Abstract

Drug and therapy screening in models of epilepsy have made considerable advances in recent years. Rodent models of seizures are widely used in drug development and have helped to evaluate several approved antiseizure drugs (ASDs) over the last four decades. However, current ASDs are only fully effective in approximately two-thirds of patients, with the remaining populations experiencing drug-refractory seizures. Advances in understanding of additional model systems have provided new avenues for screening and evaluation of novel therapies in epilepsy. Zebrafish and Drosophila systems allow for high-throughput (vs. rodent assays) screening and drug repurposing studies and can be adapted for specific genetic populations. Similarly, mouse models based on genetic mutations corresponding to human pathology can be used and manipulated to study pathophysiology and screen novel compounds. Special populations of epilepsy, such as individuals developing seizures arising from central nervous system infection, can be modeled by viral-induced epilepsy models. The application of chemically induced seizures has been streamlined into moderate throughput assays in mice subjected to intra-amygdala or intra-hippocampal kainic acid, generating mice with repetitive seizures and other behavioral characteristics consistent with temporal lobe epilepsy. Finally, patient-derived induced pluripotent stem cells offer an opportunity to study patient-derived organoids, in the context of patient-specific genetic backgrounds, where drug screening can be used as a personalized medicine approach. These new approaches to drug development offer opportunities to complement and improve upon traditional screening approaches in rodents, in an effort to improve approaches to hard-to-treat epilepsy conditions.

Introduction

Recent developments in our understanding of the etiologies of many types of epilepsy, combined with a host of new experimental model systems, have brought significant changes in the way that new therapies are developed and assessed in preclinical studies. This is therefore an exciting time in epilepsy therapy development. It is hypothesized that with sufficient knowledge of the mechanisms underlying epilepsy, the development of appropriate biomarkers (Simonato et al., 2021), and utilization of the right model systems, new therapies will be identified that go beyond simply stopping seizures and are also disease modifying. In this chapter, we will discuss several new experimental approaches, from cell-based assays to whole organisms, that are being used to assess the potential efficacy of new therapies in stopping seizures. While therapy development has traditionally referred to the development of small molecules, a variety of alternative approaches, many of which are described in numerous chapters in this edition, can be explored, developed, and validated with the model systems described in this chapter. Biologics, antisense oligonucleotides, neuromodulation approaches (e.g., electrical stimulation, focused ultrasound, transcranial magnetic stimulation, and optogenetics), diets, viral transfection, and cell-based transplantation therapies are all amenable to these novel preclinical approaches and hold significant promise for addressing the unmet clinical needs of the person with epilepsy or at risk for developing epilepsy.

Zebrafish and Other Model Organisms

Armed with the information derived from the explosion in identification of genetic mutations that underlie epilepsy, the development of the use of zebrafish larvae models has been incredibly successful in high-throughput approaches for therapy discovery. Zebrafish models offer numerous advantages in drug development, including the ability to use CRISPR-Cas9 systems to rapidly introduce epilepsy conferring mutations efficiently, rapid phenotypic characterization, and the ability to use a combination of approaches such as behavioral assays, motion detection, imaging, and electrophysiology to validate the ability of promising compounds to stop not only convulsive activity but also electrographic seizure events and comorbid behaviors. Of note, this approach can provide a rapid path for FDA-approved drugs to enter clinical trials for repurposing in the treatment of epilepsy. This was recently accomplished using a zebrafish larvae model of Dravet syndrome (DS), a catastrophic epileptic encephalopathy that often results in therapy-resistant seizures that increase the risk of sudden unexplained death in epilepsy (SUDEP). In the majority of patients, DS results as a consequence of loss-of-function mutations in the SCN1A gene, which encodes the pore-forming α-subunit of a voltage-gated sodium channel (Nav1.1) (Harkin et al., 2007). A loss-of-function missense mutation in the zebrafish ortholog, scn1Lab, was found to induce convulsive behavior and electrographic ictal-like activity in mutant larvae as early as 3 days post fertilization (dpf) and were thus amenable to screening hundreds of compounds. Through this initial ground-breaking work, Baraban and colleagues identified several FDA-approved compounds that were efficacious in blocking both the convulsive behaviors and electrographic ictal activity, which paved the way for clinical trials for clemizole, an antihistamine that also has activity at serotonergic receptors (Baraban et al., 2013; Dinday et al., 2015). Additional hits of FDA-approved drugs also act through a number of mechanisms at the serotonergic receptor, which as a consequence largely of this work, is now viewed as a novel target for the treatment of seizures in DS (Griffin et al., 2017).

