Gene therapy for epilepsy

The application of Gene Therapy towards Epilepsy, as well as other neurological diseases, is an ongoing area of research. It aims to utilize viral and non-viral vectors in the delivery of DNA to target areas for the treatment of patients before their diseases progress. In recent years gene therapy has delivered increasingly promising results in animal trials and other pre-clinical settings, leading to a broad range of research being conducted in hopes of developing viable gene therapies for neurological disorders such as epilepsy.

Epilepsy is a chronic set of neurological disorders that are characterized by seizures, affecting over 50 million people, or 0.4% - 1% of the global population.^[1][2] Currently, there is a basic understanding of the pathophysiology and its existing treatments, including medication, surgery, and dieting. While these treatments are effective for many epileptics, there are still approximately 20% - 30% of patients who don't have access to them or have developed a resistance to the antiepileptic drugs.^[3][4] As a result, many epileptics are left without any treatment options to consider, and thus there is a strong need for the development of innovative methods for treating epilepsy.

In recent studies, gene therapy has provided increasingly significant advances in the treatment of a wide range of diseases in animal and pre-clinical trials. Through the use of viral vector gene transfer, with the purpose of delivering DNA to targets for treatment, multiple neuropeptides have shown potential as targets for epilepsy treatment. Among those are adenovirus and adeno-associated virus vectors which have the properties of high and efficient transduction, ease of production in high volumes, a wide range of hosts, and extended gene expression.^[5]

Animal and pre-clinical studies have indicated that gene therapy can be an alternative to traditional medicines in treating diseases such as epilepsy. There have been many discoveries in the past decade advancing the field of gene therapy, with AAV5 being one of the most effective adeno-associated vectors (AAV) for transfection into the hippocampus.^[6] Although adeno-associated vectors require large doses for efficient gene delivery and are susceptible to existing immunities, they have proven to be most effective in epilepsy treatments due to their properties of long-term gene expression, and non-pathogenic nature. However, there still remain many challenges and safety concerns in the development and use of gene therapies.

One of the primary challenges in the approach of gene therapies is safety. Two major clinical studies have led to safety concerns, including a clinical trial in Philadelphia for ornithine transcarbamylase (OTC) deficiency, and another clinical trial in Paris for severe immunodeficiency. In the Philadelphia clinical trial, a young man died after being provided a high dose of an adeno-associated virus in the artery, where the cause of death was his inflammatory response that resulted in a massive cytokine production. In the Parisian clinical trial, semi-poetic stem cells were transduced with a corrective trans gene using a retro virus, leading to 3 out of the 20 patients developing t-cell leukemia. This was attributed to mutagenesis, which is when the DNA being integrated is placed into the wrong part of the genome. ^[7]. Between these two clinical studies, attention was brought to the need for extensive consideration of areas such as immunoresponses and insertional mutagenesis, which can be detrimental to patient safety.^[8]

Another key challenge is delivering enough DNA volume to target areas with the least resistance and insertional mutagenesis. Scaling up from the volume needed for animal trials to that needed for effective human transfection is an area of difficulty. With its size of less than 20 nm, AAV in part addresses this problem, allowing for its passage through the extracellular space, leading to widespread transfection.

Other challenges in the use of gene therapy for treating human diseases that need to be addressed in the future include: understanding how epilepsy fosters unfavorable conditions for grafts of neural precursors, developing an alternate vehicle for delivering drugs to target areas without requiring surgery, developing innovative drugs for epilepsy, deciding what DNA to transfect, and mutagenesis.^[4][8]

In finding a method for treating epilepsy, the pathophysiology of epilepsy is considered. As the seizures that characterize epilepsy are a result of excitatory signals, the logical goal for gene therapy treatment is to balance excitatory and inhibitory signals. Out of the viral approaches, the main three targets being researched currently are Somatostatin, Galanin, and Neuropetide Y (NPY). However, Adenosine and GABA/GABA Receptors are beginning to gain more momentum as well.

