Novel AAV-mediated Therapeutic Strategies for Epilepsy
Stacey Beth Foti
A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the
curriculum of Neurobiology
Chapel Hill 2008
Approved by:
Advisor: Thomas McCown
Advisor: Richard Jude Samulski
Committee Chair: Mohanish Deshmukh
Reader: Tal Kafri
Reader: Kay Lund
ii
©2008 Stacey Beth Foti
ALL RIGHTS RESERVED
iii
ABSTRACT
Stacey Beth Foti: Novel AAV-mediated Therapeutic Strategies for Epilepsy (Under the direction of Thomas McCown and Richard Jude Samulski)
Epilepsy afflicts 2.1 million people in the United States, and many patients
develop drug resistant seizures. For these patients, gene therapy may be an attractive
treatment option. Recent studies have shown that viral vector-mediated expression of
neuropeptides such as galanin and neuropeptide Y (NPY) can attenuate seizure sensitivity
and prevent seizure-induced cell death in the brain. NPY13-36 is a C-terminal peptide
fragment of NPY that activates the NPY-Y2 receptor, thought to mediate the anti-seizure
activity. Therefore we investigated if recombinant adeno-associated virus (AAV)-
mediated expression and constitutive secretion of NPY or NPY13-36 could alter limbic
seizure sensitivity. We found that AAV-mediated delivery of both NPY and NPY13-36
attenuates limbic seizures, and provides a vector technology platform for delivering
therapeutic peptide fragments with increased receptor selectivity.
To further explore the potential of this platform, we utilized a strategic approach
to deliver multiple neuropeptides simultaneously. To achieve this, we used a proteolytic
strategy such that our vectors contained a single promoter driving expression of a
cleavable chimeric fusion protein of galanin and NPY13-36. The chimeric fusion protein
contained a linker sequence capable of being cleaved by the intracellular protease furin,
and also contained a fibronectin constitutive secretion signal (FIB) sequence. We first
characterized several constructs in vitro, and determined that 1) a single FIB
iv
sequence was sufficient to cause secretion of both proteins, 2) the proteins were cleaved
from one another regardless of position relative to the cleavage sequence, 3) cleavage
efficiency of secreted proteins was 100%, 4) if the cleavage sequence was absent,
uncleaved fusion protein was secreted into the medium.
We then tested these constructs in a limbic seizure model, and showed that all of
our vectors were capable of attenuating limbic seizure sensitivity. However, no
additional efficacy was observed with the vector delivering both galanin and NPY13-36.
Surprisingly, the level of attenuation was less than with previously published single
peptide vectors, suggesting reduced translation efficiency. This body of work describes a
gene therapy vector technology platform to express and constitutively secrete single and
multiple proteins from transduced cells, and to deliver peptide fragments that are capable
of selective receptor targeting.
v
ACKNOWLEDGEMENTS
I would like to thank the current and past members of the Samulski lab for all their
encouragement and scientific discussions that were invaluable to me in this endeavor. I
would especially like to thank Vivian Choi and Doug McCarty for their mentorship and
patience when I was new to the lab. They set me down the right path! I would also like
to thank my advisors Thomas McCown and Jude Samulski for their constructive criticism
and guidance. They really helped put things in perspective, particularly when I was
getting lost in the details. Finally, I would like to thank my friends (especially Angelique
Camp and Matt Hirsch) and my family; they truly supported me through this process.
