AVS 406 The Use of Recombinant Adeno-Associated ViralFall 2009 Vectors in Gene Therapy to Treat Epilepsy Omega CantrellAbstract Galanin-focused studies Figure 7. Total time spent in seizure activity for rats in the control group is depicted in figure 7a. Total time spent in seizure activity for NPY-treated rats is depicted in figure 7b.Epilepsy is defined as being “recurrent, unprovoked seizures”, and naturally occurs in a wide In Lin et al.’s 2003 study, an rAAV vector using neuron-specific enolase (NSE) as a promoterrange of species. It affects approximately 1% of the U.S. population, and is a good target for was used with a galanin (GAL) transgene, and was injected into the dorsal hippocampus of thegene therapy by recombinant adeno-associated virus (rAAV) vectors. rAAV is capable of stably rat brain. Kainic acid was then used to induce seizure activity. As figure 3a illustrates, EEG-transferring foreign genetic material to neurons (transduction), in addition to being able to detected seizure activity was shown to decrease by 40% in GAL-treated animals compared toinfect a wide range of organisms and cell types, but, in vector form, is not pathogenic. The the rAAV-empty injected controls. Additionally, time spent in seizure activity was shown to Figure 3. Number of EEG-detected seizures is depicted in figure decrease 55%, from an average of 25 minutesneuropeptides galanin (GAL) and neuropeptide Y (NPY) are found abundantly in the central 3a. Time spent in seizure activity is depicted in figure 3b.nervous system, have strong anticonvulsant effects and are promising transgene options for spent in seizurerAAV vectors. Galanin is thought to have more of an impact than NPY. Studies have shown activity in the control Mazarati and Wasterlain (2002)that GAL is able to reduce time spent in seizures by 77% and number of EEG-detected seizures animals to an averageby 40%. Following seizures induced by electrical stimulation in the perforant path of the of 13.5 minutes spenthippocampus, the number of GAL-positive neurons was 16 times that of the control group. in seizure activity in Conclusions GAL-treated animals. In the studies conducted regarding rAAV vectors containing the GAL transgene, there was aBecause GAL was able to more significantly decrease the rate of seizure occurrence and total Lin et al. (2003) Figure 3b depicts these findings. considerable reduction in seizure activity. There was an average decrease of 77% in time spent intime spent in seizure activity, it is a better option as a transgene for rAAV in gene therapy for In Mazarati and Wasterlain’s 2002 study of galanin and its effects on the rat hippocampus, a seizure activity, as well as a 40% decrease in number of seizures observed. The experimentsepilepsy and other seizure disorders. more drastic decrease was observed. The results of this study are illustrated in figure 5. In this conducted with rAAV-NPY vectors also showed a decrease in EEG-detected seizure activities, butKey words: epilepsy, gene therapy, recombinant adeno-associated virus (rAAV), galanin study, the control group was Figure 5. Time spent in seizures, control and galanin-treated animals in red boxes. it was only a 13% decrease – not nearly as vast as that observed in experiments conducted with(GAL), neuropeptide Y (NPY), transgene shown to have undergone rAAV-GAL vectors. approximately 590 minutes in total When compared, each neuropeptide was observed to have an anticonvulsant effect, as well asIntroduction seizure activity, whereas the the ability to decrease time spent in seizure activities, decrease time between seizures, and toEpilepsy is the most common neurological disorder in humans, with a prevalence of 0.5-1.0% galanin-treated animals spent less decrease the number of seizures observed in the tested animals compared to the control.(Chandler, 2006; Ransom and Blumenfeld, 2007). It is thought to be caused by an imbalance in than 10 minutes in total seizure However, rAAV-GAL vectors were observed to have a greater effect than the rAAV-NPY vectorsneurotransmitters, causing the neuronal hyperactivity responsible for seizure episodes activity. This is a tremendous and because of this, rAAV vectors utilizing galanin as a transgene for expression and secretion in(NIH, 2004). These disorders can often be controlled with medications or surgery, but up to difference, and is far too large to neuronal cells are the best choice for gene therapy.one-third of epileptics are resistant to medication (Riban et al., 2009), and only a small attribute to chance alone. Without While these vectors have worked well in rodent models of seizures, there are still manypercentage of epileptics are eligible for surgery, which has inherent risks of potential brain obstacles that must be surpassed before this treatment can be applied to humans. These include question, galanin was able to prevent the occurrence of EEG- Mazarati and Wasterlain (2002)damage (NIH, 2004). Recombinant adeno-associated viral (rAAV) vectors are an attractive establishing an effective, minimally invasive method for vector delivery, as well as receiving detected seizures in the animals used in this experiment.target for gene therapy to treat epilepsy primarily because they are capable of stably approval from the Food and Drug Administration (FDA) for treatment. Once established, theseintroducing foreign genetic material into neurons without eliciting a response from the host Neuropeptide Y-focused studies tests may be able to help control seizures in humans suffering from epileptic disorders. rAAV-cell’s immune system (Carter, 2008). Figure 1 illustrates the rAAV genome. To Figure 1. The rAAV genome, ITRs in red boxes. mediated gene therapy is a promising new therapy, and one day will hopefully become a Richichi et al. (2004) used kainic acid, an excitatory neurotransmitter agonist, to induceengineer the vector, all coding widespread, successful treatment not just for epilepsy, but many other human seizure disorders prolonged seizures (status epilepticus, or SE) in rats. The results of this study can be seen insequences other than the as well. Gene Therapy Approaches in Neurology (2007). figure 6. A seizure onset delay of approximately 11.5 minutes was observed in NPY-treatedintrovertedterminal repeat sequences (ITRs) are removed. Figure 6. Comparison of control (rAAV-NSE-empty) rats and experimental (rAAV-NSE-NPY) rats.Figure 2 illustrates how a viral vector is constructed. Following the removal of rAAV genes, References Treatment Onset (min) Number of Time in discrete Time in status Total time in Burton, E.A., J.C. Glorioso, and D.J. Fink. 2007. Gene Therapy Approaches in Neurology. In Molecular Neurology. Ed. S.G. Waxman.Figure 2. The construction of an rAAV vector. the desired coding sequence (transgene) is added to the seizures seizures (min) epilepticus (min) seizures (min) Elsevier Academic Press. Burlington, MA. 101-123. vector, in addition to a cell-specific promoter (to restrict Carter, B.J. 2008. Adeno-Associated Virus Vectors. In Concepts in Genetic Medicine. Ed. B. Dropulic and B.J. Carter. Wiley-Liss. rAAV-NSE-empty 6.2 ±0.3 18.0±1.0 53.5±6.0 86.9±10.1 137.0±7.9 Hoboken, NJ. 61-68. expression to the targeted area), as well as a helper virus rAAV-NSE-NPY 11.5±1.8 23.0±6.0 53.4±9.7 0 53.4±9.7 Chandler, K. 2006. Canine epilepsy: What can we learn from human seizure disorders? The Veterinary Journal. 172: 207-217. to assist in replication functions (Burton et al., 2007). The Lin, E.D., C. Richichi, D. Young, K. Baer, A. Vezzani, and M.J. During. 2003. Recombinant AAV-mediated expression of galanin in rat Richichi et al. (2004) rats, compared to 6.2 minutes in the control group. There were also hippocampus suppresses seizure development. European Journal of Neuroscience. 18: 2087-2092. most common transgene choices are the neuropeptides no episodes of staus epilepticus in NPY-treated groups, whereas SE episodes lasted an average Mazarati, A.M and C.G. Wasterlain. 2002. Anticonvulsant effects of four neuropeptides in the rat hippocampus during self-sustaining galanin (GAL) and neuropeptide Y (NPY). Both are status epilepticus. Neuroscience Letters. 331: 123-127. of 87 minutes in the control group. abundantly expressed in the hippocampus of the National Institutes of Health. 2004. National Institute of Neurological Disorders and Stroke. Seizures and Epilepsy: Hope Through In Mazarati and Wasterlain’s 2002 study, there was no significant difference noted in average Research. 13-27. brain, and display a strong anticonvulsant effect in the Pieribone, V.A., Z.D. Xu, X. Zhang, and T. Hökfelt. 1998. Electrophysiologic Effects of Galanin on Neurons of the Central Nervous time in one seizure (15 minutes in the control group, compared to 13 minutes in the NPY- System. In Annals of the New York Academy of Sciences. Ed. B.M. Boland, J. Cullinan, and A.C. Fink. New York Academy of Sciences. body (Pieribone et al., 1998). This review will attempt to New York. 264-273. treated group). However, as figures 7a and 7b show, the NPY-treated rats were observed tohttp://media.wiley.com/CurrentProtocols answer the question “Which transgene (GAL or NPY) is Ransom, C.B. and H. Blumenfeld. 2007. Acquired Epilepsy: Cellular and Molecular Mechanisms. In Molecular Neurology. Ed. S.G./HG/hg1209/hg1209-fig-0001-1-full.gif have spent approximately 4 hours in total seizure activity, spanning over an 8-hour period. In Waxman. Elsevier Academic Press. Burlington, MA. 347-370. the best option for gene therapy to treat epilepsy?” the control group, rats spent approximately 10 hours in total seizure activity, spanning over a Riban, V., H.L. Fitzsimons, and M.J. During. 2009. Gene therapy in epilepsy. Epilepsia. 50(1): 24-32. 21-hour period. Richichi, C, E.D. Lin, D. Stefanin, D. Colella, T. Ravizza, G. Grignaschi, P. Veglianese, G. Sperk, M.J. During, and A. Vezzani. 2004. Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus. Journal of Neuroscience. 24(12): 3051-3059.