Gene therapy for epilepsy

Gene therapy is being studied for some forms of epilepsy.[1] It relies on viral or non-viral vectors to deliver DNA or RNA to target brain areas where seizures arise, in order to prevent the development of epilepsy or to reduce the frequency and/or severity of seizures. Gene therapy has delivered promising results in early stage clinical trials for other neurological disorders such as Parkinson's disease,[2] raising the hope that it will become a treatment for intractable epilepsy.

Gene therapy for epilepsy

Overview

Epilepsy refers to a group of chronic neurological disorders that are characterized by seizures, affecting over 50 million people, or 0.4–1% of the global population.[3][4] There is a basic understanding of the pathophysiology of epilepsy, especially of forms characterized by the onset of seizures from a specific area of the brain (partial-onset epilepsy). Although most patients respond to medication, approximately 20%–30% do not improve with or fail to tolerate antiepileptic drugs.[5][6] For such patients, surgery to remove the epileptogenic zone can be offered in a small minority, but is not feasible if the seizures arise from brain areas that are essential for language, vision, movement or other functions. As a result, many people with epilepsy are left without any treatment options to consider, and thus there is a strong need for the development of innovative methods for treating epilepsy.

Through the use of viral vector gene transfer, with the purpose of delivering DNA or RNA to the epileptogenic zone, several neuropeptides, ion channels and neurotransmitter receptors have shown potential as transgenes for epilepsy treatment. Among vectors are adenovirus and adeno-associated virus vectors (AAV), which have the properties of high and efficient transduction, ease of production in high volumes, a wide range of hosts, and extended gene expression.[7] Lentiviral vectors have also shown promise.

Clinical research

Among challenges to clinical translation of gene therapy are possible immune responses to the viral vectors and transgenes and the possibility of insertional mutagenesis, which can be detrimental to patient safety.[8] Scaling up from the volume needed for animal trials to that needed for effective human transfection is an area of difficulty, although it has been overcome in other diseases. With its size of less than 20 nm, AAV in part addresses these problems, allowing for its passage through the extracellular space, leading to widespread transfection. Although lentivectors can integrate in the genome of the host this may not represent a risk for treatment of neurological diseases because adult neurons do not divide and so are less prone to insertional mutagenesis

Viral approaches in preclinical development

In finding a method for treating epilepsy, the pathophysiology of epilepsy is considered. As the seizures that characterize epilepsy typically result from excessive and synchronous discharges of excitatory neurons, the logical goal for gene therapy treatment is to reduce excitation or enhance inhibition. Out of the viral approaches, neuropeptide transgenes being researched are somatostatin, galanin, and neuropeptide Y (NPY). However, adenosine and gamma-aminobutyric acid (GABA) and GABA receptors are gaining more momentum as well. Other transgenes being studied are potassium channels and tools for on-demand suppression of excitability (optogenetics and chemogenetics).

Adenosine

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.[6] 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.[7] 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

Galanin, found primarily within the central nervous system (limbic system, piriform cortex, and amygdala), plays a role in the reduction of long term potentiation (LTP), regulating consumption habits, as well as inhibiting seizure activity.[11] 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.[12] Conducted studies confirm 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.[6][11][13] Galanin expression can be utilized to significantly moderate and reduce seizure activity and limit seizure cell death.[11]

Neuropeptide Y

Neuropeptide Y (NPY), which is found in the autonomic nervous system, helps modulate the hypothalamus, and therefore, consumption habits.[6] Experiments have been conducted to determine the effect of NPY on animal models before and after induced seizures.[6][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.[6][14] After examining the effects of NPY on behavioral and physiological responses, it was discovered that it had no effect on LTP, learning, or memory.[14] A protocol for NPY gene transfer is being reviewed by the FDA.[13]