My parents George and Susan Foti are great motivators; my brother Andrew Foti always
knows what to say to make me feel better when I have my doubts; and my fiancé Bruce
Herzer who is my sounding board, and reminds me that I really am cut out to be a
scientist.
vi
TABLE OF CONTENTS
List of Tables ......................................................................................................................x
List of Figures .................................................................................................................... xi
Chapter 1 Introduction......................................................................................................1
I.A. Epilepsy ............................................................................................................1
I.B. Gene Therapy....................................................................................................4
I.C. AAV Biology....................................................................................................6
I.D. AAV Vectors ..................................................................................................10
I.E. Potential Gene Therapy Targets for Epilepsy .................................................13
I.F. Ex Vivo Gene Therapy.....................................................................................17
I.G. In Vivo Viral Vector-mediated Alterations in Receptor Function ..................19
I.H. In Vivo Viral Vector-mediated Expression and Secretion of Neuropeptides and Peptide Fragments.......................................................22
I.I. Combination Therapy.......................................................................................25
Chapter 2 Methods ..........................................................................................................31
II.A. rAAV Vectors: Cloning and Construction ....................................................31
II.A.1. Cloning Plasmids for rAAV-CB-FIB-NPY and rAAV-CB-FIB-NPY13-36.............................................................31
II.A.2. Cloning Plasmids for Multiple Gene Product Delivery Vectors ...........................................................................................32
II.A.3. DNA Sequencing...........................................................................33
vii
II.B. In Vitro Methods............................................................................................35
II.B.1. Cell Culture and Transfection........................................................35
II.B.2. Immunoprecipitation and Western Blotting ..................................35
II.C. rAAV Production, Purification, and Characterization...................................36
II.D. In Vivo Methods ............................................................................................44
II.D.1. Experimental Animals...................................................................44
II.D.2. rAAV Vector Microinjection ........................................................44
II.D.3. In Vivo Detection of AAV-Derived NPY or NPY13-36...............46
II.D.4. In Vivo Detection of AAV-Derived GFP ......................................47
II.D.5. Kainic Acid Treatment ..................................................................47
Chapter 3 Results .............................................................................................................48
III.A. rAAV-Mediated Expression and Constitutive Secretion of NPY or NPY13-36 Suppresses Seizure Activity In Vivo ...................................48
III.B. A Strategic Approach for Delivering Multiple Gene Products Using a Single AAV Vector ......................................................................51
III.B.1. In Vitro Characterization of Dual Reporter Gene Product Delivery Vectors...............................................................53
III.B.2. In Vitro Characterization of Double FIB Multiple Gene Product Delivery Vectors .....................................................59
III.B.3. In Vivo Characterization of Double FIB Multiple
Gene Delivery Vectors...................................................................62
III.B.4. In Vitro Characterization of Single FIB Multiple Gene Product Delivery Vectors .....................................................65
III.B.5. In Vivo Functional Test of Single FIB Multiple Gene Product Delivery Vectors .....................................................68
Chapter 4 Discussion .......................................................................................................73
viii
IV.A. rAAV-Mediated Expression and Constitutive Secretion of NPY or NPY13-36 Suppresses Seizure Activity In Vivo ......................73
IV.B. A Strategic Approach for Delivering Multiple Gene Products Using a Single AAV Vector ......................................................................78
Chapter 5 The Future of AAV-mediated Gene Therapy in Brain: Obstacles and Opportunities...................................................................83
V.A. Vector Delivery.............................................................................................83
