AVS 406 Review Paper


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Review paper written for AVS 406 - "The Use of Recombinant Adeno-Associated Viral Vectors in Gene Therapy to Treat Epilepsy".

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AVS 406 Review Paper

  1. 1. The Use of Recombinant Adeno-Associated Viral Vectors in Gene Therapy to Treat Epilepsy Omega CantrellAbstract Epilepsy is defined as being recurrent, unprovoked seizures, and naturally occurs in awide range of species. It affects approximately 1% of the U.S. population, and is a goodtarget for gene therapy by recombinant adeno-associated virus (rAAV) vectors. rAAV iscapable of stably transferring foreign genetic material to neurons (transduction), inaddition to being able to infect a wide range of organisms and cell types, but, in vectorform, is not pathogenic. The neuropeptides galanin (GAL) and neuropeptide Y (NPY) arefound abundantly in the central nervous system, have strong anticonvulsant effects andare promising transgene options for rAAV vectors. Galanin is thought to have more of animpact than NPY. Studies have shown that GAL is able to reduce time spent in seizuresby 77% and number of EEG-detected seizures by 40%. Following seizures induced byelectrical stimulation in the perforant path of the hippocampus, the number of GAL-positive neurons was 16 times that of the control group. Because GAL was able to moresignificantly decrease the rate of seizure occurrence and total time spent in seizureactivity, it is a better option as a transgene for rAAV in gene therapy for epilepsy andother seizure disorders.Key words: epilepsy, gene therapy, recombinant adeno-associated virus (rAAV),galanin (GAL), neuropeptide Y (NPY), transgene 1
  2. 2. Introduction Normal brain function involves neuron-to-neuron transmission, causing signals fromthe brain to be sent to the rest of the body. For this to occur, sodium channels open, Figure 1. Normal brain wave patterns. allowing sodium (Na+) ions to enter. This operates on a negative feedback loop, such that potassium acts to cease signaling by closing the sodium channels. As Figure 1 illustrates, on an electroencephalograph (EEG), normal brain patterns appear as small http:/www.epilepsy.org.au/images/ElectroEn waves. When abnormal brain waves are seen cephalogram.png on an EEG, a seizure disorder is usually toblame (NIH, 2004; Hains, 2006). Epilepsy is a widely occurring seizure disorder in many species, including rats, dogs,and cats, and is the most common acquired neurological disorder in humans (Chandler,2006). It has been defined as recurrent Figure 2. Brain wave patterns observed during an epileptic seizure.unprovoked seizures, and has a prevalence of0.5-1.0% in humans, with a higher rate ofoccurrence in the underdeveloped world(Ransom and Blumenfeld, 2007).There aremany different kinds of epilepsy, and thesedisorders are diagnosed based on many factors. http://brain.fuw.edu.pl/~suffa/SW/SW_patt.gif 2
  3. 3. Figure 2 illustrates EEG-detected abnormalities, and other factors include age of onset,characteristics of the seizure, and seizure induction stimuli (Chandler, 2006). As can beseen in figure 2, epileptic brain waves appear as high spikes in rapid succession on anEEG. Seizure intensity is determined by the height of the spikes, coupled with the rate ofsuccession. Higher, more rapid spikes are indicative of a more severe seizure (Hains,2006). Epilepsy is thought to be caused by an imbalance in neurotransmissions (Vezzani,2004). As mentioned before, normal neurotransmissions are facilitated by a negativefeedback loop between sodium, which opens the channels, and potassium, which closesthe channels. In a normal brain, neurons fire approximately 30 times each second. Duringa seizure, neuronal firing can occur as many as 500 times each second (NIH, 2004).Normally, when excitatory neurotransmitters such as glutamate are released, neuronalfiring occurs. Once this reaches a certain level (which varies among individuals, andaccording to the type of signaling that is occurring), another negative feedback loopoccurs. When this happens, an inhibitory neurotransmitter such as γ-aminobutyric acid(GABA) serves to stop the action of the excitatory neurotransmitters and cease neuronalfiring (NIH, 2004). Because of this, another common theory is that either a high level ofexcitatory neurotransmitters or low level of inhibitory neurotransmitters are responsiblefor the abnormal neuronal firing that causes seizures to occur. Having just one seizure does not mean a person has epilepsy. In order to beconsidered for a diagnosis of epilepsy, a person must have had two or more seizures.There are two main categories of epilepsy – focal (or partial), which affects only one partof the brain. This includes temporal lobe epilepsy, which, according to Ransom and 3