While there are significant advantages to screening for ASDs in zebrafish, like all model systems, there are also limitations in the utility of this approach. For example, as recently noted (Griffin et al., 2021), if only behavioral motor assays are used for phenotypic analysis, in the absence of electrophysiology or calcium imaging, phenotypes may not be accurately assessed. In addition, as is true for mouse models, phenotypic penetrance of seizures may be different than in humans because of gene-gene interactions, developmental regulation of gene expression, and lack of cortical development (Griffin et al., 2021). Nevertheless, the ability to use zebrafish in high-throughput assays has put them at the forefront of drug discovery in epilepsy and other disorders (Shcheglovitov et al., 2021).

Drosophila melanogaster Models of Epilepsy

As has been done in zebrafish, Drosophila have also been used to generate mutations found in the orthologs of human epilepsy-conferring genes. Drosophila breed rapidly, have numerous conserved genes, are easy to genetically manipulate, and can provide high-throughput screening opportunities. Drug administration is straightforward and can be achieved by administration in food, and the ability to induce seizures can then be evaluated in the presence and absence of ASDs. For a comprehensive review of the use of Drosophila to model developmental epileptic encephalopathies (DEEs), see Takai et al. (2020). While to date, no novel compounds or targets have been identified using Drosophila as a model organism, the ease with which basic mechanisms can be identified, coupled with high-throughput approaches and ease of genetic manipulations, make it likely that this model system, like zebrafish, can also yield promising novel approaches to therapy discovery.

Mouse Models of Genetic Epilepsy and Therapy Development

As is the case with model organisms, induced pluripotent stem cells (iPSCs), and other in vitro neuronal preparations, it is now straightforward to introduce epilepsy-conferring mutations into mice, a commonly used species for preclinical therapy development. The highly conserved nature of proteins between mice and humans, a complex cortex, and the ability to monitor seizure activity with video electroencephalogram (EEG) combine to provide the research community with tractable animal models to not only determine the mechanisms underlying seizures because of specific mutations but also to develop targeted therapies for the treatment of seizures. As next-generation sequencing has identified epilepsy-conferring mutations in more than 80 genes, the number of animal models has also risen dramatically, and several recent reviews comprehensively summarize the face validity of many of the available models (Fallah et al., 2020; Griffin et al., 2018; Maljevic et al., 2017; Wang et al., 2021). Mouse models for Fragile X syndrome, Rett syndrome, CDKL5 deficiency disorder, tuberous sclerosis, and those for numerous DS mutations have been shown to have spontaneous seizures and relevant comorbidities (Fallah et al., 2020; Wong et al., 2016). With respect to ASD preclinical pharmacology, perhaps the most common mouse genetic models used to date are those with DS mutations. Mouse models with a variety of DS-conferring mutations are available, wherein mice are observed to have hyperthermia-induced seizures, spontaneous seizures, behavioral comorbidities, and SUDEP (Griffin et al., 2018). Hyperthermia-induced seizures allow for precise timing of drug delivery (Fig. 64–2), as seizures are induced within minutes of a rise in core body temperature and have been used extensively in several different mouse models of DS to evaluate the ability of different prototype and investigational compounds to prevent seizures (Anderson et al., 2019, 2022; Cao et al., 2012; Hawkins et al., 2017a, 2017b, 2021; Kaplan et al., 2017; Kharouf et al., 2020; Silenieks et al., 2019).

Figure 64–2.

A diagram of the Dravet mouse hyperthermia assay. Seizures are induced when there is a rise in the core body temperature of a Dravet syndrome mouse. This model can be used to evaluate the ability of a compound to increase the temperature at which an (more...)