V.B. Vector Safety, Tolerability, and Efficacy......................................................85
V.B.1. Vector Targeting............................................................................85
V.B.1.a. Capsid-mediated Vector Targeting.................................86
V.B.1.b. Cell Type-specific Promoter-mediated Vector Targeting ....................................................90
V.B.1.c. Inducible and Conditional Promoter-mediated Vector Targeting ....................................................92
V.B.2. Cis-acting Regulatory Elements....................................................93
V.B.3. Immune Response .........................................................................95
V.C. Conclusions and Progress of Current Clinical Trials in Brain ....................100
Appendices......................................................................................................................104
Appendix A. Plasmids Generated and Characterized ..........................................104
Appendix B. Multiple Gene Product Delivery Vectors .......................................107
Appendix C. Nerve Growth Factor (NGF) and Glial Cell Derived Neurotrophic Factor (GDNF).........................................107
Appendix D. Publications ....................................................................................112
Appendix D.1. Adeno-associated Virus-Mediated Expression and Constitutive Secretion of NPY or NPY13-36 Suppresses Seizure Activity In Vivo ................................113
Appendix D.2. Viral Vector Gene Therapy ............................................122
ix
References .......................................................................................................................145
x
LIST OF TABLES
Table 1-1 Current AAV-mediated Gene Therapy Clinical Trials for Neurological Diseases..................................................................................7
Table 1-2. Potential Therapeutic Targets that Influence Seizure Behavior .......................16
Table 2-1. PCR Primers for Multiple Gene Product Delivery Vectors .............................34
Table 5-1. Current Open Gene Therapy Trials for Neurological Diseases......................101
Table 6-1. Plasmids Generated and Characterized ..........................................................104
xi
LIST OF FIGURES
Figure 1-1. Different Types of Epilepsy Surgery ................................................................3
Figure 1-2 AAV Latent and Lytic Life Cycles ....................................................................9
Figure 1-3. Comparing wtAAV to rAAV..........................................................................11
Figure 1-4. Ex Vivo versus In Vivo Viral Gene Delivery...................................................18
Figure 1-5. Strategies to Co-express Gene Products .........................................................26
Figure 2-1. Dot Blot, Standard Curve, and Titer Calculations for AAV2-CB-FIB-NPY and AAV2-CB-FIB-NPY13-36 ..............................37
Figure 2-2. Dot Blot, Standard Curve, and Titer Calculations for AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP, AAV2-CB-FIB-EGFP, and AAV2-CB-EGFP ..........................................38
Figure 2-3. Dot Blot, Standard Curve, and Titer Calculations for Multiple Gene Product Delivery Vectors ..................................................39
Figure 2-4. Infectious Center Assay of AAV2-CB-FIB-GAL- RKRRKR-EGFP........................................................................................40
Figure 2-5. Infectious Center Assay of AAV2-CB-FIB-EGFP- RKRRKR-GAL..........................................................................................41
Figure 2-6. Infectious Center Assay of AAV2-CB-FIB-GAL-EGFP ...............................42
Figure2-7. Infectious Center Assay of AAV2-CB-FIB-GAL- RKRRKR-NPY13-36 ................................................................................43
Figure 2-8. Coronal Section of the Rat Brain Depicting the Location of Vector Microinjection............................................................................45
Figure 3-1. AAV Vectors that Express and Constitutively Secrete NPY or NPY13-36..............................................................................................49
Figure 3-2. The Effects of AAV-FIB-NPY and AAV-FIB-NPY13-36 Vectors on the Expression of Limbic Seizure Behaviors ..........................50
Figure 3-3. The In Vivo Presence of FIB-NPY and FIB-NPY13-36 mRNA 1 Week after Vector Infusion into the Piriform Cortex .............................52
xii
Figure 3-4. AAV Vectors Characterized in Initial Multiple Gene Product Delivery Studies ...........................................................................54
Figure 3-5. Luciferase Assay on Concentrated Medium ...................................................56 Figure 3-6. Luciferase Antibody Protein Analysis of Luciferase-GFP
Multiple Gene Product Delivery Constructs..............................................57 Figure 3-7. GFP Antibody Protein Analysis of Luciferase-GFP Multiple