  4. 4. Blumenfeld (2007), is more common in adults, and is often resistant to medical therapy.Generalized seizures affect both of the brain’s hemispheres simultaneously (NIH, 2004).As detailed previously, a seizure occurs when an imbalance in neurotransmitters causessodium channels in the brain to remain open, resulting in abnormal neuronal firing. Thisabnormal firing results in symptoms of the many different types seizures, includingmuscle spasms, repeated movements (also called automatisms), loss of consciousness,and/or temporary loss of muscle tone. These disorders are commonly controlled with medications that operate in a variety ofways, and are prescribed according to the type or types of seizures the patient has. Acommon mode of action for an antiepileptic medication is to either close or block thesodium channels in the brain, preventing neuronal hyperactivity. In doing this, thesedrugs can help to control seizures, but are not guaranteed to prevent seizure occurrence inall who take them. According to Riban et al. (2009), about one-third of diagnosedepileptics suffer from a form of the disease resistant to anticonvulsant drugs. In thesemore severe cases, surgery may be necessary. This is often a last resort for doctors, and is Figure 3. The human brain,usually done only if a patient continues to suffer from arrow pointing to the corpus callosum.seizures after years of treatmentwith different types ofanticonvulsant drugs. TheNational Institutes of Healthrecognizes only three categoriesof seizures that can be treatedsuccessfully with surgery, one of http://static.guim.co.uk/sys- images/Guardian/Pix/pictures/2009/4/6/1239055 717363/Cross-section-of-the-huma-001.jpg 4
  5. 5. these being focal seizure disorders, which includes temporal lobe epilepsy, and has a 64%success rate when compared with treatment with prescription medication alone (NIH,2004). Surgery should not be taken lightly, however, as it often involves either removalof an area in the brain where seizures are observed to occur most frequently (seizurefocus area), or, in more severe cases, cutting the corpus callosum (illustrated in figure 3).This disconnects the two hemispheres of the brain, and does not stop those seizuresfocused in one area of the brain (focal seizures), but merely prevents them fromspreading across the whole brain (NIH, 2004). Because there are so few options forepilepsy treatment, and such a high incidence of medication-resistant epilepsies, genetherapy – especially when mediated by recombinant adeno-associated virus (rAAV) – isconsidered to be a promising new option for those who suffer from a more severe seizuredisorder. To better understand how rAAVs are used for gene therapy, it is necessary tounderstand how a vector is constructed. A virus operates by first infecting a host cell,then replicating its own genome inside a host cell. This amplifies viral infection in thehost organism, and after the host cells are lysed, these viruses can further infect more hostcells, resulting in a widespread infection. Viral vectors are genetically engineered in thesense that their life cycles are manipulated such that the beneficial stages responsible for Figure 4. Genome of an AAV vector, introverted terminal repeat sequences (ITRs) in red boxes. Gene Therapy Approaches in Neurology (2007). genomic replication are retained, whilethe deleterious stages responsible for viral replication and cell lysis are eliminated(Burton et al., 2007). Figure 4 depicts the relatively small (~4.5kb) rAAV genome, which 5
