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

1. The Use of Recombinant Adeno-Associated Viral Vectors in Gene Therapy to Treat
Epilepsy
Omega Cantrell
Abstract
Epilepsy is defined as being recurrent, unprovoked seizures, and naturally occurs in a
wide range of species. It affects approximately 1% of the U.S. population, and is a good
target for gene therapy by recombinant adeno-associated virus (rAAV) vectors. rAAV is
capable of stably transferring foreign genetic material to neurons (transduction), in
addition to being able to infect a wide range of organisms and cell types, but, in vector
form, is not pathogenic. The neuropeptides galanin (GAL) and neuropeptide Y (NPY) are
found abundantly in the central nervous system, have strong anticonvulsant effects and
are promising transgene options for rAAV vectors. Galanin is thought to have more of an
impact than NPY. Studies have shown that GAL is able to reduce time spent in seizures
by 77% and number of EEG-detected seizures by 40%. Following seizures induced by
electrical 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 more
significantly decrease the rate of seizure occurrence and total time spent in seizure
activity, it is a better option as a transgene for rAAV in gene therapy for epilepsy and
other seizure disorders.
Key words: epilepsy, gene therapy, recombinant adeno-associated virus (rAAV),
galanin (GAL), neuropeptide Y (NPY), transgene
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2. Introduction
Normal brain function involves neuron-to-neuron transmission, causing signals from
the 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 to
blame (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 of
0.5-1.0% in humans, with a higher rate of
occurrence in the underdeveloped world
(Ransom and Blumenfeld, 2007).There are
many different kinds of epilepsy, and these
disorders are diagnosed based on many factors.
http://brain.fuw.edu.pl/~suffa/SW/SW_patt.gif
2

