Melanie Tallent, PhD - US grants

DESCRIPTION (provided by applicant): AMPA glutamate receptors mediate the majority of fast excitatory neurotransmission in the brain. Four AMPA receptor subunits exist, GluR1-GluR4, with functional channels containing various combinations of these four subunits. In hippocampal and cortical pyramidal neurons, AMPA receptors are comprised largely of GluR1/GluR2 and GluR2/GluR3 heteromeric tetramers. Dysregulation of AMPA receptor expression has been reported in many neurological disorders, including epilepsy. AMPA receptors are alternatively spliced, with the best-characterized splice variants being the flip and flop isoforms. AMPA receptors containing flip vs. flop cassettes have distinct channel properties. GluR1 flip and flop variants have similar kinetics, but the flip isoform shows greater sensitivity to glutamate, increasing synaptic gain. Expression of flip channels is associated with greater vulnerability to excitotoxicity and hyperexcitability. Increases in GluR1 flip to flop ratio in hippocampus and cortex are found in epileptic tissue in humans and animal models and may contribute to development of epilepsy (epileptogenesis) and seizure susceptibility. Thus normalizing aberrant splicing represents a novel therapeutic strategy for preventing epileptogenesis. Splice modulating oligonucleotides (SMOs) have unique chemistries and distinct advantages over classic antisense oligonucleotides and siRNA, and are in clinical trials for treating muscular dystrophy and spinal muscular atrophy. We have developed an SMO that specifically and potently reduces GluR1 flip in vivo. We have also shown that knockdown of GluR1 flip with its selective SMO protects against seizures in a neonatal epilepsy model and can prevent post-seizure hyperexcitability associated with epileptogenesis. The goals of this application are to continue to test our novel SMO in mouse models of epileptogenesis in both neonates and adults, to determine if it protects against development of chronic seizures and their deleterious cognitive comorbidities. Our studies are the first to target modulation of alternative splicing as a therapeutic approach for preventing the development of epilepsy, a critical unmet need. Further, our approach represents a new platform technology in epilepsy therapeutics that is applicable to many additional gene targets. PUBLIC HEALTH RELEVANCE: In spite of the recent large increase in the number of drugs available to treat epilepsy, there are no drugs in clinical use that prevent the development of this disease in vulnerable populations. We have developed a novel compound that acts at the level of gene transcription to normalize excitability in the epileptic brain and prevents changes in the brain associated with the development of epilepsy. Our proposed research is designed to validate this compound as a drug for preventing epilepsy in both infants and adults.

? DESCRIPTION (provided by applicant): Dravet Spectrum disorders resulting from SCN1A loss-of-function mutations include febrile seizures, generalized epilepsy with febrile seizure plus (GEFS+), and Dravet syndrome (severe myoclonic epilepsy of infancy or SMEI), in order of increasing severity. In the most severe cases, progressive developmental and behavioral impairments manifest along with the recurrent and varied seizure episodes that advance to include multiple seizure types by age 2. In many patients, seizures are resistant to currently available antiepileptic drugs. Thus, there is a significant and urgent need for the development of novel approaches to therapy. SCN1 channel function in inhibition is functionally opposed by the related SCN8 channel. Thus, a validated and logical strategy for rebalancing the deficit of inhibitory input caused by SCN1A loss-of-function mutation is to specifically reduce SCN8-mediated excitation. VGS channel alpha subunits undergo several alternative splicing events which regulate the inhibitory and excitatory balance of sodium currents in the CNS. SCN8A subunits are naturally alternatively spliced at two specific sites of interest which control channe function or kinetics. A novel strategy to reduce SCN8A- mediated excitation and seizures associated with SCN1A loss-of-function mutations as a treatment for DS is to direct splicing at each of these sites by developing compounds called splice modulating oligonucleotides (SMOs). SMOs are a class of synthetic RNA based compounds that sterically block or weaken interactions between elements of the splice machinery and the pre-mRNA. SMOs are ideal but under-developed drug candidates as they bind to their targets with exceptional potency, specificity, and negligible off-target effects. SMOs targeting SCN8A splicing will designed in silico and refined for potency and specificity in vivo (Aim 1) leading to an SMO drug candidate for each splice site. Each SMO candidate will be characterized by dose-response in normal mice at P10-42 days, and SMO dose-effect on flurothyl-induced seizure threshold evaluated in normal mice (Aim 2). Finally, SMOs will be evaluated as therapeutics in SNN1A R1648H mutant mice (Aim 3), for effects on longevity, motor function (ataxia, tremor) and flurothyl-induced seizure susceptibility. Safety and efficacy of SMO treatment will further be assessed for both positive and adverse effects on behavior in adult heterozygous SCN1A R1648H mice, which have a normal lifespan. The strategy of specifically reducing only the Na+ channel (SCN8A) that counterbalances SCN1A input should be more efficacious and be much less likely to cause unwanted effects than using sodium channel blockers which antagonize multiple VGS channels. The ultimate goal is to develop these SMOs as potential therapeutic for the treatment of Dravet Syndrome and related disorders in patients resistant to currently available pharmacotherapies.