  6. 6. codes for just two genes: rep and cap, and has an introverted terminal repeat sequence(ITRs) at each end of its single-stranded DNA (Carter, 2008). To construct a vector, repand cap are first removed and then replaced with a coding sequence of a similar length(4.5kb), while the ITRs are retained as “stops” for the genetic code (Burton et al., 2007).The removal of rep has two purposes: the first – and the most obvious reason – is to makeroom for the coding sequence intended for therapy, and the second being to make rAAVsincapable of attaching to human DNA. While not a problem with animals tested in thelaboratory, the rep gene allows rAAVs to adhere to a site on human DNA chromosome19 (Carter, 2008). Because this is a potential risk, rep is removed. Recombinant adeno-associated virus (rAAV) is an example of a viral vector – agenetically engineered virus (Burton et al., 2007) – and has garnered attention in recentyears as a promising tool in gene therapy to potentially treat disorders such as epilepsy. Figure 5. An adeno-associated virus and its structural components. rAAV has several positive attributes for gene therapy, the most notable being the ability to stably introduce foreign genetic material into neurons (called Gene Therapy Approaches in Neurology (2007).transduction) (Carter, 2008) and the ability to remain in vivo as a latent form for longperiods of time (Samulski et al., 1999). Figure 5 is an illustration of an adeno-associatedvirus. While it is not able to carry a large amount of genetic material, Dong et al. (1998)have reported that recombinant AAV (rAAV) has a genome of approximately 4.5 kilo-base pairs (kb) in length, and consequently, can hold a coding sequence of up to 5kb 6
  7. 7. when the its genes are removed (Carter, 2008). This appears to be enough to packagemost complementary DNA (cDNA) sequences (Burton et al., 2007). These vectors arenot capable of replication, which means that it will not be able to spread farther than thesite of injection (Vezzani, 2004). The rAAV vector also has the ability to infect a widevariety of cell types (as well as host organisms), and this vector should be coupled with acell-specific promoter (such as a neuronal promoter) to restrict gene expression to thedesired area of infection (Vezzani, 2004). The AAV vector is incapable of replication, meaning that, when its genes have beenremoved, it is not able to replicate the newly-inserted genome in a host cell without aid.Therefore, a helper virus is required for AAV to replicate in vivo. Typically, this virus isan adenovirus, which is how this vector came to be known as an “adeno-associatedvirus”. However, a herpes simplex virus can also be used (Burton et al., 2007). With ahelper, AAV is now known as a recombinant adeno-associated virus (rAAV), and viralreplication can now occur, but does so only in the cell nucleus. It typically attaches tonon-mitotic (non-dividing) cells, neurons being a primary example (Carter, 2008). Thegenetic material carried by the vector is then transferred to the host cell, and rAAVs arenoted for the ability to stably transfer foreign genetic material to a host cell with littlereaction by the host cell’s immune system (Vezzani, 2004). Because it is very small, anrAAV vector is capable of infecting a wide range of cell type in a variety of organisms. Acell-specific promoter should also be used, so as to restrict gene expression to a particulararea or specific cell type (Carter, 2008). The most common method of rAAV production is referred to as the triple plasmidtransfection method, and involves the transfer of three plasmids to the vector (Burton et 7
  8. 8. al., 2007). These plasmids consist of the following: (1) packaging signals and thetransferred coding sequence, (2) a code which expresses the functions of an rAAV’s repand cap gene functions, and (3) a code expressing the helper virus (adenovirus or herpessimplex virus) helper functions (Burton et al., 2007). This vector is then best deliveredwithin the parts of the organ essential to its function, referred to as an intraparenchymalinjection. This must be done in small volumes (1-10 μl), and at a low flow rate (0.2-0.4μl/minute). The particles then appear to diffuse to the target cells, but because of both thecell-specific promoter in the vector and rAAVs’ limited replication, transduction istypically limited to only a few millimeters from the injection site (Burton et al., 2007).This results in a limited efficacy in areas requiring large volumes, but nonethelessremains the best way to deliver the treatment to date. The most common choices for coding sequences to replace the rAAV vector’sgenome (called transgenes) are galanin (GAL) and neuropeptide Y (NPY). Galanin is a Figure 6. The human brain, with the hippocampus highlighted in blue. neuropeptide composed of 29 amino acids. It is called a neuropeptide because it is concentrated in the central nervous system (CNS), is expressed only in neuronal cells, and is highly expressed in neurons in the http://upload.wikimedia.org/wikipedia/commons/2/2e/Gray7 39-emphasizing-hippocampus.png region of the brain known as thehippocampus (see figure 6)(Pieribone et al., 1998). In the CNS, galanin acts as aninhibitor for excitatory neurotransmitters. Neuropeptide Y (NPY) is a 36 amino acid 8
