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Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies. 5th edition. New York: Oxford University Press; 2024. doi: 10.1093/med/9780197549469.003.0076
Epilepsy is a disease with diverse etiology; however, greater than 70%–80% of epileptic syndromes are thought to be associated with some genetic abnormality. Because of our fast-expanding genetic knowledge in epilepsy, we are approaching a new era of clinical management. We have an increased understanding of the underlying pathogenesis of genetic epilepsy as well as novel targets for precision therapies. Gene therapy, including gene transfer therapy, gene silencing, and gene editing, are promising new approaches to genetic epilepsy which have the potential to treat more than just the seizure manifestation of these devastating disorders. This chapter briefly reviews the role of genetic testing in epilepsy and introduces the application of gene therapy to genetic epilepsy syndromes as well as epileptogenesis more broadly.
Epilepsy is best defined as a chronic neurological disease with the propensity for the brain to generate recurrent unprovoked seizures. Although there are characteristics that affect incidence and prevalence rates, epilepsy does not discriminate, affecting regions worldwide, as well as all age groups, sexes, races, and social classes. The incidence rate of epilepsy is approximately 40–70 per 100,000 person-years (Sander, 2003) with incidence rates being higher in the youngest and the oldest age groups (Beghi, 2020; Fiest et al., 2017). The lifetime prevalence of epilepsy is estimated at 4–8 per 1,000 persons with it affecting up to 3 million people in the United States alone (Helbig, 2018).
Epilepsy is a disease with diverse etiology; however, greater than 70%–80% of epileptic syndromes are thought to be associated with some genetic abnormality (Dunn, 2018). As the etiology of epilepsy is the strongest prognostic factor for seizure recurrence, identifying and better understanding the gene–disease relationship is quickly moving to the forefront of investigation (Helbig, 2018). The concept of a genetic basis for epilepsy originated in the early 1900s when Herman Bernhard Lundborg published on the genetics of progressive myoclonic epilepsy (Lundborg, 1903), a hypothesis which was later solidified (Ottman et al., 1989; Annegers et al., 1982). Within the last two decades, there has been considerable progress toward a deeper understanding of epilepsy genetics. With the advent of next-generation sequencing (NGS), novel genetic findings are implicating other disease-causing mechanisms beyond the channelopathy hypothesis, including mTOR signaling, chromatin remodeling, transcriptional regulation, and synaptic vesicle trafficking (Myers, 2015).
Genetic testing was historically reserved for familial epilepsies or those refractory to antiseizure medications. In 2010, the International League Against Epilepsy published a report citing “more than 20 genes with a major effect on susceptibility to idiopathic epilepsies” (Ottman et al., 2010). Just over a decade later, there are hundreds of genetic etiologies associated with epilepsy, and genetic testing has become an integral piece of the evaluation of a patient with unprovoked seizures (Chen and Mefford, 2021). The diagnostic yield for any genetic test is generally going to be higher in those patients with early-onset seizures, neurodevelopmental comorbidities, family history of epilepsy, and medication resistance. Targeted testing with NGS has a reported 30%–40% diagnostic yield in patients with a family history of epilepsy and those with early-onset seizures (Lee, 2020). If a patient has dysmorphic features or global developmental delay, it may be prudent to begin with a chromosomal microarray (CMA) which will detect copy number variants that may be missed by NGS alone. Compared to other tests, NGS provides the advantage of being able to evaluate multiple genes at the same time, which may be more efficient than single gene testing. In a study by Gursoy et al., the rate of diagnosis was 46.2% with epilepsy gene panel testing in comparison to 15.4% with targeted single gene sequencing (Gursoy, 2016). For patients with nondiagnostic results on CMA and panel testing, whole-exome sequencing (WES) may be revealing. The overall diagnostic yield for exome-based testing in pediatric epilepsy has been reported between 15% and 28%, with the highest yield in early-onset epilepsies (Lassuthova, 2018; Howell, 2018). If the cost of WES and whole-genome sequencing (WGS) continues to decrease, the diagnostic algorithm may evolve to the earlier use of such testing strategies.