A DS mouse model is now in use in the screening paradigm of the contract site of the epilepsy therapy screening program (ETSP) as both hyperthermia-induced seizures as well as spontaneous seizures are observed in the Scn1a^A1783V/WT mouse. This represents the first time in the history of the program wherein a genetic model of human epilepsy is in use. The Scn1a^A1783V/WT is a conditional knock-in, loss-of-function mutation with both hyperthermia-induced and spontaneous seizures that reliably persist into adulthood. Thus, this mouse represents a useful model of DS in which to screen for novel therapies. The effect of a battery of prototype ASDs on hyperthermia-induced seizures in this mouse model was recently published (Pernici et al., 2021a). While clobazam, levetiracetam, and tiagabine were found to effectively shift the temperature at which seizures occur to significantly higher temperatures, indicating potential therapeutic value, the sodium channel blocker, carbamazepine, shifted the temperature at which seizures occur to a lower temperature, which is consistent with findings in patients and other mouse models of DS (Anderson et al., 2019; Hawkins et al., 2017a). Numerous other ASDs tested in this model had no effect on the temperature at which seizures occur, indicating that this model represents a refractory model of hyperthermia-induced seizures and may be useful in finding novel investigational compounds. Additionally, a combination dosing approach, wherein clobazam and valproic acid are administered to mice, along with an additional investigational compound, seeks to mimic the recommended clinical treatment algorithm for patients with DS (Cross et al., 2019). Under those testing conditions, stiripentol was efficacious in combination, whereas it was without effect when administered alone in this assay. This suggests that the combined use of clobazam and valproic acid with an add-on investigational compound may be a useful strategy for screening novel therapies in this refractory mouse model of DS. The complex pharmacology of such a treatment regimen would necessitate the use of pharmacokinetic evaluation, especially before proceeding to subchronic dosing regimens.

As has been noted previously (Griffin et al., 2018), it is currently unclear what relationship exists between evoked hyperthermia-induced seizures and true epilepsy, where spontaneous, unprovoked seizures occur. Therefore, pharmacology studies in animal models wherein spontaneous seizures are observed may prove useful as a secondary screen following identification of active compounds in hyperthermia-induced seizures. To that end, the contract site of the ETSP has also evaluated spontaneous seizures that occur in both male and female Scn1a^A1783/WT mice (Pernici et al., 2021b). Male Scn1a^A1783V/WT mice exhibit significantly increased seizure frequency (1.1 ± 0.7 seizures/day) as compared to female mice (0.7 ± 0.6 seizures/day), and the seizures often occur in clusters, with the potential to have several days between seizures. These baseline seizure frequency data have allowed us to perform a power analysis to determine the optimal length of time to treat mice with ASDs. We estimate that to detect a 50% reduction in seizures and achieve 80% power at a 95% significance level, in a combined cohort of males and females, we will need an n = 24 mice with 10 days of treatment. This accounts for both seizure clustering and low frequency of seizures. Given the capacity of the contract site of the ETSP, these labor-intensive video EEG experiments can be performed to test the most promising novel compounds for the ability to prevent spontaneous seizures in this mouse model of DS. A limitation of these subchronic experiments, however, is that there is a need for high-quality pharmacokinetic data to inform treatment regimens for 10 days of administration to mice. Numerous injections per day are not always feasible or practical, and not all compounds are orally available if delivered in food (Anderson et al., 2022). Nevertheless, subchronic administration may reveal potential adverse effects that are not always observable following single acute administration and can provide important evidence regarding the ability of a novel compound to reduce spontaneous seizures.