Somatostatin

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 modulates the firing rates of pyramidal cells 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.[15] The theory in utilizing somatostatin is that if pyramidal cells are eliminated, then the feed forward, otherwise known as inhibition, is lost. Somatostatin containing interneurons carry the neurotransmitter 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.[6][14]

Potassium channels

Kv1.1 is a voltage-gated potassium channel encoded by the KCNA1 gene. It is widely expressed in the brain and peripheral nerves, and plays a role in controlling the excitability of neurons and the amount of neurotransmitter released from axon terminals. Successful gene therapy using lentiviral delivery of KCNA1 has been reported in a rodent model of focal motor cortex epilepsy[16]. The treatment was well tolerated, with no detectable effect on sensorimotor coordination. Gene therapy with a modified potassium channel delivered using either a non-integrating lentivector that avoids the risk of insertional mutagenesis or an AAV has also been shown to be effective in other models of epilepsy [17].

Optogenetics

A potential obstacle to clinical translation of gene therapy is that viral vector-mediated manipulation of the genetic make-up of neurons is irreversible. An alternative approach is to use tools for on-demand suppression of neuronal and circuit excitability. The first such approach was to use optogenetics. Several laboratories have shown that the inhibitory light-sensitive protein Halorhodopsin can suppress seizure-like discharges in vitro as well as epileptic activity in vivo.[18][19][20][21] A draw-back of optogenetics is that light needs to be delivered to the area of the brain expressing the opsin. This can be achieved with laser-coupled fiber-optics or light-emitting diodes, but these are invasive.

Chemogenetics

An alternative approach for on-demand control of circuit excitability that does not require light delivery to the brain is to use chemogenetics. This relies on expressing a mutated receptor in the seizure focus, which does not respond to endogenous neurotransmitters but can be activated by an exogenous drug. G-protein coupled receptors mutated in this way are called Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Success in treating epilepsy has been reported using the inhibitory DREADD hM4D(Gi), which is derived from the M4 muscarinic receptor.[22] AAV-mediated expression of hM4D(Gi) in a rodent model of focal epilepsy on its own had no effect, but when activated by the drug clozapine N-oxide it suppressed seizures. The treatment had no detectable side effects and is, in principle, suited for clinical translation. Olanzapine has been identified as a full and potent activator of hM4D(Gi)[23]. A 'closed-loop' variant of chemogenetics to stop seizures, which avoids the need for an exogenous ligand, relies on a glutamate-gated chloride channel which inhibits neurons whenever the extracellular concentration of the excitatory neurotransmitter glutamate rises[24].

CRISPR

A mouse model of Dravet syndrome has been treated using a variant of CRISPR that relies on a guide RNA and a dead Cas9 (dCas9) protein to recruit transcriptional activators to the promoter region of the sodium channel gene Scn1a in interneurons[25].

Non-viral approaches

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 home in 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.[26][27]

Future implications

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, neuropeptide y, potassium channels, optogenetics and chemogenetics for epilepsy. As the field of gene therapy continues to grow and show promising results for the treatment of epilepsy among other diseases, additional research needs to be done in ensuring patient safety, developing alternative methods for DNA delivery, and finding feasible methods for scaling up delivery volumes.[28][29]