  9. 9. neuropeptide that is found abundantly in the CNS, and is also found in peripheral nervoustissue. (Dumont and Quirion, 2006). Like galanin, NPY acts by inhibiting excitatoryneurotransmitters and is found abundantly in the hippocampus. Both galanin and NPYhave been shown to dramatically increase in concentration after a seizure (Scharfman andGray, 2006), and have been shown to elicit an anticonvulsant effect in the body. Two types of studies of transgenes paired with rAAV vectors will be discussed: thosewith galanin (GAL), and those with neuropeptide Y (NPY).Galanin (GAL)-focused studiesLin et al. (2003) The work of Lin et al. (2003) utilized the hippocampus of adult male rats, withexperimental rats receiving an injection of Figure 7. The dorsal portion of the hippocampus (encircled in red).rAAV-GAL and controls receivingan injection of rAAV-empty(rAAV, without the galanintransgene). To restrict expression toneuronal cells, neuron-specificenolase (NSE) was used as a promoter in the http://www.brainybehavior.com/blog/wp- content/uploads/2008/11/gray747.pngtransgene-carrying rAAV vector. Figure 7illustrates the dorsal hippocampus, the section of the rats’ brains injected bilaterally witheither the rAAV-GAL or rAAV-empty vector. Later, these rats were subjected to seizuresto determine efficacy of the vector. Seizure analysis in these rats was determined viaelectroencephalogram (EEG) detection. To accomplish this, the animals were implantedwith electrodes and a guide cannula 2.5 months after the initial injection of rAAV-NSE- 9
  10. 10. Figure 8. Effects of rAAV-empty and rAAV-GAL onnumber of observed seizures in rats.GAL or rAAV-empty (no transgene present). Following implantation, the rats were given kainic acid to induce seizure activity, and were then monitored on the EEG and analyzed for number of seizures as well as time spent in seizure activity, as detected by EEG analysis by a blind party. Figure 8 Lin et al. (2003)depicts the 40% decrease in the amount of seizures observed, with an average of25 seizures noted in the control group compared to an average of 15 seizures in ratsinjected with rAAV-NSE-GAL. As shown in figure 9, there was a 55% decrease in totaltime spent in kainic acid-induced seizure activity. To determine galanin expression,another group of rats were injected unilaterally in the right dorsal hippocampus withrAAV-NSE-GAL. These rats were not subjected to seizures, but were killed 2.5 months Figure 9. Effects of rAAV-empty and rAAV-GAL on total after vector injection. Upon time in seizure activity. analysis of the hippocampal areas of these animals, it was found that there was a higher amount of GAL in the right dorsal side (injected Lin et al. (2003) with rAAV-NSE-GAL) than in the non-injected (left dorsal) side ofthe hippocampus.Mazarati et al. (1998) Mazarati et al. (1998) also studied rAAV-NSE-GAL in the rat hippocampus. Thisstudy was done on 8-10 week old rats, and was included as part of a later study of theeffects of four neuropeptides – including NPY – in the rat hippocampus during aprolonged seizure, referred to as status epilepticus (SE). The animals were subjected to 10
  11. 11. Figure 10. The perforant path of the hippocampus (in blue).either electricalstimulation in theperforant path toinduce statusepilepticus (SE). Asits name implies, perforant path stimulation (PPS) http://www.nature.com/neuro/journal/ v10/n3/images/nn0307-271-F1.gifoccurs in the perforant path of the hippocampus(see figure 10). This area is neuron-dense, and acts as the inputpathway in the hippocampus (MRC, 2003). As a result, excess stimulation here willresult in prolonged seizure activity. For quantitative analysis, the rats were implanted Figure 11. The dentate gyrus of the with a bipolar stimulating electrode in the perforant hippocampus (encircled in red). path, as well as a bipolar recording electrode in the dentate gyrus of the hippocampus, encircled in red in figure 11. Subjects received an injection of varying concentration of galanin either 30 minutes before the beginning http://www.brainybehavior.com/blog/wp -content/uploads/2008/11/gray747.png of PPS or 30 minutes after its conclusion.Controls received an injection of 0.9% NaCl. Figure 12. The effects of galanin on time spent in seizure activity between controls and GAL-treated rats.Figure 12 illustrates the findingsthat those rats treated with higherconcentrations of galanin 30minutes prior to PPS were 11 Mazarati et al. (1998)