While there are numerous examples of compounds that are effective in mouse models of seizures and epilepsy failing to ultimately reach the clinic, rodent testing has identified numerous compounds that are now approved for clinical use as epilepsy treatments (Kehne et al., 2017). Additionally, those approved compounds were often first identified using naïve animals with acute seizures induced by electrical stimulation, such as the maximal electroshock seizure (MES) and 6 Hz tests. What we currently do not know is whether using genetic mouse models for therapy discovery will yield clinically successful drugs in discrete patient populations. However, this hypothesis will certainly be tested going forward. Although it is not currently feasible to generate mouse models of all relevant epilepsy-conferring mutations (indeed, hundreds of pathogenic mutations are associated with DS for SCN1A alone; Harkin et al., 2007), recent advances in the use of model systems and iPSCs can provide high-throughput screening approaches that can narrow the search for viable molecular targets and novel therapies for the treatment of epilepsy that arises as a consequence of genetic mutations (Griffin et al., 2021; Shcheglovitov et al., 2021). Once targets and compounds and therapeutic approaches are identified, further testing in genetic rodent models of epilepsy may provide important evidence supporting the development of these approaches and move them closer to use in the clinic.

Theiler’s Murine Encephalomyelitis Virus Mouse Model

Viral infections can produce seizures and, in some cases, promote the development of epilepsy (Eeg-Olofsson, 2003; Ficker et al., 1998; Pardo et al., 2014). Injection of Theiler’s murine encephalomyelitis virus (TMEV) intracortically in mice has been used as a model of viral-induced seizures and temporal lobe epilepsy (TLE) (Libbey et al., 2008; Stewart et al., 2010a, 2010b). Both spontaneous and handling-induced seizures occur following central infection with the Daniels strain of TMEV (Broer et al., 2016; Patel et al., 2019; Stewart et al., 2010a; Waltl et al., 2018a, 2018b). Further, this mouse model has been used as a means to screen compounds with antiseizure and anti-inflammatory mechanisms of action (Barker-Haliski et al., 2015; Cusick et al., 2013; Metcalf et al., 2021; Patel et al., 2019), and it has been incorporated into the screening program for the National Institute of Neurological Disorders and Stroke (NINDS) ETSP. Seizures resulting from central nervous system (CNS) infection may result from central inflammation and/or fever (Pardo et al., 2014). While seizures may be treated with traditional ASDs, drugs with anti-inflammatory mechanisms may also be used. Therefore, this animal model offers the opportunity to not only differentiate ASDs for their efficacy against infection-related seizures, but it may also help to identify compounds with unique mechanisms.

Using a standard screening approach wherein mice are subjected to twice-daily drug injections followed by handling sessions and quantification of behavioral seizures, several medications have been evaluated. Typically, handling-induced seizures begin 2–3 days following infection, peaking around 5 days post infection and stop by 8–10 days post infection (Fig. 64–1A). Spontaneous seizures have also been observed to occur in the weeks following initial infection (Fig. 64–1B). However, the rate of spontaneous seizures observed is relatively low and requires large numbers of mice monitored under continuous video-EEG in order to evaluate potential therapies for their effects on spontaneous seizures in this model. However, handling-induced seizures occur at a high rate (50%–70% of mice infected) (Metcalf et al., 2021) and are amenable to screening. ASDs administered during the acute infection period (day 3–7 post infection) with varying mechanisms of action were effective in reducing seizures in this model. When available, the median effective dose (ED₅₀) in the 6 Hz seizure model was used to inform dose selection, and when efficacy was observed at well-tolerated doses, additional doses were administered in order to evaluate dose–response relationships. Sodium channel blockers were differentially effective, with lacosamide and phenytoin significantly reducing seizures, whereas carbamazepine and lamotrigine were ineffective (Metcalf et al., 2021). Similarly, compounds acting on GABA were also differentially effective, with tiagabine and phenobarbital reducing seizures, whereas clonazepam was ineffective (Metcalf et al., 2021). Gabapentin, levetiracetam, topiramate, and valproic acid were also effective in reducing handling-induced seizures (Metcalf et al., 2021). However, while several ASDs reduced handling-induced seizures in this model, none were able to completely eliminate seizures.

Figure 64–1.

Mouse model of viral-induced epilepsy. A. C57BL/6J mice are intracerebrally injected with Theiler’s murine encephalomyelitis virus (TMEV), a neurotropic virus member of the Picornaviridae family that can cause encephalitis in infected mice. Between (more...)