Adenosine is an inhibitory nucleoside that doubles up as a neuromodulator, aiding in the modulation of brain function. It has anti-inflammatory properties, in addition to neuroprotective and anti-epileptic properties ^[4]. The most prevalent theory is that upon brain injury there is an increased expression of the adenosine kinase (ADK). The increase in adenosine kinase results in an increased metabolic rate for adenosine nucleosides. Due to the decrease in these nucleosides that possess anti-epileptic properties, and the overexpression of the ADK, seizures are triggered, potentially resulting in the development of epileptogenesis.^[5] Studies have shown that ADK overexpression results from astrogliosis following a brain injury, which can lead to the development of epileptogenesis. While ADK overexpression leads to increased susceptibility to seizures, the effects can be counteracted and moderated by adenosine.^[9] Based on the properties afforded by adenosine in preventing seizures, in addition to its FDA approval in the treatment of other ailments, such as tachycardia and chronic pain, adenosine is an ideal target for the development of anti-epileptic gene therapies.^[10]

Galanin, found primarily within the CNS (limbic system piriform cortex, and amygdala), plays a role in the reduction of long term potentiation, regulating consumption habits, as well as inhibiting seizure activity [10]. Introduced back in the 1990s, by Mazarati et al., galanin has been shown to have neuroprotective and inhibitory properties. Through the use of mice that are deficient in GalR1 receptors, a picrotoxin-kindled model was utilized to show that galanin plays a role in modulating and preventing hilar cell loss as well as decreasing the duration of induced seizures.^[11] Recent studies conducted have confirmed these findings of preventing hilar hair cell loss, decreasing the number and duration of induced seizures, increasing the stimulation threshold required to induce seizures, and suppressing the release of glutamate that would increase susceptibility to seizure activity.^[4][12][13] Galanin expression can be utilized to significantly moderate and reduce seizure activity and limit seizure cell death.^[12]

Neuropeptide Y, which is found in the autonomic nervous system, helps modulate the hypothalamus, and therefore, consumption habits [source]. A number of experiments have been conducted to determine the effect of NPY on animal models before and after induced seizures.^[4][14] To evaluate the effect prior to seizures, one study inserted vectors 8 weeks prior to kindling, showing an increase in seizure threshold. In order to evaluate the effects after epileptogenesis was present, the vectors were injected into the hippocampus of rats after seizures were induced. This resulted in a reduction of seizure activity. These studies established that NPY increased the seizure threshold in rats, arrested disease progression, and reduced seizure duration.^[4][14] After examining the effects of NPY on behavioral and physiological responses, it was discovered that it had no effect on long term potentiation (LTP), learning, or memory.^[14] Given the existing research and stable results, a protocol for NPY gene transfer is being reviewed by the FDA ^[13]

Somatostatin is a neuropeptide and neuromodulator that plays a role in the regulation of hormones as well as aids in sleep and motor activity. It is primarily found in interneurons that modulate pyramidal cells’ firing rates primarily at a local level. They feed-forward inhibit pyramidal cells. In a series of studies where somatostatin was expressed in a rodent kindling model, it was concluded that somatostatin resulted in a decreased average duration for seizures, increasing its potential as an anti-seizure drug.^[7] The theory in utilizing somatostatin is that if you eliminate pyramidal cells, then the feed forward, otherwise known as inhibition, is lost. Somatostatin containing interneurons carry the neurotransmitter gamma-aminobutryic acid (GABA), which primarily hyperpolarizes the cells, which is where the feed forward theory is derived from. The hope of gene therapy is that by overexpressing somatostatin in specific cells, and increasing the GABAergic tone, it is possible to restore balance between inhibition and excitation.^[4][14]

Magnetofection is done through the use of super paramagnetic iron oxide nanoparticles coated with polyethylenimine. Iron oxide nanoparticles are ideal for biomedical applications in the body due to their biodegradable, cationic, non-toxic, and FDA-approved nature. Under gene transfer conditions, the receptors of interest are coated with the nanoparticles. The receptors will then hone and travel to the target of interest. Once the particle docks, the DNA is delivered to the cell via pinocytosis or endocytosis. Upon delivery, the temperature is increased ever so slightly, lysing the iron oxide nanoparticle and releasing the DNA. Overall, the technique is useful for combatting slow vector accumulation and low vector concentration at target areas. The technique is also customizable to the physical and biochemical properties of the receptors by modifying the characteristics of the iron oxide nanoparticles.^[15][16]

The use of gene therapy in treating neurological disorders such as epilepsy has presented itself as an increasingly viable area of ongoing research with the primary targets being somatostatin, galanin, and neuropeptide y for epilepsy. Although the field of gene therapy has been growing and has shown promising results for the treatment of epilepsy among other diseases, research still needs to be done in ensuring patient safety, developing alternative methods for DNA delivery, and feasible methods for scaling up delivery volumes. In addition, more quantitative methods of measuring the effectiveness of potential therapies should be considered including, but not limited to the method of kindling, duration of the latent period, and seizure frequency.^[17]