With the rapid expansion of genetic understanding of epilepsy, we are approaching a new era of clinical management. In addition to providing “closure” of the diagnostic odyssey for families, we are also limiting superfluous testing in search of a cause and providing more precise prognostication that may impact families’ reproductive decisions. But of paramount importance is the profound effect on therapeutic possibilities. By targeting specific pathophysiology’s, precision medicine is swiftly becoming a reality (Berkovic, 2015). Today, identification of an SCN1A variant in Dravet syndrome can prevent inappropriate therapeutic interventions (i.e., sodium channel drugs) and treatment with nicotine patch can alleviate seizure burden in patients with nAChR gene variants (Fox, 2020). Future therapies have the potential to affect an even bigger impact through disease modification by targeting the underlying pathophysiology.
Traditionally, drugs to treat epilepsy have been approved based upon demonstration of at least a 50% reduction in seizure burden in a placebo-controlled randomized clinical trial (Auvin et al., 2019; Brock et al., 2021). Each drug is approved independently with a narrow indication and without data to demonstrate comparative efficacy of other therapeutics on the market. Further, there is a large unmet need to treat other symptoms that accompany genetic epilepsy syndromes such as the cognitive, neuropsychiatric, and developmental disabilities (Snoeijen-Schouwenaars et al., 2021). Early initiation of antiseizure treatment and seizure control had no effect on psychosocial dysfunction and neuropsychiatric comorbidities (Sager et al., 2021).
With increasingly precise genetic diagnoses have come increasingly precise therapeutic approaches, such as stiripentol for Dravet syndrome (Pejcic et al., 2021; Carvill et al., 2021). Precision therapeutic approaches aim to intervene at the core pathogenesis of a genetic disorder and are transitioning the field of epilepsy management from one of symptom management (e.g., seizure reduction) to one of disease-modifying therapy. As such, the traditional clinical trial design with a primary outcome measure of seizure reduction is likely to inadequately capture the full spectrum of benefit of new precision therapies in epilepsy (Brock et al., 2021). Clinical trials in rare disease often utilize alternative clinical trial designs or novel approaches to the selection of clinical endpoints, such as multidimensional and composite endpoints (Brock et al., 2021; Kempf et al., 2018; Abrahamyan et al., 2016). Finally, a deep understanding of the natural history of each disorder, including incorporation of disease-relevant biomarkers and outcome measures with input from all stakeholders, will be required to adequately design clinical trials in genetic epilepsies (Brock et al., 2021; Armstrong and Marsh, 2021).
Gene therapy is a therapeutic strategy used to modify genes to treat both congenital and acquired disease and is a potential strategy for some genetic epilepsy syndromes (Zhang and Wang, 2021; Dunbar et al., 2018). It was first conceptualized in the 1960s with the discovery of the genetic code and initial studies demonstrating the ability to transfer exogenous genetic information into cells (Friedmann, 1992). Subsequent advancements in genetic engineering technologies helped propel the idea of gene therapy to reality, with the first clinical trial beginning in 1990 for treatment of severe combined immunodeficiency (Blaese et al., 1995) and the first approval by a regulatory agency in 2003 for treatment of squamous cell carcinoma (Pearson et al., 2004). Now there are multiple approved gene therapies available for certain cancers and inherited genetic diseases that are safe and effective at ameliorating disease symptoms and slowing disease progression (Umesawa et al., 2020).
In this chapter, we will provide an overview of gene therapy, including a brief explanation of three types of gene therapy, an overview of viral vectors, and current limitations and challenges in the field. Finally, we review potential applications of gene therapy in epilepsy.
With advancements in technologies to manipulate the genome, three major categories of gene therapy have emerged: gene replacement, gene silencing, and gene editing. Gene replacement aims to replace a nonfunctional gene that causes disease with a functional copy of the gene into cells (Deverman et al., 2018). Diseases due to loss-of-function mutations are ideal candidates for gene replacement because the protein product is either nonfunctional or has attenuated activity. An inherited retinal disease due to mutations in RPE65 is one such example and has an approved gene replacement therapy, Luxturna® (Gao et al., 2020).