Gene Product Delivery Constructs.............................................................58 Figure 3-8. Double FIB Multiple Gene Product Delivery Vectors....................................60
Figure 3-9. In Vitro Protein Analysis of Double FIB Multiple Gene Product Delivery Constructs ...................................................................................61
Figure 3-10. In Vivo Fluorescence Pattern in Rat Cortex after Infusion of AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP, AAV2-CB-FIB-EGFP, or AAV2-CB-EGFP.............................................63
Figure 3-11. The Effects of AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP on the Expression of Limbic Seizure behaviors .............................................64
Figure 3-12. AAV Vectors Characterized in Final Multiple Gene Product Delivery Studies.........................................................................................66
Figure 3-13. In Vitro Characterization of Fluorescence Patterns, Cleavage, and Secretion of Proteins Derived from Multiple Gene Product Delivery Vectors...........................................................................67
Figure 3-14. Immunofluorescence of Neurons in the Piriform Cortex That Have Been Transduced with GFP-containing Vectors ..............................69
Figure 3-15. The In Vivo Presence of FIB-GAL-RKRRKR-NPY13-36 mRNA 1 Week After Vector Infusion into the Piriform Cortex ...............70
Figure 3-16. The Effects of Multiple Gene Product Delivery Vectors on Limbic Seizure Behavior ...........................................................................71
Figure 4-1. Regulated versus Constitutive Secretion in Neurons ......................................76
Figure 6-1. Immunoprecipitation Followed by SDS-PAGE and Coomassie Staining of Multiple Gene Product Delivery Vectors for Mass Spectrometry...................................................................................108
xiii
Figure 6-2. NGF and GDNF ELISA................................................................................111
Chapter 1
Introduction
I.A. Epilepsy
About 2.1 million people in the United States are living with epilepsy (Hirtz,
Thurman et al. 2007), with a world wide prevalence of 1 percent (Hauser, Hesdorffer et
al. 1990), making epilepsy one of the most common neurological diseases. Epilepsy
syndromes can be classified into two major categories based on the origin of seizure
activity. In generalized epilepsy the seizure originates bilaterally from the cerebral
hemispheres and typically involves the whole brain. For partial or focal epilepsy, the
area of seizure genesis is localized to one or more defined areas termed foci, although it
may spread to involve the entire brain.
Temporal lobe epilepsy (TLE), is a partial epilepsy where seizure genesis occurs
in temporal lobe structures such as the hippocampus and amygdala, and is the most
frequent and severe form of adult focal epilepsy (Cohen, Navarro et al. 2002). Patients
with TLE can have seizures that cause them to lose consciousness. If the seizures go
untreated, they can lead to severe brain damage. Having epilepsy is not only debilitating,
it can also be a significant economic burden. The estimated annual economic cost of
epilepsy in the United States including the direct cost of treatment and the indirect costs
such as lost productivity and wages is 12.5 billion dollars (Shafer and Begley 2000).
There is a critical need for effective epilepsy treatment. Currently, the preferred
treatment method for epilepsy is to administer anti-epileptic drugs (AEDs) to arrest the
seizures or at least reduce their frequency and intensity. If seizures are left untreated,
they can increase in duration and subsequently cause more activity-induced pathology in
the brain. The AEDs attempt to prevent seizure activity by four major mechanisms: (1)
increasing gamma-aminobutyric acid (GABA)-ergic actions which are predominantly
inhibitory; (2) decreasing excitatory glutamatergic transmission; (3) decreasing voltage-
dependant calcium release; and (4) decreasing voltage-dependant sodium conductance
(Ure and Perassolo 2000).
Since seizures seem to result from hyperexcitable networks of neurons, the AEDs
are designed to enhance inhibitory neurotransmission and hyperpolarize neurons.
Unfortunately, even with new AEDs available, the number of drug resistant epilepsies
has not decreased (Loscher and Leppik 2002). Patients who continue to exhibit
intractable seizures or those who develop intolerable side effects while on AEDs are
considered to have medically refractory epilepsy. Unfortunately, 30–40% of all patients
with epilepsy have medically intractable seizures, and only half of these patients are
candidates for surgery (Shafer, Hauser et al. 1988; Engel, Levesque et al. 1992; Sander
1993; Siegel 2004), which is currently one of the few treatment options for medically
refractory patients. Additionally, 20% of the patients who do undergo temporal lobe
surgery will still have seizures post-operatively (Spencer, Spencer et al. 1984).