Project Summary/Abstract Alzheimer`s Disease (AD), is a severe neurodegenerative disorder characterized by progressive memory loss, language deficits, personality changes, and dementia, most prevalent in patents over 65. Current treatments only have modest and temporary effects on cognitive decline, highlighting the need for novel treatments which can halt the progression of AD. While familiar AD is caused by autosomal dominant mutations in amyloid precursor protein and presenilin, the majority of AD cases are considered sporadic with a combination of both genetic and environmental risk factors. However, amyloid-beta (A?) plaques, hyper-phosphorylated tau neurofibrillary tangles (NTs), and severe gliosis are all neurodegenerative hallmarks common to all forms of AD. The receptor for advanced glycation end-products (RAGE) is upregulated in the brains of AD patients and interacts directly with A? in neuronal, immune and vascular cells. RAGE signaling can also induce hyper-phosphorylation of tau and is implicated in the pathogenesis of co-morbid risk factors for sporadic AD (e.g. cardiovascular disease and diabetes). Thus, RAGE is a prime target for therapeutic development in both familial and sporadic AD. The RAGE pre-mRNA yields mutually exclusive alternative splice variants that are either, membrane- bound and capable of signaling (mbRAGE), or truncated and endogenously secreted to clear RAGE ligands without signaling (esRAGE). In AD mice, overexpression of mbRAGE accelerated the pathologic, cognitive, and behavioral hallmarks of AD, and infusion of synthetic esRAGE decreased A? and NT pathology as well as improved learning/memory and synaptic function. Thus, a novel strategy to reduce RAGE signaling is develop compounds called splice modulating oligonucleotides (SMOs) capable of controlling pre-mRNA alternative splicing to decrease expression of mbRAGE and simultaneously increase expression of esRAGE. SMOs are a class of synthetic RNA based compounds that sterically block or weaken interactions between elements of the splicing machinery and the pre-mRNA. SMOs bind to their targets with exceptional potency, specificity, long duration of action and negligible off-target effects. Previous pharmacological approaches to target ligand- RAGE interactions have been hampered by off-target effects or a short half-life, and none have the dual mechanism of both increasing natural esRAGE and decreasing mbRAGE isoform expression proposed here. SMOs targeting RAGE will be designed in silico and refined for potency and specificity, first in vitro cell culture (Aim 1) to select lead SMO drug candidates. Each SMO candidate will then be characterized and further optimized for safety and potency in vivo in RAGE transgenic mice (Aim 2). Finally, SMOs will be evaluated as therapeutics in an acute streptozotocin (STZ) induced model of AD for prevention/reversal of cognitive deficits and tau hyper-phosphorylation (Aim 3). The ultimate goal is to develop a lead RAGE SMO as potential therapeutic for the treatment of AD.