References

1. Walker MC, Schorge S, Kullmann DM, Wykes RC, Heeroma JH, Mantoan L (September 2013). "Gene therapy in status epilepticus" (PDF). Epilepsia. 54 Suppl 6: 43–5. doi:10.1111/epi.12275. PMID 24001071.
2. Palfi S, Gurruchaga JM, Ralph GS, Lepetit H, Lavisse S, Buttery PC, et al. (March 2014). "Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial". Lancet. 383 (9923): 1138–46. doi:10.1016/S0140-6736(13)61939-X. PMID 24412048.
3. Hirose G (May 2013). "[An overview of epilepsy: its history, classification, pathophysiology and management]". Brain and Nerve = Shinkei Kenkyu No Shinpo. 65 (5): 509–20. PMID 23667116.
4. Sander JW, Shorvon SD (November 1996). "Epidemiology of the epilepsies". Journal of Neurology, Neurosurgery, and Psychiatry. 61 (5): 433–43. doi:10.1136/jnnp.61.5.433. PMC 1074036. PMID 8965090.
5. Pati S, Alexopoulos AV (July 2010). "Pharmacoresistant epilepsy: from pathogenesis to current and emerging therapies". Cleveland Clinic Journal of Medicine. 77 (7): 457–67. doi:10.3949/ccjm.77a.09061. PMID 20601619. S2CID 8184157.
6. Weinberg MS, McCown TJ (June 2013). "Current prospects and challenges for epilepsy gene therapy". Experimental Neurology. 244 (Special): 27–35. doi:10.1016/j.expneurol.2011.10.003. PMC 3290712. PMID 22008258.
7. Naegele JR, Maisano X, Yang J, Royston S, Ribeiro E (May 2010). "Recent advancements in stem cell and gene therapies for neurological disorders and intractable epilepsy". Neuropharmacology. 58 (6): 855–64. doi:10.1016/j.neuropharm.2010.01.019. PMC 2838966. PMID 20146928.
8. Giacca M (2010). Gene Therapy. New York: Springer. pp. 284–86. ISBN 978-88-470-1642-2.
9. Boison D (December 2006). "Adenosine kinase, epilepsy and stroke: mechanisms and therapies". Trends in Pharmacological Sciences. 27 (12): 652–8. doi:10.1016/j.tips.2006.10.008. PMID 17056128.
10. Boison D, Stewart KA (December 2009). "Therapeutic epilepsy research: from pharmacological rationale to focal adenosine augmentation". Biochemical Pharmacology. 78 (12): 1428–37. doi:10.1016/j.bcp.2009.08.005. PMC 2766433. PMID 19682439.
11. McCown TJ (July 2006). "Adeno-associated virus-mediated expression and constitutive secretion of galanin suppresses limbic seizure activity in vivo". Molecular Therapy. 14 (1): 63–8. doi:10.1016/j.ymthe.2006.04.004. PMID 16730475.
12. Mazarati AM, Halászi E, Telegdy G (August 1992). "Anticonvulsive effects of galanin administered into the central nervous system upon the picrotoxin-kindled seizure syndrome in rats". Brain Research. 589 (1): 164–6. doi:10.1016/0006-8993(92)91179-i. PMID 1384926.
13. Löscher W, Gernert M, Heinemann U (February 2008). "Cell and gene therapies in epilepsy--promising avenues or blind alleys?". Trends in Neurosciences. 31 (2): 62–73. doi:10.1016/j.tins.2007.11.012. PMID 18201772. S2CID 33488218.
14. Simonato M (September 2014). "Gene therapy for epilepsy". Epilepsy & Behavior. 38: 125–30. doi:10.1016/j.yebeh.2013.09.013. PMID 24100249. S2CID 18881057.
15. Zafar R, King MA, Carney PR (February 2012). "Adeno associated viral vector-mediated expression of somatostatin in rat hippocampus suppresses seizure development". Neuroscience Letters. 509 (2): 87–91. doi:10.1016/j.neulet.2011.12.035. PMID 22245439. S2CID 34166460.
16. Wykes RC, Heeroma JH, Mantoan L, Zheng K, MacDonald DC, Deisseroth K, et al. (November 2012). "Optogenetic and potassium channel gene therapy in a rodent model of focal neocortical epilepsy". Science Translational Medicine. 4 (161): 161ra152. doi:10.1126/scitranslmed.3004190. PMC 3605784. PMID 23147003.