Even though surgical techniques have become more refined, several factors still
limit success. The highest surgical success rate is achieved if the epileptogenic tissue can
be directly removed using procedures like focus surgery, anterior temporal lobectomy,
and total lobe resection (See Figure 1-1). Lower success is achieved when the area of
2
3
Figure 1-1. Different Types of Epilepsy Surgery (modified from Siegel 2003). A. Temporal lobectomy, B. Selective amygdalo-hippocampectomy, C. Frontal lobectomy, and D. Anatomical hemispherectomy. Arrows show where brain has been resected. Surgery is currently the only alternative for the 30% of patients who develop intractable epilepsy. Gene therapy represents a potential treatment that is less invasive.
4
seizure genesis overlaps with tissues that serve important sensory or motor functions. For
these patients, preventing seizure spread by surgically interrupting the hyperexcitable
circuits is accomplished through procedures like amygdalo-hippocampectomy and
corpuscollosectomy. Surgical complications such as visual field deficits, transient or
persistent hemiparesis, infections, epidural hematoma, dysphasia, global memory deficits,
and transient psychosis or depression may also limit success (Siegel 2004). Although
current surgical procedures can be beneficial to patients, more effective and potentially
less invasive treatment strategies need to be designed.
Theoretically, a gene therapy approach to seizure suppression is an attractive
alternative treatment option for focal epilepsy. Focal epilepsy arises from a
circumscribed and hyperexcitable network of neurons whose synchronous electrical
discharge creates seizure activity. Since the area of seizure genesis is confined, the
localized nature of in vivo gene therapy should prove capable of influencing a site that
initiates seizure activity, therefore preventing the cascade leading to seizure spread. One
significant advantage of localized gene delivery is the limited exposure of the rest of the
brain and body to the therapeutic compounds, thereby minimizing the potential for
unwanted side effects. Furthermore, gene therapy allows for localized de novo synthesis
of the therapeutic compounds in the brain, surmounting the difficulty of delivering
therapeutic compounds with short half-lives across the otherwise impermeable blood
brain barrier.
I.B. Gene Therapy
The primary goal of gene therapy is to provide long term and effective treatment
of a disease by delivering therapeutic genes into the patient’s target cells. Gene therapy
5
offers the possibility that chronic disease progression (which occurs with medically
intractable seizures) can be halted and the symptoms can be controlled, substantially
increasing the quality of life for the patient. Using a virus as a means to deliver
therapeutic genes circumvents many of the problems that non-viral vector-based gene
therapy is subject to, such as inefficient gene transfer and transient gene expression
(Huang, Hung et al. 1999). In order to complete their lifecycle, viruses must efficiently
deliver their genes into the nucleus of a host cell. Thus, viruses can be exploited as
excellent gene delivery tools by substituting therapeutic genes in place of viral genes.
Focal epilepsy would be very amenable to viral gene therapy particularly because
viruses like adenovirus (Ad), lentivirus, and adeno-associated virus (AAV) are capable of
infecting post-mitotic neurons. This provides direct access to the cells responsible for
generating and propagating the seizure activity; and which are otherwise difficult to
genetically manipulate. Ad is a non-enveloped, double stranded DNA virus that causes
mild upper respiratory tract infections in humans. Ad vectors have a large packaging
capacity (up to 37kb), can infect both dividing and quiescent cells, and remains episomal
after infection, with a very low probability of randomly integrating into the host cell’s
chromosomes (Schiedner, Morral et al. 1998; Harui, Suzuki et al. 1999). The use of Ad
vectors in the brain is controversial because it has been reported to cause vector-
associated toxicity and inflammation (Wood, Charlton et al. 1996; Thomas, Birkett et al.
2001; McMenamin, Lantos et al. 2004). In addition, Ad-mediated gene expression is
unstable in the brain over time (Thomas, Birkett et al. 2001; McMenamin, Lantos et al.
2004).
6
Lentiviral vectors (HIV based) are effective in transducing neurons, do not seem
to cause cytotoxicity upon central nervous system (CNS) infection, and are capable of
long term transgene expression (Blomer, Naldini et al. 1997; Bosch, Perret et al. 2000).