TMEV infection in mice results in substantial elevations in cytokines (Kirkman et al., 2010; Libbey et al., 2011), and compounds that reduce inflammation may reduce seizures in this model. Further, counteracting the innate immune response in this model that contributes to seizures represents a unique antiseizure mechanism that may not be observed in other rodent models (e.g., acute electrically or chemically induced seizures). Nonsteroidal anti-inflammatory drugs (diclofenac, ibuprofen), steroids (prednisone, dexamethasone), the cyclooxygenase-2 inhibitor celecoxib, and the microglial inhibitor minocycline were administered during the acute infection period. Doses used had previously been observed to reduce inflammation or inflammation-related behaviors (e.g., pain responses) in other studies. Of these, a mild effect was observed with celecoxib and prednisone, and moderate efficacy was observed with dexamethasone and minocycline (Barker-Haliski et al., 2016; Metcalf et al., 2021).

In summary, TMEV infection and handling-induced seizures represents a unique model for screening compounds with antiseizure and/or anti-inflammatory mechanisms of action. Efficacy conferred on handling-induced seizures may be followed with confirmatory studies using video-EEG. However, since video-EEG studies are much more labor intensive, this approach should be reserved for the most promising compounds. ASDs reducing seizures in this model are likely effective due to their primary modes of action, although some may directly or indirectly reduce inflammation and thereby reduce seizures in this model. For example, levetiracetam was effective in this model across a wide range of doses and is also known to reduce inflammation (Stienen et al., 2011; Thone et al., 2012). Additional studies would be required, however, to differentiate antiseizure and anti-inflammatory effects for compounds where both potential mechanisms are present. Anti-inflammatory compounds evaluated thus far have generally been those with broad mechanisms. However, TMEV infection produces elevated reactive oxygen species, TGF-β, IL-6, TNFα, and IL-1β (Cusick et al., 2013; Kaufer et al., 2018; Kirkman et al., 2010; Libbey et al., 2008, 2010, 2011; Patel et al., 2017), and compounds acting specifically on these processes and cytokines may prove useful in reducing behavioral seizures and long-term outcomes (Bhuyan et al., 2015; Loewen et al., 2016; Wilcox et al., 2014). Identification of compounds with pronounced efficacy in this model may provide guidelines for treatment not only of infection-induced epilepsy but also seizure conditions where inflammation is pronounced.

Intra-Amygdala Kainate and Intra-Hippocampal Kainate Mouse Models

As discussed elsewhere in this text, delivery of the chemoconvulsant kainate (KA) to rodents induces status epilepticus (SE) and the subsequent development of mesial TLE (MTLE). KA can be administered via several routes (e.g., systemic, intra-nasal, intra-hippocampal, and intra-amygdala), each possessing unique experimental advantages and disadvantages, resulting in varying homology to the neuropathological features of MTLE in humans (Rusina et al., 2021). Both systemic administration of KA to rats and intra-hippocampal microinjection of KA to mice produce models of MTLE that are actively being used by the ETSP to evaluate investigational compounds for their effects on pharmacoresistant spontaneous recurrent seizures (SRSs) and hippocampal paroxysmal discharges (HPDs), respectively (Thomson et al., 2020; Wilcox et al., 2020). However, these models are not a panacea for all of the features of MTLE, nor do they represent the only models tractable enough to be useful in the further differentiation of investigational compounds under the auspices of the ETSP. Accordingly, additional KA-mediated SE models are continuing to be evaluated for inclusion in the ETSP pharmacoresistance drug screening efforts.