However, gene replacement can be an ineffective and potentially toxic strategy in diseases caused by gain-of-function mutations or disorders with dominant-negative pathology, where the mutated gene is functional and masks or overrides the wild-type gene. Alternatively, gene silencing can be used to prevent the expression of the mutated dominant gene and prompt normal expression of the healthy gene (Deverman et al., 2018). The two most widely used gene silencing technologies include antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) (Watts and Corey, 2012). ASOs are synthetic single-stranded DNA molecules that can have multiple functions: (1) inhibit translation of a targeted mRNA by acting as steric blockers, (2) modulate splicing of a targeted mRNA, and/or (3) activate RNase H, which degrades the targeted mRNA. Creative ASO designs that result in splice-switching have developed into approved gene therapies for both spinal muscular atrophy (Chiriboga, 2017) and a severe form of Duchenne muscular dystrophy (Lim et al., 2017). siRNAs are synthetic double-stranded RNA molecules that silence genes through the RNA interference (RNAi) pathway which degrades endogenous mRNA (Watts and Corey, 2012). Like ASOs, siRNAs have been approved for treatment of certain diseases, including acute hepatic porphyria (Balwani et al., 2020) and polyneuropathies due to hereditary transthyretin-mediated amyloidosis (Adams et al., 2018).
Finally, gene editing allows for precise control of genetic manipulation via site-directed mutagenesis (Maeder and Gersbach, 2016). This method can use engineered endonucleases—zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and most recently, clustered regularly interspaced short palindromic repeat-associated 9 (CRISPR-Cas9) nucleases—to generate targeted double-stranded DNA breaks, which when repaired can introduce specific nucleotide insertions, deletions, or alterations. Base editing is another gene editing tool, which does not induce double-stranded DNA breaks like the endonucleases mentioned above to make base pair changes (Porto et al., 2020). Gene editing technology is in more preliminary stages of development compared to gene replacement and silencing; however, its potential influence for treatment of disease is tremendous due to the versatility and specificity of the technology for precision medicine. Many preclinical studies have demonstrated initial proof of concept suggesting that gene editing could be developed into clinical treatments for a range of disease, including epilepsy. For example, CRISPR activation targeting the Kcna1 promotor reduced seizure frequency and improved cognitive deficits in a mouse model of temporal lobe epilepsy (Colasante et al., 2020), and there is an open-label clinical trial assessing the safety and efficacy of a gene-editing product in patients with leber congenital amaurosis (NCT03872479). Unlike gene replacement and silencing, gene editing produces a permanent change to the host genome (Maeder and Gersbach, 2016).
Therapeutic entities frequently use modified viruses, termed viral vectors, to transfer foreign genetic material into cells (Bulcha et al., 2021). While viral vectors are discussed here, nonviral vectors, like inorganic nanoparticles, can also be used (Chen et al., 2016). The common viral vectors used for gene therapy include retroviruses, like gammaretrovirus (γ-retrovirus) and lentivirus, herpes simplex virus type 1 (HSV-1), adenovirus (Ad), and adeno-associated virus (AAV) (Robbins and Ghivizzani, 1998). Each of these viral vectors has distinguishing characteristics, advantages, and limitations, which are outlined in Table 76–1.
Viral Vectors Used in Gene Therapy.
An ideal viral vector would be safe, target the cell and tissue type of disease relevance, and have a high transduction efficiency with appropriate transgene expression. Safety is essential and has been a prominent research focus due to a history of adverse clinical outcomes, including fatalities and incidences of T-cell leukemia, in the late 1990s and early 2000s (Raper et al., 2002; Hacein-Bey-Abina et al., 2003; Howe et al., 2008). Subsequent research was integral to the development of modified viral vectors, which have a better safety profile. While safety is still addressed in studies, much of the current research on viral vectors also focuses on vector tropism, transduction efficiency, transgene expression, and methods of delivery with the goal of generating viral vectors optimized for the disease and gene of interest.
Viral vectors can either be (1) integrating, where the foreign genetic material inserts itself into the host genome and is subsequently passed down to every daughter cell, or (2) nonintegrating, where the foreign genetic material remains episomal (e.g., extrachromosomal) in the cell cytoplasm (Bulcha et al., 2021). In general, integrating vectors include retroviruses, whereas nonintegrating viruses include AAV, Ad, and HSV-1. Nonintegrating vectors can be advantageous over integrating vectors for gene therapy because of reduced concern for oncogenic insertional mutagenesis. Newer generations of retroviruses, including nonintegrating lentiviruses (NILs) that have a nonfunctional integrase, have been developed to address this limitation and show an improved safety profile (Yanez-Munoz et al., 2006).