However, since lentiviral vectors randomly integrate into the host cell’s genome, they can
cause insertional mutagenesis. These random insertions may disrupt or activate cellular
genes including oncogenes. Recently, class 1 non-integrative lentiviral vectors derived
from HIV type 1 were developed and characterized in brain tissue (Philippe, Sarkis et al.
2006; Yanez-Munoz, Balaggan et al. 2006). These vectors can be further modified to be
self-inactivating (Miyoshi, Blomer et al. 1998), a combination that substantially reduces
the risks of insertional mutagenesis and replication-competence, thereby generating safer
lentiviral vectors for gene therapy in the brain.
AAV vectors which are based on a non-pathogenic and replication deficient virus
offer the safest method of viral gene delivery in the brain. In addition, AAV vectors are
capable of long term stable gene expression and do not elicit an immune response upon
infection, making it the viral vector of choice in clinical trials for neurological diseases
(see Table 1-1 and http://www.wiley.co.uk/wileychi/genmed/clinical/).
I.C. AAV Biology
Wild type AAV (wtAAV) is a non-enveloped single stranded DNA virus
belonging to the Parvovirus family. Although the virus is prevalent in the human
population (about 80% of humans are seropositive to AAV2 (Tobiasch, Rabreau et al.
1994), infection is not associated with any known disease (Blacklow, Hoggan et al.
1968). Because of this, the virus is said to be nonpathogenic. AAV is classified as a
dependovirus because it is replication deficient, and cannot cause a productive infection
7
Table 1-1. Current AAV-mediated Gene Therapy Clinical Trials for Neurological Diseases. This data is current to July 2007. For more details on these clinical trials please see: http://www.wiley.co.uk/wileychi/genmed/clinical/ Trial ID: Title: Gene Disease
US-469 Subthalamic GAD Gene Transfer in Parkinson’s Disease Patients Who Are Candidates for Deep Brain Stimulation.
Glutamic acid decarboxylase 65-67 (GAD 65-67)
Parkinson's Disease
US-593 A Phase I Open-Label Safety Study of Intrastriatal Infusion of Adeno-Associated Virus Encoding Human Aromatic L-amino Acid Decarboxylase (AAV-hAADC-2) in Subjects with Advanced Parkinson's Disease
Aromatic L-amino Acid Decarboxylase (AADC)
Parkinson's Disease
US-623 A Phase I/II, Dose-Escalating, Randomized and Controlled Study to Assess the Safety, Tolerability, and Efficacy of CERE-110 [Adeno-Associated Virus (AAV)-based, Vector-Mediated Delivery of beta-Nerve Growth Factor (NGF)] in Subjects with Mild to Moderate Alzheimer's Disease
Nerve growth factor (NGF)
Alzheimer's Disease
US-669 Hippocampal NPY Gene Transfer in Subjects with Intractable Temporal Lobe Epilepsy
Neuropeptide Y (NPY)
Epilepsy
US-689 A Phase I, Open-Label Study of CERE-120 (Adeno-Associated Virus Serotype 2 [AAV2]-Neurturin [NTN]) to Assess the Safety and Tolerability of Intrastriatal Delivery to Subjects with Idiopathic Parkinson's Disease
Neurturin (NTN)
Parkinson's Disease
US-788 Multicenter, Randomized, Double-Blind, Sham Surgery-Controlled Study of CERE-120 (Adeno-Associated Virus Serotype 2 [AAV2]-Neurturin [NTN]) to Assess the Efficacy and Safety of Bilateral Intraputamenal (IPu) Delivery in Subjects With Idiopathic Parkinson's Disease
Neurturin (NTN)
Parkinson's Disease
8
unless facilitated by a helper virus such as Ad or Herpes Simplex virus (HSV) (Buller,
Janik et al. 1981; Bauer and Monreal 1986). When helper viruses are absent and the host
cell is not subjected to DNA damaging agents or metabolic inhibitors, AAV will establish
a latent infection within the cell (Yalkinoglu, Heilbronn et al. 1988). This latent infection
will occur by site-specific integration of AAV into the host genome at human
chromosome 19q13.3qter AAVS1 site (Samulski, Zhu et al. 1991; Muzyczka 1992), by
AAV persistence as an episome within the nucleus (Yue and Duan 2003; McCarty,
Young et al. 2004), or by random integration at chromosomal double-strand breaks
(Miller, Petek et al. 2004). AAV is stable in this latent state and can persist for years
until rescued by helper virus co-infection, which reinitiates the lytic cycle (Cheung,
Hoggan et al. 1980) (see Figure 1-2).