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 be
seen in figure 2, epileptic brain waves appear as high spikes in rapid succession on an
EEG. Seizure intensity is determined by the height of the spikes, coupled with the rate of
succession. 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 negative
feedback loop between sodium, which opens the channels, and potassium, which closes
the channels. In a normal brain, neurons fire approximately 30 times each second. During
a seizure, neuronal firing can occur as many as 500 times each second (NIH, 2004).
Normally, when excitatory neurotransmitters such as glutamate are released, neuronal
firing occurs. Once this reaches a certain level (which varies among individuals, and
according to the type of signaling that is occurring), another negative feedback loop
occurs. When this happens, an inhibitory neurotransmitter such as γ-aminobutyric acid
(GABA) serves to stop the action of the excitatory neurotransmitters and cease neuronal
firing (NIH, 2004). Because of this, another common theory is that either a high level of
excitatory neurotransmitters or low level of inhibitory neurotransmitters are responsible
for the abnormal neuronal firing that causes seizures to occur.
Having just one seizure does not mean a person has epilepsy. In order to be
considered 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 part
of the brain. This includes temporal lobe epilepsy, which, according to Ransom and
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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 causes
sodium channels in the brain to remain open, resulting in abnormal neuronal firing. This
abnormal firing results in symptoms of the many different types seizures, including
muscle 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 of
ways, and are prescribed according to the type or types of seizures the patient has. A
common mode of action for an antiepileptic medication is to either close or block the
sodium channels in the brain, preventing neuronal hyperactivity. In doing this, these
drugs can help to control seizures, but are not guaranteed to prevent seizure occurrence in
all who take them. According to Riban et al. (2009), about one-third of diagnosed
epileptics suffer from a form of the disease resistant to anticonvulsant drugs. In these
more 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 treatment
with different types of
anticonvulsant drugs. The
National Institutes of Health
recognizes only three categories
of seizures that can be treated
successfully 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. 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 removal
of an area in the brain where seizures are observed to occur most frequently (seizure
focus 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 seizures
focused in one area of the brain (focal seizures), but merely prevents them from
spreading across the whole brain (NIH, 2004). Because there are so few options for
epilepsy treatment, and such a high incidence of medication-resistant epilepsies, gene
therapy – especially when mediated by recombinant adeno-associated virus (rAAV) – is
considered to be a promising new option for those who suffer from a more severe seizure
disorder.
To better understand how rAAVs are used for gene therapy, it is necessary to
understand 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 the
host organism, and after the host cells are lysed, these viruses can further infect more host
cells, resulting in a widespread infection. Viral vectors are genetically engineered in the
sense 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, while
the 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
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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, rep
and 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 make
room for the coding sequence intended for therapy, and the second being to make rAAVs
incapable of attaching to human DNA. While not a problem with animals tested in the
laboratory, the rep gene allows rAAVs to adhere to a site on human DNA chromosome
19 (Carter, 2008). Because this is a potential risk, rep is removed.
Recombinant adeno-associated virus (rAAV) is an example of a viral vector – a
genetically engineered virus (Burton et al., 2007) – and has garnered attention in recent
years 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 long
periods of time (Samulski et al., 1999). Figure 5 is an illustration of an adeno-associated
virus. 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. when the its genes are removed (Carter, 2008). This appears to be enough to package
most complementary DNA (cDNA) sequences (Burton et al., 2007). These vectors are
not capable of replication, which means that it will not be able to spread farther than the
site of injection (Vezzani, 2004). The rAAV vector also has the ability to infect a wide
variety of cell types (as well as host organisms), and this vector should be coupled with a
cell-specific promoter (such as a neuronal promoter) to restrict gene expression to the
desired area of infection (Vezzani, 2004).
The AAV vector is incapable of replication, meaning that, when its genes have been
removed, 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 is
an adenovirus, which is how this vector came to be known as an “adeno-associated
virus”. However, a herpes simplex virus can also be used (Burton et al., 2007). With a
helper, AAV is now known as a recombinant adeno-associated virus (rAAV), and viral
replication can now occur, but does so only in the cell nucleus. It typically attaches to
non-mitotic (non-dividing) cells, neurons being a primary example (Carter, 2008). The
genetic material carried by the vector is then transferred to the host cell, and rAAVs are
noted for the ability to stably transfer foreign genetic material to a host cell with little
reaction by the host cell’s immune system (Vezzani, 2004). Because it is very small, an
rAAV vector is capable of infecting a wide range of cell type in a variety of organisms. A
cell-specific promoter should also be used, so as to restrict gene expression to a particular
area or specific cell type (Carter, 2008).
The most common method of rAAV production is referred to as the triple plasmid
transfection method, and involves the transfer of three plasmids to the vector (Burton et
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8. al., 2007). These plasmids consist of the following: (1) packaging signals and the
transferred coding sequence, (2) a code which expresses the functions of an rAAV’s rep
and cap gene functions, and (3) a code expressing the helper virus (adenovirus or herpes
simplex virus) helper functions (Burton et al., 2007). This vector is then best delivered
within the parts of the organ essential to its function, referred to as an intraparenchymal
injection. This must be done in small volumes (1-10 μl), and at a low flow rate (0.2-0.4
μl/minute). The particles then appear to diffuse to the target cells, but because of both the
cell-specific promoter in the vector and rAAVs’ limited replication, transduction is
typically 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 nonetheless
remains the best way to deliver the treatment to date.
The most common choices for coding sequences to replace the rAAV vector’s
genome (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 the
hippocampus (see figure 6)(Pieribone et al., 1998). In the CNS, galanin acts as an
inhibitor for excitatory neurotransmitters. Neuropeptide Y (NPY) is a 36 amino acid
8

9. neuropeptide that is found abundantly in the CNS, and is also found in peripheral nervous
tissue. (Dumont and Quirion, 2006). Like galanin, NPY acts by inhibiting excitatory
neurotransmitters and is found abundantly in the hippocampus. Both galanin and NPY
have been shown to dramatically increase in concentration after a seizure (Scharfman and
Gray, 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: those
with galanin (GAL), and those with neuropeptide Y (NPY).
Galanin (GAL)-focused studies
Lin et al. (2003)
The work of Lin et al. (2003) utilized the hippocampus of adult male rats, with
experimental rats receiving an injection of Figure 7. The dorsal portion of the
hippocampus (encircled in red).
rAAV-GAL and controls receiving
an injection of rAAV-empty
(rAAV, without the galanin
transgene). To restrict expression to
neuronal cells, neuron-specific
enolase (NSE) was used as a promoter in the
http://www.brainybehavior.com/blog/wp-
content/uploads/2008/11/gray747.png
transgene-carrying rAAV vector. Figure 7
illustrates the dorsal hippocampus, the section of the rats’ brains injected bilaterally with
either the rAAV-GAL or rAAV-empty vector. Later, these rats were subjected to seizures
to determine efficacy of the vector. Seizure analysis in these rats was determined via
electroencephalogram (EEG) detection. To accomplish this, the animals were implanted
with electrodes and a guide cannula 2.5 months after the initial injection of rAAV-NSE-
9