Recombinant AAV (rAAV) is currently the most prominent vector used for gene delivery to the central nervous system due to its safety and high efficiency (Wang et al., 2019). Wild-type AAV (wtAAV), a helper-dependent parvovirus, was first discovered in 1965 as a contaminant of an Ad stock (Atchison et al., 1965; Hoggan et al., 1966). Interestingly, multiple serotypes of wtAAV are present in a significant portion of the human population, but they do not cause disease (Verdera et al., 2020; Boutin et al., 2010). This uncommon nonpathological characteristic of wtAAV is beneficial in terms of safety for a gene therapy viral vector, but it also means that many individuals, specifically those with preexisting immunity against AAV, require special consideration when receiving AAV gene therapies (Vandamme et al., 2017). Research focused on strategies to circumvent neutralizing antibodies against AAV are being explored (Gray et al., 2013; Bertin et al., 2020).
The AAV capsid is nonenveloped and contains a single-stranded DNA genome with a packaging capacity of 4.5 kilobases (Naso et al., 2017). wtAAV carries two genes, rep and cap, which are necessary for genome replication and packaging, in between hairpin structures, known as inverted terminal repeats (ITRs), which are critical for packaging and proper function of the viral genome (Fig. 76–1). Due to rep, wtAAV can integrate into chromosome 19 and other parts of the host genome at low frequencies; however, rAAV, which has rep and cap replaced by the gene of interest and necessary gene expression elements, lacks the natural ability of wtAAV to integrate into the genome, further supporting a high safety profile for this viral vector (McCarty et al., 2004).
Transduction efficiency of AAV can be increased by introducing a double-stranded genome (Wang et al., 2003; McCarty et al., 2001, 2003). The double-stranded (e.g., self-complementary) version of the packaged DNA genome reduces the packaging capacity to 2.2 kilobases (Fig. 76–1), but it has approximately 10- to 100-fold increased transduction efficiency and enhanced transgene expression compared to the single-stranded version due to the elimination of the second-strand synthesis step required before transcription ensues. The small packaging capacity of AAV, for both single-stranded and self-complementary versions, is one of its biggest disadvantages compared to other viral vectors.
Wild-type (WT) and recombinant AAV vector genome configurations. The genome of wtAAV contains DNA sequences encoding replication (rep) and capsid (cap) proteins. Any DNA of appropriate length can be packaged, but basic designs for gene replacement vectors (more...)
Despite the small packaging capacity limitation, rAAV has many advantages for viral vector gene therapy (Naso et al., 2017). In addition to a high safety profile, AAV can infect dividing and nondividing cells and produces stable gene expression in postmitotic cells such as neurons (Podsakoff et al., 1994; Russell et al., 1994). There are multiple AAV serotypes and over a hundred AAV variants which have diverse tropisms (Wu et al., 2006). The AAV serotype 9 is a favored choice for widespread central nervous system disease because it targets both neurons and glia (Foust et al., 2009; Hammond et al., 2017), in addition to cells in peripheral tissues, including skeletal muscle, lung, liver, and heart (Zincarelli et al., 2008). Additionally, AAV only causes mild immune responses, which is often addressed with concurrent immunosuppression (Vandamme et al., 2017).Viral Vector Delivery
There are two general techniques of delivering foreign genetic material using viral vectors for gene therapy applications, known as ex vivo and in vivo gene therapy (High and Roncarolo, 2019). In ex vivo gene therapy, the therapeutic target gene is transduced into cells of a patient in vitro and those cells are subsequently reintroduced to the patient. A recent preclinical study used an ex vivo strategy to deliver a glial cell line-derived neurotrophic factor, which reduced the number of spontaneous recurrent seizures in epileptic rats (Nanobashvili et al., 2019). In contrast, in vivo gene therapy involves administration of the therapeutic target gene directly to the patient, and it is the modality used in the approved gene therapy, Luxturna®. The remainder of this section focuses on in vivo gene therapy.