  12. 12. observed to have spent 95% less time in seizure activity than the controls. Animals werekilled at varying time intervals following the conclusion of PPS to determine theconcentration of GAL-infected neurons in and around the injection site. Figure 13illustrates the concentration of GAL-positive neurons in control and GAL-treated rats. 24hours after PPS, an average of 16 GAL-positive neurons were found in a slice of thehippocampus of GAL-treated rats, while none were found in non-GAL treated rats at any Figure 13. Difference in GAL-positivetime following the conclusion of neurons after SSSE.PPS. However, 3 days after PPS,this number has graduallydecreased to an average of 8 GAL-positive neurons in GAL-treatedrats, and decreased to 6 GAL-positive neurons 7 days after the conclusion of PPS. In Mazarati et al. (1998) Mazarati and Wasterlain’s 2002 study of Figure 14. Difference in total time spent in seizure activity between control and GAL-treated rats. rAAV-GAL, it was found that, compared to 590 minutes spent in seizure activity in the rAAV-empty control, total time spent in seizures for rAAV-GAL treated animals was Mazarati and Wasterlain (2002)under 10 minutes, as is illustrated in figure 14. 12
  13. 13. Haberman et al. (2003) In another study of the effects of galanin, Haberman et al. Figure 15. The inferior collicular cortex (in yellow) of the (2003) found that it is able to reduce seizure intensity. This brainstem. experiment used a vector constructed with a fibronectin secretory signal sequence (FIB) as its promoter. The rats used in this study were implanted with a stimulating electrode, and the inferior collicular cortex (as shown in figure 15, highlighted in yellow) of these animals’ brainstems was infused with either rAAV-FIB-GAL (experimental) or rAAV-GAL (control). Four days after treatment, seizure threshold was determined, and the rats were re-examined once per week for 4 weeks thereafter. In this period, http://upload.wikimedia.org/wikipedia/ commons/0/00/Gray685.png it was found that the threshold for seizure genesiswas 60% higher in those animals treated with rAAV-FIB-GAL. Following this, theanimals were given water with doxycycline, Figure 16. Seizure threshold comparison of control group and rAAV-FIB-GAL-treated rats.which returned the threshold tobaseline levels within 1 week.After doxycycline was removed,rAAV-FIB-GAL-treated rats wereobserved to have a graduallyincreasing threshold for seizuregenesis, with a threshold Haberman et al. (2003)approximately 30% higher in 13
  14. 14. galanin-treated rats than those in the control group. Figure 16 illustrates these results. Todetermine if treatment was able to prevent seizures, animals were injected with eitherrAAV-FIB-GAL, rAAV-GAL, or received no treatment at all. In vitro, rAAV-FIB-GALor rAAV-GAL was introduced into HEK 293 cells. Twenty-four hours later, the mediawere analyzed via ELISA for the presence of galanin. The rAAV-GAL cells showed nodetectable amount of GAL, indicating that it was not expressed or secreted in the cells,but those cells infected with rAAV-FIB-GAL showed a significant amount (32ng/mL).Neuropeptide Y (NPY)-focused studiesRichichi et al. (2004) Other studies focused on the effects of rAAV-delivered neuropeptide Y (NPY) in therat brain, more specifically, the hippocampus. Richichi et al. (2004) conducted anexperiment on adult male rats using rAAV-NSE-NPY. Neuron-specific enolase (NSE)functioned as a cell-specific promoter in this vector. Two subtypes (called serotypes anddetermined by protein markers on the cell surface) of rAAV were used in this experiment– serotype 2, and a mixture of serotypes 1 and 2 (called serotype 1/2). The hippocampusof these rats was injected bilaterally with the vector on each side. Eight weeks later, theanimals were implanted with electrodes and cannulas, and 4 days after implantation,received a unilateral injection of kainic acid in either the dorsal hippocampus to induce Figure 17. Difference in observed seizure activity and onset time of seizures. Richichi et al. (2004) seizure activity. Seizure activity was measured viaelectroencephalogram (EEG) analysis by a blind party, and was done before injecting the 14