17. Snowball A, Chabrol E, Wykes RC, Shekh-Ahmad T, Cornford JH, Lieb A, et al. (April 2019). "Epilepsy Gene Therapy Using an Engineered Potassium Channel". The Journal of Neuroscience. 39 (16): 3159–3169. doi:10.1523/JNEUROSCI.1143-18.2019. PMC 6468110. PMID 30755487.
18. Tønnesen J, Sørensen AT, Deisseroth K, Lundberg C, Kokaia M (July 2009). "Optogenetic control of epileptiform activity". Proceedings of the National Academy of Sciences of the United States of America. 106 (29): 12162–7. Bibcode:2009PNAS..10612162T. doi:10.1073/pnas.0901915106. PMC 2715517. PMID 19581573.
19. Wykes RC, Heeroma JH, Mantoan L, Zheng K, MacDonald DC, Deisseroth K, et al. (November 2012). "Optogenetic and potassium channel gene therapy in a rodent model of focal neocortical epilepsy". Science Translational Medicine. 4 (161): 161ra152. doi:10.1126/scitranslmed.3004190. PMC 3605784. PMID 23147003.
20. Krook-Magnuson E, Armstrong C, Oijala M, Soltesz I (2013-01-01). "On-demand optogenetic control of spontaneous seizures in temporal lobe epilepsy". Nature Communications. 4: 1376. Bibcode:2013NatCo...4.1376K. doi:10.1038/ncomms2376. PMC 3562457. PMID 23340416.
21. Paz JT, Davidson TJ, Frechette ES, Delord B, Parada I, Peng K, et al. (January 2013). "Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury". Nature Neuroscience. 16 (1): 64–70. doi:10.1038/nn.3269. PMC 3700812. PMID 23143518.
22. Kätzel D, Nicholson E, Schorge S, Walker MC, Kullmann DM (May 2014). "Chemical-genetic attenuation of focal neocortical seizures". Nature Communications. 5: 3847. Bibcode:2014NatCo...5.3847K. doi:10.1038/ncomms4847. PMC 4050272. PMID 24866701.
23. Weston M, Kaserer T, Wu A, Mouravlev A, Carpenter JC, Snowball A, et al. (April 2019). "Olanzapine: A potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics". Science Advances. 5 (4): eaaw1567. Bibcode:2019SciA....5.1567W. doi:10.1126/sciadv.aaw1567. PMC 6469940. PMID 31001591.
24. Lieb A, Qiu Y, Dixon CL, Heller JP, Walker MC, Schorge S, Kullmann DM (September 2018). "Biochemical autoregulatory gene therapy for focal epilepsy". Nature Medicine. 24 (9): 1324–1329. doi:10.1038/s41591-018-0103-x. PMC 6152911. PMID 29988123.
25. Colasante G, Lignani G, Brusco S, Di Berardino C, Carpenter J, Giannelli S, et al. (January 2020). "dCas9-Based Scn1a Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice". Molecular Therapy. 28 (1): 235–253. doi:10.1016/j.ymthe.2019.08.018. PMC 6952031. PMID 31607539.
26. Arsianti M, Lim M, Khatri A, Russell P, Amal R (2008). "Promise of Novel Magnetic Nanoparticles for Gene Therapy Application: Synthesis, Stabilisation, and Gene Delivery". Chemeca 2008: Towards a Sustainable Australasia: 734.
27. Scherer F, Anton M, Schillinger U, Henke J, Bergemann C, Krüger A, et al. (January 2002). "Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo". Gene Therapy. 9 (2): 102–9. doi:10.1038/sj.gt.3301624. PMID 11857068.
28. Krook-Magnuson E, Soltesz I (March 2015). "Beyond the hammer and the scalpel: selective circuit control for the epilepsies". Nature Neuroscience. 18 (3): 331–8. doi:10.1038/nn.3943. PMC 4340083. PMID 25710834.
29. Kullmann DM, Schorge S, Walker MC, Wykes RC (May 2014). "Gene therapy in epilepsy-is it time for clinical trials?". Nature Reviews. Neurology. 10 (5): 300–4. doi:10.1038/nrneurol.2014.43. PMID 24638133. S2CID 16544426.