AAV virions are comprised of a linear single-stranded DNA genome of 4.7 kb,
which is encapsidated by a non-enveloped icosahedral shell, measuring approximately 22
nm in diameter. The elegant simplicity of the genome is astounding, with only two genes
rep (replication) and cap (capsid) encoding nonstructural and structural proteins
respectively. From a single open reading frame (ORF) the rep gene encodes four
replication proteins (Rep78, Rep 68, Rep 52, and Rep 40), which collectively are
responsible for site-specific integration, DNA nicking, and helicase activity. Rep
proteins also respond to intracellular cues (like the presence of helper virus) to regulate
the AAV promoters that drive expression of its genome (Pereira, McCarty et al. 1997).
The cap gene encodes three overlapping proteins (VP1, VP2, and VP3) which differ from
each other only by their N terminus (Rose, Maizel et al. 1971; Berns and Linden 1995).
Together they assemble into the capsid which is made up of 60 subunits with
9
Figure 1-2 AAV Latent and Lytic Life Cycles. In the absence of helper virus, AAV latently integrates into host chromosomes, with 70% of the genomes being targeted to the human chromosome 19q13.3qter AAVS1 site. If the cells are then superinfected with a helper virus, AAV enters its lytic phase and initiates virus replication. AAV can also immediately enter its lytic phase upon helper virus co-infection, resulting in the replication of both AAV and the helper virus.
wtAAV
19q13.3qter
Ad
Helper Virus Co-infection
Cell Lysis
Helper Virus Infection
Cell Lysis
Latent Infection
wtAAV
19q13.3qter 19q13.3qter 19q13.3qter
Ad
Helper Virus Co-infection
Cell Lysis
Helper Virus Infection
Cell Lysis
Latent Infection
10
approximately a 1:1:20 ratio and T=1 icosahedral symmetry (Rose, Maizel et al. 1971;
Muzyczka 1992). The rep and cap genes are flanked by the AAV inverted terminal
repeats (TR), which are the only cis-acting elements required for genome replication and
packaging (Samulski, Chang et al. 1987; Samulski, Chang et al. 1989).
I.D. AAV Vectors
Since the AAV TR is the only required cis-element for replication and
encapsidation, the two viral genes (rep and cap) can be removed and replaced by a
transgene expression cassette (see Figure 1-3). As long as rep and cap are provided in
trans, AAV vectors can be produced and subsequently purified. Building on experiments
by Ferrari et al., (1996) which demonstrated that Ad helper function could be conferred
using noninfectious Ad DNA, pioneering work was done by Xiao, Li, and Samulski,
allowing production of high titer AAV vectors in the absence of contaminating helper
viruses (Ferrari, Samulski et al. 1996; Ferrari, Xiao et al. 1997; Xiao, Li et al. 1998).
Consequently these vectors are safer for clinical trial applications. In addition, there are
approximately 300 nucleotides of viral DNA present in the AAV vector expression
cassette (the TR), and this sequence is not translated into viral proteins. Perhaps this lack
of translated viral proteins reduces the risk of AAV vector genomes eliciting a host cell
immune response, and may contribute to the longevity of transgene expression (longer
than 3 years in humans) (Duan, Sharma et al. 1998; Jiang, Pierce et al. 2006).