The route of administration is integral to the success of an effective in vivo gene therapy, and it is an area of ongoing research. Routes commonly utilized today include direct injection into a target organ (e.g., brain parenchyma), intravenous (IV), intrathecal (IT), and intracisternal magna (ICM) (Gray et al., 2010b). In the case of epilepsy, treating the central nervous (CNS) system is paramount, and any effective gene therapy must either cross the blood–brain barrier (BBB) or be directly delivered to the CNS. Exciting new strategies to induce transient breaks in the BBB to allow site-specific delivery of the gene therapy are being explored (e.g., focused ultrasound) (Timbie et al., 2015). There is also the possibility of engineering novel AAV vector technology, to take advantage of transient disruption of the BBB that occurs following a seizure, to specifically target areas of seizure damage after an IV injection of the vector (Gray et al., 2010a).
Though there is promise in gene therapy, significant challenges are present. This is not an exhaustive list, but it highlights current limitations faced in the field with a focus on AAV-mediated gene therapy applications in epilepsy.
Currently, the ability to regulate transgene expression to mimic physiological levels with viral-mediated gene replacement therapy is limited. For dose-sensitive genes, where too much or too little gene expression can have negative consequences, complex gene regulatory elements may be needed (Goverdhana et al., 2005). Currently, the easiest, but least controllable option is to use specific promoters that drive approximate levels of expression in the cell and tissue type of interest. Another option to regulate transgene expression is the use of micro RNA (miRNA) targeting sites which regulate gene expression through posttranscriptional mRNA processing (Sinnett et al., 2021). Gene editing approaches will conceivably generate more appropriate physiological gene expression than a gene replacement approach, as the host gene is directly manipulated, keeping all endogenous gene regulation mechanisms intact.
When designing a gene therapy trial, the timing of gene replacement is often critical for success, and preclinical studies consistently demonstrate that earlier delivery of the healthy gene yields a greater prospect of benefit, and there may be a point at which a disease has advanced beyond that which can be rescued by gene replacement (Gray, 2016). In the case of epilepsy, genetic testing is only pursued after the diagnosis of epilepsy is made; thus, the feasibility of treating before symptom onset would require a paradigm shift in diagnostic approaches.
Accessibility and affordability of gene therapies has been a perpetual obstacle that will need to be addressed quickly as more gene therapies are approved. The FDA-approved, AAV-mediated gene therapies, Zolgensma® to treat spinal muscular atrophy, and Luxturna®, to treat retinal dystrophy, cost $2.1 million and $850,000, respectively. The high cost is justified because gene therapy is a one-time treatment with the potential for substantial savings from even higher costs of the traditional long-term intensive medical interventions without the gene therapy treatment (Garrison et al., 2021). However, current payment structures and insurance agencies are not built for extremely high one-time payments, prompting the need for alternative payment mechanisms, such as outcomes-based pricing and payment over time (Jorgensen and Kefalas, 2021). With relatively few approved gene therapy products on the market, factors driving the high cost include research and development costs, high drug manufacturing costs, and more generalized risk associated with a new class of drug product.
In this section, we will review three applications of gene therapy in epilepsy using the illustrative examples of gene replacement in Rett syndrome, gene editing and manipulation in Dravet syndrome, and viral-mediated supplementation of neuroactive substances to reduce epileptogenesis.
Gene replacement is the method of gene therapy in which a healthy copy of a human gene is packaged into a vector and delivered directly to the patient. We will discuss the example of Rett syndrome (RTT) as an application of gene replacement in a genetic epilepsy syndrome.
In brief, Rett syndrome (RTT) is a neurodevelopmental disorder most often caused by a pathogenic variant in methyl-CpG-binding protein 2 (MECP2) on the X chromosome. Hallmark symptoms include early normal to only mildly delayed development followed by a period of regression and characteristic hand stereotypies whose onset is often coincident with the regression period (Neul et al., 2008). RTT provides an excellent case study when considering the importance of the timing and dosing of gene replacement. While the ideal scenario would be gene replacement in utero, the diagnosis is not made until after the onset of clinical symptoms. In the case of RTT, the predicted optimal treatment timing would be after diagnosis but before the predictable plateau phase, after which it is possible that protein product replacement would no longer produce appreciable benefit (Sinnett and Gray, 2017; Gadalla et al., 2017).