10. Figure 8. Effects of rAAV-empty and rAAV-GAL on
number 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 of
25 seizures noted in the control group compared to an average of 15 seizures in rats
injected with rAAV-NSE-GAL. As shown in figure 9, there was a 55% decrease in total
time spent in kainic acid-induced seizure activity. To determine galanin expression,
another group of rats were injected unilaterally in the right dorsal hippocampus with
rAAV-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 of
the hippocampus.
Mazarati et al. (1998)
Mazarati et al. (1998) also studied rAAV-NSE-GAL in the rat hippocampus. This
study was done on 8-10 week old rats, and was included as part of a later study of the
effects of four neuropeptides – including NPY – in the rat hippocampus during a
prolonged seizure, referred to as status epilepticus (SE). The animals were subjected to
10

11. Figure 10. The perforant path of the hippocampus (in blue).
either electrical
stimulation in the
perforant path to
induce status
epilepticus (SE). As
its name implies, perforant path stimulation (PPS) http://www.nature.com/neuro/journal/
v10/n3/images/nn0307-271-F1.gif
occurs in the perforant path of the hippocampus
(see figure 10). This area is neuron-dense, and acts as the input
pathway in the hippocampus (MRC, 2003). As a result, excess stimulation here will
result 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 findings
that those rats treated with higher
concentrations of galanin 30
minutes prior to PPS were
11
Mazarati et al. (1998)

12. observed to have spent 95% less time in seizure activity than the controls. Animals were
killed at varying time intervals following the conclusion of PPS to determine the
concentration of GAL-infected neurons in and around the injection site. Figure 13
illustrates the concentration of GAL-positive neurons in control and GAL-treated rats. 24
hours after PPS, an average of 16 GAL-positive neurons were found in a slice of the
hippocampus of GAL-treated rats, while none were found in non-GAL treated rats at any
Figure 13. Difference in GAL-positive
time following the conclusion of neurons after SSSE.
PPS. However, 3 days after PPS,
this number has gradually
decreased to an average of 8 GAL-
positive neurons in GAL-treated
rats, 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. 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 genesis
was 60% higher in those animals treated with rAAV-FIB-GAL. Following this, the
animals were given water with doxycycline, Figure 16. Seizure threshold comparison of
control group and rAAV-FIB-GAL-treated rats.
which returned the threshold to
baseline levels within 1 week.
After doxycycline was removed,
rAAV-FIB-GAL-treated rats were
observed to have a gradually
increasing threshold for seizure
genesis, with a threshold
Haberman et al. (2003)
approximately 30% higher in
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14. galanin-treated rats than those in the control group. Figure 16 illustrates these results. To
determine if treatment was able to prevent seizures, animals were injected with either
rAAV-FIB-GAL, rAAV-GAL, or received no treatment at all. In vitro, rAAV-FIB-GAL
or rAAV-GAL was introduced into HEK 293 cells. Twenty-four hours later, the media
were analyzed via ELISA for the presence of galanin. The rAAV-GAL cells showed no
detectable 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 studies
Richichi et al. (2004)
Other studies focused on the effects of rAAV-delivered neuropeptide Y (NPY) in the
rat brain, more specifically, the hippocampus. Richichi et al. (2004) conducted an
experiment 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 and
determined 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 hippocampus
of these rats was injected bilaterally with the vector on each side. Eight weeks later, the
animals 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 via
electroencephalogram (EEG) analysis by a blind party, and was done before injecting the
14

