and the nearly 3 year open label extension revealed no cases of valvulopathy or pulmonary hypertension as of March 2018. Beyond seizures, the caregiver’s global impression of change was “much improved” or “very much improved” in 55% of patients on high dose and 41% of patients on low dose. The Behavioral Rating Inventory of Executive Function (BRIEF) index showed a significant improvement in the high dose group, which could be related to seizure reduction or direct action of fenfluramine itself. Zogenix expects to submit their New Drug Application (NDA) to the FDA in the first quarter of 2019.
Alex Nord, PhD, of the University of California at Davis, received a 2015 DSF Research Grant to study regulatory mechanisms of Scn1a expression in a mouse model of Dravet syndrome. He presented his work, which focused on the functional requirement for one specific regulatory element near Scn1a, named “h1b,” that were discovered in 2007. Selectively deleting this regulatory element affected Scn1a expression in the mice, and deleting both copies of h1b was lethal. These studies have demonstrated the h1b element is required for Scn1a expression in the brain, with studies on how h1b regulates Scn1a expression ongoing.
George Richerson, MD, PhD, of the University of Iowa, presented his work on a respiratory mechanism of SUDEP in a mouse model of Dravet syndrome. The mice, modeled from a human SCN1A truncating mutation, suffer from a high rate of SUDEP, apparently due to seizure-induced respiratory arrest, usually by age 60 days. His team monitored mice during spontaneous seizures and hyperthermic seizure induction and found that breathing stops first, followed by slowed heartrate (bradycardia). In addition, the mice breathe less efficiently for long periods after a seizure, and most SUDEP and nonfatal seizures occur in the mice at night. They often have a flurry of seizures prior to SUDEP, often after a period of relative seizure infrequency. While a ketogenic diet reduced SUDEP in the mice by about 50%, it did not reduce nonfatal seizures, and they breathed neither deeper nor faster. Additionally, the mice have prolonged apneas associated with bradycardia (slow heart rate).
Chad Frasier, PhD, of the University of Michigan, presented his work using cardiomyocytes (individual heart cells) that, when grown in a dish, contract upon external stimuli and are responsible for the repolarization phase and propagation of the action potential. Although SCN5A is the most predominantly expressed sodium channel in the heart, the others (including SCN1A) are present as well. Using cells taken from 4 human patients, they created cardiomyocytes (iPSCs) that are patient-specific. The cells in the dish “beat” synchronously at a significantly faster rate than the controls. They are also prone to beating twice after stimulation instead of just once, like the controls. To double check the reliability of the model, they took one of the healthy control cells and induced a mutation, and when those cells were developed into cardiomyocytes, they exhibited the same abnormalities in sodium current, despite there being no history of cell damage from seizures or Dravet syndrome, confirming that haploinsufficiency (lacking ½ of the healthy SCN1A genes) is the cause of the abnormalities.
Dr. Baraban closed the evening with a brief discussion about the future of Dravet syndrome research and asked the professionals in attendance to consider, as gene therapy science progresses, where might be the best place to test these therapies, and which models might prove most useful.
DSF expresses our sincerest gratitude to Dr. Parent, Dr. Isom, and Dr. Baraban, and thanks the speakers and all of the attendees who took time from their busy AES schedule to dedicate the evening to our Dravet syndrome Community!