  15. 15. animals with kainic acid, and up to 3 hours following the injection. As shown in figure17, time for seizure onset was delayed almost twofold in both serotypes of rAAV-NSE-NPY, with an average of 11.5 minutes observed in serotype 1/2, compared to an averageof 6.2 minutes in the control group. In these subjects, those treated with serotype 1/2 hadno EEG-detected episodes of status epilepticus (SE), but in the control group, SE lasted atleast 60 minutes.Mazarati and Wasterlain (2002) Mazarati and Wasterlain (2002) conducted a study of the effects of fourneuropeptides in the rat hippocampus, one of these neuropeptides being GAL, andanother being NPY. This study used 8-10 week old male rats, which were implanted witha bipolar stimulating electrode in the perforant path of the hippocampus (see figure 10),as well as a combination of a bipolar recording electrode and guide cannula in the dentategyrus of the hippocampus (see figure 11). These animals were subjected to 30 minutes ofperforant path stimulation (PPS) 7-10 days later, to induce a state of SSSE. Recordings ofbrain activity were made via EEG by a blind party during PPS and 24 hours after itsconclusion. Spike distribution for this activity was measured in 30 minute periods, andseizure activity was assessed according to total time spent in seizure activity, timebetween the end of PPS and occurrence of the last seizure, and time spent in seizureactivity in a 1 hour period during a state of prolonged seizure activity (called self-sustaining status epilepticus, or SSSE). Ten minutes after the conclusion of PPS, thedentate gyrus of the rats were injected with NPY. Controls were injected with the samedose of 0.9% NaCl. As illustrated by figure 18, there was no significant difference notedin time spent in EEG-detected seizure activity between the control (average time in 15
  16. 16. seizure activity: 15 minutes) and NPY-treated group (average time in seizure activity: 13minutes). NPY-treated animals were found to have spent 4 hours total in seizure activity,and SSSE decreased to less than 20 minutes in these animals.Discussion Mazarati et al. (1998) showed that, when injected Figure 18. Time in seizures following PPS treatment.into the dentate gyrus, galanin has a seizure-protectingeffect. In this situation, galanin prevents theinitiation of self-sustaining status epilepticus(SSSE). When injected after perforant pathstimulation was performed, galanin was ableto stop the maintenance phase of anestablished SSSE, an accomplishment evenanticonvulsant drugs could not achieve.Similarly, Lin et al. (2003) showed that whenrAAV-NSE-GAL was used, an over- Mazarati and Wasterlain (2002)expression of GAL by the vector was able todrastically reduce seizure activity brought about by an intrahippocampal injection ofkainic acid, in addition to producing a strong anticonvulsant effect. Neuropeptide Y (NPY) in neuronal cells causes a decrease in both seizuresusceptibility and epileptogenesis, and therefore, the use of rAAV-NSE-NPY for over-expression of NPY in rat hippocampal cells was used to inhibit seizures andepileptogenesis (Richichi et al., 2004). In these rats, kainic acid-induced, EEG-detectedepisodes of prolonged seizure activity (status epilepticus, or SE) were not observed in 16
  17. 17. NPY-treated rats, but episodes lasting at least 60 minutes were observed in the controlgroup. A delay in onset of seizures was observed in these animals, with NPY-treated ratshaving an average delay time of approximately 11.5 minutes on average, while theaverage delay for the control group was observed to be 6.2 minutes, almost half that ofthe experimental group. Animals that received an injection of kainic acid showed adecrease in EEG-detected seizure activity induced by the injection, due to rAAV vector-mediated NPY over-expression. EEG-detected episodes of SE were eliminated in ratsexpressing the NPY transgene in multiple areas of the hippocampus. Richichi et al.(2004) concluded that the ability of NPY to inhibit the release of excitatoryneurotransmitters from brain cells may be the cause of the decrease in seizures noted inrats injected with rAAV-NSE-NPY. In the hippocampus, NPY has inhibitory properties, acting as an anticonvulsant.According to Mazarati and Wasterlain (2002), mice that lack NPY are more inclined todevelop seizures. In their study, Mazarati and Wasterlain (2002) were able to show that,after injecting the dentate gyrus in rats with rAAV-delivered NPY, SSSE episodes weresignificantly attenuated, though only a 2-minute difference in time spent in seizures wasobserved between the experimental and control groups. This indicates that there was nosignificant difference in time spent in seizures when comparing NPY-treated rats to thesaline-injected controls.Conclusions In order to determine the most effective method of rAAV-mediated gene therapy,many aspects must be considered. These include the level of expression of the transgeneincluded in the vector, length of time spent in seizure activity, length of time between 17