Dravet Spectrum disorders resulting from SCN1A loss-of-function (LOF) mutations include febrile seizures, generalized epilepsy with febrile seizure plus (GEFS+), and Dravet Syndrome (DS), in order of severity. DS symptoms begin in infancy and lead to progressive developmental and behavioral impairments along with characteristic recurrent and varied seizures. In many patients, seizures are resistant to currently available antiepileptic drugs and uncontrolled seizures are associated with an increased incidence of SUDEP (sudden unexplained death in epilepsy). Thus, there is a significant and urgent need for the development of novel drug therapies. SCN1A-containing Nav1.1 channels functionally oppose the related SCN8A-containing Nav1.6 channels. Notably, introducing an SCN8A LOF mutation into a SCN1A LOF DS mouse model will ?rescue? the DS phenotype, ameliorating seizures and early death. Therefore, specifically reducing SCN8A-mediated excitation should rebalance the deficit of inhibitory input caused by SCN1A loss-of-function mutations and cause fewer adverse effects than sodium channel blockers which non-selectively antagonize multiple Na+ channels. SCN8A subunits are naturally alternatively spliced to produce a non-functional isoform. Thus, we developed novel compounds called splice modulating oligonucleotides (SMOs) that direct SCN8A pre-mRNA splicing to produce less functional isoforms of SCN8A protein. SMOs are a class of synthetic RNA based compounds that sterically block or weaken interactions between elements of the splice machinery and the pre- mRNA with exceptional potency, specificity, and negligible off-target effects at efficacious doses in CNS. SCN8A SMOs were designed in silico and refined for potency and specificity in vivo to identify two top SMO drug candidates. Preliminary testing of both candidate SMOs in a DS mouse model with an SCN1A LOF mutation largely eliminated seizures during a critical period and robustly increased survival. From these two candidates a lead SCN8A SMO will be selected to move forward into further pre-clinical testing. Non-GLP toxicology and pharmacodynamics testing will be performed in rats by the same intrathecal (i.t.) delivery method expected to be used in clinical trials. The single maximal tolerable dose (MTD) and no observed adverse effects (NOAEL) will be determined, followed by assessment of MTD and NOAEL at multiple doses (up to 4 weekly doses), and lastly studies to understand the duration of action at the MTD and NOAEL doses (Aim 1). Additional testing will be performed in DS mice to establish the MTD and the minimally effective SMO dose (MED) that reduces seizures, improves survival, and does not cause adverse motor effects or induce anxiety behaviors associated with SCN8A knockdown in mice (Aim 2). These studies are designed to continue pre-clinical testing in preparation for formal GLP-toxicology studies. The ultimate goal is to develop an SCN8A SMO as potential therapeutic for the treatment of Dravet Syndrome.

Project Summary/Abstract Dravet Spectrum disorders resulting from SCN1A loss-of-function (LOF) mutations include febrile seizures, generalized epilepsy with febrile seizure plus (GEFS+), and Dravet Syndrome (DS) in order of severity. DS symptoms begin in infancy and lead to progressive developmental and behavioral impairments along with characteristic recurrent and varied seizures. In many patients, seizures are resistant to currently available antiepileptic drugs and uncontrolled seizure activity is associated with an increased incidence of SUDEP (sudden unexplained death in epilepsy). Thus, there is a significant and urgent need for the development of novel drug therapies. SCN1A-containing Nav1.1 channels functionally oppose the related SCN8A-containing Nav1.6 channels. We have developed a novel therapeutic oligonucleotide candidate, LSP-SCN8A, that works by reducing levels of SCN8A by directing its splicing to a non-functional isoform. LSP-SCN8A greatly reduces seizures and increases lifespan in a mouse DS model. The goals of the Phase 1 portion of the study will be to perform neurophysiological characterization of LSP- SCN8A in DS mice by cellular electrophysiology and video-EEG. In the Phase 2 component, we will perform additional dose-range studies for LSP-SCN8A in DS mice, confirm splicing activity of LSP-SCN8A in human cells, and generate a PD lot of the LSP-SCN8A SMO for non-GLP dose-finding studies in juvenile rats and primates in preparation for formal GLP-toxicology studies. We will also prepare for and conduct the pre-IND meeting with the FDA. The ultimate goal of our company is to fully develop LSP-SCN8A as a therapeutic to treat DS.