The intra-amygdala kainate (IAK) model, originally developed in rats (Ben-Ari et al., 1979, 1980) and adapted for mice in the early 2000s (Araki et al., 2002), has been used by a number of laboratories to investigate neuropathology, epileptogenesis, and MTLE resulting from focal microinjection of KA (Iori et al., 2017; Jimenez-Pacheco et al., 2013; Kondratiuk et al., 2015; Li et al., 2008, 2013a; Welzel et al., 2020). Similar to the systemic KA rat model, microinjection of KA in the mouse amygdala leads to SE, a seizure-free latent period, and ultimately the development of behavioral and electrographic SRSs. However, unlike models that employ systemic injections of KA and lead to widespread damage, IAK microinjection in mice causes limited and primarily unilateral hippocampal damage similar to human MTLE (Mouri et al., 2008). Furthermore, IAK mouse models offer several advantages that make them particularly well suited for assessing the efficacy of antiseizure therapies (e.g., SRSs manifest after a short latent period, occur with a relatively stable frequency, and mice require smaller quantities of often precious experimental compounds) (Jimenez-Pacheco et al., 2016; West et al., 2022). For these reasons, a mouse variant of the IAK microinjection model of MTLE has been developed (Fig. 64–3), pharmacologically characterized using standard ASDs, and adopted by the ETSP (West et al., 2022). Using five ASDs representing three mechanistic classes, this model was shown to produce SRSs that are resistant to two sodium channel-inhibiting ASDs (phenytoin and carbamazepine) and partially sensitive to GABA receptor-modulating ASDs (diazepam and phenobarbital) or a mixed-mechanism ASD (valproate). Although a more comprehensive examination of the effects of additional ASDs, including additional dose–response relationships combined with pharmacokinetic evaluations of plasma and brain concentrations, will be necessary in order to fully appraise this model, these data alone make a compelling case for the designation of SRSs in these mice as pharmacoresistant.

Figure 64–3.

Microinjection of kainic acid (KA) into the mouse amygdala induces status epilepticus (SE) and the subsequent development of spontaneous recurrent seizures (SRS). A. Schematic showing coordinates for injection cannula and CA1 electrode implantation sites. (more...)

Similar to the IAK model, the intra-hippocampal kainate (IHK) model shares the principal advantage of creating a focal lesion with limited and primarily unilateral damage akin to human MTLE. Microinjection of KA into the hippocampus has been performed in rats (Schwarcz et al., 1978), mice (Bouilleret et al., 1999), guinea pigs (Carriero et al., 2012), and cats (Tanaka et al., 1992). Furthermore, KA can be injected into either the dorsal or ventral hippocampus (Zeidler et al., 2018). As discussed elsewhere in this text, a unique feature of the IHK mouse model is the occurrence of relatively high-frequency electrographic seizures in the forms of high-voltage sharp waves (HVSWs) and hippocampal paroxysmal discharges (HPDs) (Klee et al., 2017; Twele et al., 2017). The high frequency of these electrographic events facilitates the screening of novel investigational ASDs because these experiments do not require multiday continuous video-EEG evaluations as required when measuring drug effects on the lower-frequency behavioral (electroclinical) SRSs. That said, the pharmacological sensitivity of behavioral seizures in IHK models is still of great interest, but using these models for this purpose has been challenging. For instance, while the rat IHK model produces frequent behavioral SRSs (Klee et al., 2017; Rattka et al., 2013), the evaluation of precious investigational compounds, which may have limited supply, is expensive (if at all possible) in relatively large rats. Conversely, mice require less quantity of a compound in order to perform subchronic administration experiments, but behavioral SRSs in the IHK mouse model have historically been reported to be infrequent and highly variable (Bielefeld et al., 2017; Klee et al., 2017; Welzel et al., 2020). Promising with regards to the mouse IHK model is a recent report by Lisgaras and Scharfman that describes a new implementation of the mouse IHK model exhibiting robust and frequent (2–4 per day) behavioral SRSs (Lisgaras et al., 2022). It is expected that this version of the IHK mouse, with its higher frequency electroclinical SRSs, may be adequate for the evaluation of their SRS sensitivities to classical and novel pharmacological interventions.