  18. 18. seizure activity, and number of seizures observed. By analyzing rAAV-mediatedexperiments involving one or both of the neuropeptides of interest, NPY and GAL can becompared and contrasted, and a conclusion regarding efficacy can be more easilydetermined. In the studies conducted regarding rAAV vectors containing the GAL transgene, therewas a considerable reduction in seizure activity. There was an average decrease of 77%in time spent in seizure activity, as well as a 40% decrease in number of seizuresobserved. There was also found to be a large quantity of galanin expression and secretionin cells in and around the injection site in the hippocampus, with 16 times more galanin-positive cells found in galanin-treated animals than in the control group. The experimentsconducted with rAAV-NPY vectors also showed a decrease in EEG-detected seizureactivities, but it was only a 13% decrease – not nearly as vast as that observed inexperiments conducted with rAAV-GAL vectors. When compared, each neuropeptide was observed to have an anticonvulsant effect, aswell as the ability to decrease time spent in seizure activities, decrease time betweenseizures, and to decrease the number of seizures observed in the tested animals comparedto the control. However, rAAV-GAL vectors were observed to have a greater effect thanthe rAAV-NPY vectors and because of this, rAAV vectors utilizing galanin as atransgene for expression and secretion in neuronal cells are the best choice for genetherapy. While these vectors have worked well in rodent models of seizures, there are stillmany obstacles that must be surpassed before this treatment can be applied to humans.These include establishing an effective, minimally invasive method for vector delivery, 18
  19. 19. as well as receiving approval from the Food and Drug Administration (FDA) fortreatment. Once established, these tests may be able to help control seizures in humanssuffering from epileptic disorders. AAV-mediated gene therapy is a promising newtherapy, and will hopefully one day become a widespread, successful treatment for notjust epilepsy, but many other human seizure disorders.ReferencesBurton, E.A., J.C. Glorioso, and D.J. Fink. 2007. Gene Therapy Approaches inNeurology. In Molecular Neurology. Ed. S.G. Waxman. Elsevier Academic Press.Burlington, MA. 101-123.Carter, B.J. 2008. Adeno-Associated Virus Vectors. In Concepts in Genetic Medicine.Ed. B. Dropulic and B.J. Carter. Wiley-Liss. Hoboken, NJ. 61-68.Chandler, K. 2006. Canine epilepsy: What can we learn from human seizure disorders?The Veterinary Journal. 172: 207-217.Dong, J.Y., P.D. Fan, and R.A. Frizell. 1996. Quantitative analysis of the packagingcapacity of recombinant adeno-associated virus. Human Gene Therapy. 7: 2102-2112.Dumont, Y. and R. Quirion. 2006. An overview of neuropeptide Y: pharmacology tomolecular biology and receptor localization. In NPY Family of Peptides in Neurobiology,Cardiovascular and Metabolic Disorders: from Genes to Therapeutics. Ed. Z. Zukowskaand G.Z. Feuerstein. Birkhaüser Verlag. Basel, Switzerland. 7-33.Haberman, R.P., R.J. Samulski, and T. J. McCown. 2003. Attenuation of seizures andneuronal death by adeno-associated virus vector galanin expression and secretion. NatureMedicine. 9(8): 1076-1080.Haberman , R.P., T.J. McCown, and R.J. Samulski. 1998. Inducible long-term geneexpression in brain with adeno-associated virus gene transfer. Gene Therapy. 5: 1604-1611.Hains, B. 2006. Brain Disorders. Chelsea House Publishers. Philadelphia. 37-38.Lin, E.D., C. Richichi, D. Young, K. Baer, A. Vezzani, and M.J. During. 2003.Recombinant AAV-mediated expression of galanin in rat hippocampus suppressesseizure development. European Journal of Neuroscience. 18: 2087-2092. 19
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