15. animals with kainic acid, and up to 3 hours following the injection. As shown in figure
17, 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 average
of 6.2 minutes in the control group. In these subjects, those treated with serotype 1/2 had
no EEG-detected episodes of status epilepticus (SE), but in the control group, SE lasted at
least 60 minutes.
Mazarati and Wasterlain (2002)
Mazarati and Wasterlain (2002) conducted a study of the effects of four
neuropeptides in the rat hippocampus, one of these neuropeptides being GAL, and
another being NPY. This study used 8-10 week old male rats, which were implanted with
a 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 dentate
gyrus of the hippocampus (see figure 11). These animals were subjected to 30 minutes of
perforant path stimulation (PPS) 7-10 days later, to induce a state of SSSE. Recordings of
brain activity were made via EEG by a blind party during PPS and 24 hours after its
conclusion. Spike distribution for this activity was measured in 30 minute periods, and
seizure activity was assessed according to total time spent in seizure activity, time
between the end of PPS and occurrence of the last seizure, and time spent in seizure
activity 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, the
dentate gyrus of the rats were injected with NPY. Controls were injected with the same
dose of 0.9% NaCl. As illustrated by figure 18, there was no significant difference noted
in time spent in EEG-detected seizure activity between the control (average time in
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16. seizure activity: 15 minutes) and NPY-treated group (average time in seizure activity: 13
minutes). 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-protecting
effect. In this situation, galanin prevents the
initiation of self-sustaining status epilepticus
(SSSE). When injected after perforant path
stimulation was performed, galanin was able
to stop the maintenance phase of an
established SSSE, an accomplishment even
anticonvulsant drugs could not achieve.
Similarly, Lin et al. (2003) showed that when
rAAV-NSE-GAL was used, an over-
Mazarati and Wasterlain (2002)
expression of GAL by the vector was able to
drastically reduce seizure activity brought about by an intrahippocampal injection of
kainic acid, in addition to producing a strong anticonvulsant effect.
Neuropeptide Y (NPY) in neuronal cells causes a decrease in both seizure
susceptibility and epileptogenesis, and therefore, the use of rAAV-NSE-NPY for over-
expression of NPY in rat hippocampal cells was used to inhibit seizures and
epileptogenesis (Richichi et al., 2004). In these rats, kainic acid-induced, EEG-detected
episodes of prolonged seizure activity (status epilepticus, or SE) were not observed in
16

17. NPY-treated rats, but episodes lasting at least 60 minutes were observed in the control
group. A delay in onset of seizures was observed in these animals, with NPY-treated rats
having an average delay time of approximately 11.5 minutes on average, while the
average delay for the control group was observed to be 6.2 minutes, almost half that of
the experimental group. Animals that received an injection of kainic acid showed a
decrease 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 rats
expressing the NPY transgene in multiple areas of the hippocampus. Richichi et al.
(2004) concluded that the ability of NPY to inhibit the release of excitatory
neurotransmitters from brain cells may be the cause of the decrease in seizures noted in
rats 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 to
develop 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 were
significantly attenuated, though only a 2-minute difference in time spent in seizures was
observed between the experimental and control groups. This indicates that there was no
significant difference in time spent in seizures when comparing NPY-treated rats to the
saline-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 transgene
included in the vector, length of time spent in seizure activity, length of time between
17

18. seizure activity, and number of seizures observed. By analyzing rAAV-mediated
experiments involving one or both of the neuropeptides of interest, NPY and GAL can be
compared and contrasted, and a conclusion regarding efficacy can be more easily
determined.
In the studies conducted regarding rAAV vectors containing the GAL transgene, there
was 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 seizures
observed. There was also found to be a large quantity of galanin expression and secretion
in 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 experiments
conducted with rAAV-NPY vectors also showed a decrease in EEG-detected seizure
activities, but it was only a 13% decrease – not nearly as vast as that observed in
experiments conducted with rAAV-GAL vectors.
When compared, each neuropeptide was observed to have an anticonvulsant effect, as
well as the ability to decrease time spent in seizure activities, decrease time between
seizures, and to decrease the number of seizures observed in the tested animals compared
to the control. However, rAAV-GAL vectors were observed to have a greater effect than
the rAAV-NPY vectors and because of this, rAAV vectors utilizing galanin as a
transgene for expression and secretion in neuronal cells are the best choice for gene
therapy.
While these vectors have worked well in rodent models of seizures, there are still
many 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. as well as receiving approval from the Food and Drug Administration (FDA) for
treatment. Once established, these tests may be able to help control seizures in humans
suffering from epileptic disorders. AAV-mediated gene therapy is a promising new
therapy, and will hopefully one day become a widespread, successful treatment for not
just epilepsy, but many other human seizure disorders.
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