In addition to timing of the treatment, the level of protein expression must also be considered. In traditional gene replacement, the subsequent expression levels are not highly controlled, so the potential of overexpression toxicity must be considered. In the case of RTT, the classical phenotype results from the loss of functional MeCP2 protein; however, the related and often more severe MECP2 duplication disorder results from excess MeCP2 protein (del Gaudio et al., 2006). Further, a significant number of patients with RTT have detectable levels of residual MeCP2 protein, including those with the truncating variant R294X causing a hypomorphic state, potentially raising the risk of generating toxicity from supra-physiological levels of MeCP2 protein (Bassuk, 2021; Gadalla et al., 2013).
Early preclinical data concentrated on homozygous knockout male mice, in whom traditional gene replacement extended survival. Once attention turned to female heterozygous mice, with mosacisim between wild-type expressing cells and variant cells mediated by random and at times skewed x-inactivation, traditional gene replacement continued to extend the lifespan, but overexpression symptoms began to be observed, particularly motoric and associative learning defects as well as increased anxiety symptoms (Chao and Zoghbi, 2012). Similar effects were noted in female mice with truncating or hypomorphic states (Collins et al., 2021; Vermudez et al., 2021). These observations led to the realization that gene therapy would need to involve some type of regulatory element to prevent transgene overexpression on a cell-by-cell basis, as demonstrated by the design of the miR-Responsive Auto-Regulatory Element (miRARE). Inclusion of the miRARE element within the miniMECP2 construct provided the same efficacy without the dose-dependent toxicity seen in previous studies delivering unregulated MECP2, and it serves as a potential new gene regulation approach for gene transfer therapy in other dose-sensitive genes (Sinnett et al., 2021).
Although gene therapy in epilepsy is still in its infancy, promising advances in our understanding of genetics and the development of novel technologies offer exciting possibilities. As mentioned previously, gene editing technology has the advantage of being versatile and specific, potentially retaining endogenous gene expression controls with reduced risk for genotoxicity. Dravet syndrome (DS) is a monogenic disease presenting as a developmental epileptic encephalopathy. It is caused by a change in one copy of the SCN1A sodium channel gene in >80% of cases, therefore leading to insufficient number of NaV1.1 channels at the neuronal surface (Higurashi, 2021). Recent technological advances are generating optimism about gene editing for monogenic epilepsies, and DS has been targeted as one in which to investigate such therapy.
The large size of the SCN1A gene limits the ability to utilize AAV-mediated gene transfer therapy. Early preclinical studies are investigating the use of larger viruses (e.g., Adenovirus, Lentivirus) as well as utilizing a dual-AAV approach to allow for SCN1A gene transfer (Kay, 2001). Alternatively, in a pivotal study Colasante and colleagues demonstrated that a modified version of the CRISPR-Cas9 system can be utilized to restore NaV1.1 protein expression without cleaving the target DNA (Colasante, 2020). Finally, another unique and gene-specific treatment approach in DS has been investigated whereby targeted augmentation of nuclear gene output (TANGO) technology was used to develop an antisense oligonucleotide (ASO) that was able to increase expression of the wild-type SCN1A allele to compensate for the loss of mutant allele expression, both in vivo (e.g., mouse model) and in vitro (e.g., cultured human cells) (Han, 2020).
In addition to monogenic epileptic syndromes, epigenetic modifications of gene expression also play an important role in epileptogenesis in genetic epilepsies (Boison and Rho, 2020). Other gene products may confer a benefit to seizure reduction, aside from gene replacement approaches. Substances such as adenosine, galanin, and neuropeptide Y may be delivered using gene therapy technology to reduce epileptogenesis more broadly.
Adenosine is a purine ribonucleoside and integral component of ATP. Adenosine acts as an endogenous anticonvulsant and seizure terminator through a negative feedback loop to reduce energy consumption when supplies are low (Boison, 2016). Acutely during a seizure, adenosine levels rise and adenosine kinase (ADK) levels drop transiently, resulting in a reduction in DNA methylation and increased transcription and expression of pro-epileptogenic genes (Boison, 2016). Seizures induce an inflammatory cascade which can cause overexpression of ADK, hypermethylation of DNA, and relative depletion of adenosine levels (Boison, 2016). Supplementation of adenosine, including through an AAV-mediated manipulation of adenosine, to the epileptic brain of rats is sufficient to prevent kindling epileptogenesis (Boison, 2009; McCown, 2010; Boison and Rho, 2020).