Induced Pluripotent Stem Cells and the Future

Pluripotent stem cells derived from patients have emerged as a means to study neuronal excitability and pathogenesis in a patient-specific manner. Traditional screening models in rodents are generally performed using relatively homogenous animal populations (e.g., inbred mouse strains) and do not reproduce any patient-specific pathophysiology contributing to seizures. By contrast, iPSC-derived organoids may offer a patient-specific opportunity to study epilepsy and antiseizure pharmacology (Shcheglovitov et al., 2021). Patient fibroblasts obtained from skin biopsies, for example, are subjected to a mixture of regulatory factors (e.g., Yamanaka factors) that induce pluripotency and then neural tissue generation (Grainger et al., 2018). iPSCs can be differentiated into a number of different cell types, including, in the context of epilepsy, neurons and glial cells (Cunningham et al., 2014; Krencik et al., 2011; Liu et al., 2013b; Maroof et al., 2013; Yu et al., 2014). For example, in addition to neurons and interneurons (Kim et al., 2014; Liu et al., 2013b), several studies have demonstrated that stem-cell-derived astrocytes reproduce physiologic functions of this cell type, including synaptic localization, gliotransmission, and calcium propagation (Hill et al., 2016; Shaltouki et al., 2013). Further, the heterogeneity of iPSC cultures recapitulates key network phenotypes, including a balance between excitation and inhibition and drug responsiveness (Ishii et al., 2017; Pei et al., 2016; Tukker et al., 2018). Further, iPSCs incorporate the patient’s genetic background, allowing for evaluation of different mutations types (variants), such as those that occur in DS. Several laboratories have generated iPSCs for patients with DS (Isom, 2017; Liu et al., 2013c; Sun et al., 2016, 2018; Tanaka et al., 2018; Tidball et al., 2020), which may lead to bench-to-bedside screening and therapy (Tidball et al., 2020). Thus, this approach can be used to both identify physiologic changes inherent with specific mutations and variants, such as changes in ion channel responses (Livide et al., 2015; Ricciardi et al., 2012; Tidball et al., 2020), and screen-specific treatments.

Under certain growing conditions, iPSCs will self-organize into spheroid structures (brain organoids) and, given sufficient time and growth conditions, become reminiscent of brain structures such as the hippocampus (Yu et al., 2014). Brain organoids are a monolayer in culture and consist of heterogenous cell populations of cells that can be further isolated and studied under various experimental conditions. For example, iPSC-derived forebrain neurons from patients with DS have been used to study ion channel conductance and demonstrate hyperexcitable phenotypes (Jiao et al., 2013; Liu et al., 2013c). Furthermore, three-dimensional brain organoids supported by hydrogels or other matrices have been generated from human iPSCs, recapitulating distinct brain structures and functions (Hirose et al., 2020).

Finally, advances in genome editing technology have further enhanced the utility of iPSCs and brain organoids. For example, the use of CRISPR/Cas9 approaches has allowed for reprogramming of iPSC lines for various purposes and the development of isogenic cell lines (e.g., cell lines where the mutation of interest is absent) (Tidball et al., 2017). Isogenic controls can be a critical missing piece from initial studies using patient-derived iPSC approaches, as they are required for proper interpretation of outcomes (Rowe et al., 2019). Therefore, gene editing has not only filled this gap but will continue to inform iPSC organoid studies.

Summary and Conclusions

While traditional animal models of seizures (e.g., MES, 6 Hz, and kindling models) have helped to identify and develop numerous clinically available ASDs over the last four decades, there continues to be an unmet clinical need to treat seizures in the nearly 37% of patients who remain refractory to existing therapies (Chen et al., 2018). The advent of the genetic revolution has greatly increased our understanding of the etiology of many types of epilepsy, and the ability to introduce pathogenic gene variants into a number of organisms, such as zebrafish and mice, provides the research community with an array of new assays to identify novel therapeutics. In addition, the use of spontaneously seizing animals to screen for these therapies has greatly changed the nature of preclinical evaluation of drugs. While these labor-intensive studies often require large numbers of animals to sufficiently power experiments, some groups have suggested utilizing multicenter preclinical studies to increase throughput and rigor and reproducibility (Pitkanen et al., 2019). Overall, it is hypothesized that the use of more etiologically relevant in vivo and in vitro assays will aid in the discovery of new therapies to treat those people with refractory epilepsy. Time will tell if this hypothesis holds.

Acknowledgments

Disclosure Statement

References

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