Galanin is a neuropeptide that is highly expressed in the temporal lobes and is released during a seizure (Sorensen and Kokaia, 2013). Galanin attenuates long-term potentiation to suppress seizure activity through inhibition of hippocampal release of acetylcholine and glutamate (McCown, 2009; Simonato, 2014). Animal studies demonstrate that AAV-mediated exogenous administration of galanin to the hippocampi, inferior colliculus, and piriform cortex lead to an increased seizure threshold and reduced frequency and severity of kainite-induced seizures (Haberman et al., 2003; Lin et al., 2003). Finally, AAV-mediated galanin delivery in a model of fully kindled rats also demonstrated seizure reduction with constitutive galanin secretion (McCown, 2006).
Neuropeptide Y (NPY) is an endogenous peptide that is overexpressed in the brain during seizures and can attenuate seizures through inhibition of glutamate release and reducing neuronal excitability through activation of the Y1, Y2, and Y5 receptors (Vezzani et al., 1999; Cattaneo et al., 2020). NPY-knockout mice have an increased susceptibility to seizures, a rat model with overexpression of NPY had a reduced seizure-threshold, and there is a natural upregulation of NPY in interneurons in animal models of epilepsy as well as humans with temporal lobe epilepsy (Sorensen and Kokaia, 2013). Direct hippocampal injection of recombinant AAV expressing NPY (rAAV-NPY) may be an alternative to surgical resection for intractable temporal lobe epilepsy. In a rat model of chronic epilepsy, this approach resulted in a significant reduction in seizure frequency and reduced epileptic progression in comparison to untreated animals (Noe et al., 2008). Further, studies have demonstrated that rAAV-NPY can successfully transduce human neuronal cell lines and may reduce anxiety in addition to seizures when injected into the thalamus or somatosensory cortex (Patricio et al., 2018; Powell et al., 2018). Finally, there is ongoing work exploring dual-gene vector delivery of NPY in combination with critical Y-type receptors that is showing promising results of electrographic seizure reduction in an animal model of TLE (Cattaneo et al., 2020).
In conclusion, gene therapy is a promising novel therapeutic approach for focal, generalized, and genetic epilepsy. Genetic testing availability has expanded the landscape of epilepsy syndromes linked to single-gene disorders and opened the opportunity for precision therapies targeting the underlying pathophysiology of each epilepsy syndrome or identifying common pathways shared among multiple disorders. Novel therapies, such as gene therapy, will require novel approaches to clinical trial design and robust clinical trial readiness programs equipped to identify disease-relevant biomarkers and clinical outcome measures that go beyond seizure reduction. Finally, while the available technology is promising, future studies can build upon this foundation to improve the specificity and efficiency of viral vectors, introduce regulatory elements to control gene expression levels, and develop novel modalities to manipulate gene expression through techniques such as silencing RNAs, engineered minigenes, and regulation of upstream or downstream regulators that could address multiple syndromes simultaneously.
Epilepsy is a disease with diverse etiology; however, greater than 70% to 80% of epileptic syndromes are thought to be associated with some genetic abnormality.
The highest diagnostic yield for genetic testing is in patients with early-onset seizures, neurodevelopmental comorbidities, and significant family history of epilepsy and medication resistance.
Gene therapy includes a variety of techniques, including viral-mediated gene transfer therapy, gene expression regulation, and gene editing.
Current limitations of gene therapy include the inability to control gene expression levels with precision, challenges with timing and delivery of gene therapy, and the costs of gene therapy.
Gene therapy has the potential to be a disease-modifying therapy which reduces seizures in addition to reducing the morbidity of associated symptoms of genetic epilepsy including neurodevelopmental disability and neuropsychiatric symptoms.
This is an open access publication, available online and distributed under the terms of a Creative Commons Attribution-Non Commercial-No Derivatives 4.0 International licence (CC BY-NC-ND 4.0), a copy of which is available at https://creativecommons.org/licenses/by-nc-nd/4.0/. Subject to this license, all rights are reserved.
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