Along with its lack of immunogenicity and pathogenicity, AAV vectors have a
broad tissue tropism (e.g. brain, liver, muscle) and can transduce both mitotic and
quiescent cells (During, Samulski et al. 1998; Song, Morgan et al. 1998; Ye, Rivera et al.
1999; Acland, Aguirre et al. 2001; Flotte 2001), reinforcing the suitability of rAAV for
11
Figure 1-3. Comparing wtAAV to rAAV. A. The wtAAV genome encodes two viral genes, the rep and cap, and is flanked by terminal repeats (TR). The four Rep proteins: Rep78, Rep68, Rep52 and Rep40 are responsible for site-specific integration, DNA nicking, and helicase activity. All four proteins are transcribed from the p5 and p19 promoters and by alternative splicing. The three viral structural proteins VP1, VP2, and VP3 share the same C-terminus and form trimers and pentamers that ultimately come together and encapsidate the single stranded genome. They are transcribed from the p40 promoter and by alternative splicing. B. Since the AAV terminal repeats are the only cis-elements required for viral vector (a.k.a. recombinant AAV or rAAV) production, rep and cap can be removed and replaced by a transgene expression cassette. As long as rep and cap are provided in trans, AAV vectors can be produced and subsequently purified.
wtAAV
A.
p19 p40 polyA
Rep Cap
Vp2
Vp1
Vp3
Rep 78
Rep 52
Rep 40
Rep 68
p5
Promoter Transgene
rAAV
B.
polyA
12
clinical trials in brain. Several AAV serotypes have been tested in the brain, although not
every serotype was tested at the same time in the same model. While there is some
variability, some generalizations can be made: capsids from AAV9, AAV8, AAV7, and
AAV5 cause transduction in neurons (and a few glia) farthest from the site of injection
(Klein, Dayton et al. 2006; Taymans, Vandenberghe et al. 2007; Klein, Dayton et al.
2008), followed by AAV1 (Burger, Gorbatyuk et al. 2004) which still transduces neurons
farther away than AAV2, which seems to have the most localized effects. AAV4 appears
to transduce ependymal cells exclusively, severely limiting its use as a vector for
neurological diseases (Davidson, Stein et al. 2000). Furthermore, with the discovery of
novel serotypes and the ability to engineer new capsid mutations by rational design and
directed evolution approaches, it is now possible to enhance viral transduction and
targeting (see Chapter 5 for detailed discussion).
Despite its many advantages, there are a few drawbacks to using AAV vectors.
Perhaps the primary concern is the vector’s limited packaging capacity (approximately
5kb). AAV vectors can package genomes up to 6kb, but infectivity drops dramatically
when the genome is larger than that of wtAAV (Grieger and Samulski 2005). However,
there are elegant ways of surmounting this obstacle, such as shortening and optimizing
the components of the expression cassette (promoter, therapeutic gene sequence, and
polyA), or employing a split vector system. A split vector system divides the coding
sequence of the transgene in two, such that each one is packaged in a separate vector
which relies upon co-infection, and the ability of AAV vectors to concatamerize with one
another to allow gene expression (Duan, Yue et al. 2003).
13
Another concern that potentially limits AAV vector efficiency is the ability and
speed with which the infected cells orchestrate viral genome second strand synthesis.
Since traditional AAV vectors are single stranded, the DNA must be converted to a
double stranded form before gene expression can be initiated, and the process is
dependent upon the host cell-mediated DNA synthesis (Ferrari, Samulski et al. 1996;
Fisher, Gao et al. 1996). One way to overcome this obstacle was pioneered by McCarty
et al, and involves generating a self-complimentary AAV vector (scAAV) that upon
uncoating could fold back on itself through intramolecular base pairing, creating a
duplexed form (McCarty, Monahan et al. 2001; McCarty, Fu et al. 2003; Wang, Ma et al.
2003). Once duplexed, the efficiency with which transcriptionally active double stranded
molecules are formed is greatly enhanced. Not only does this scAAV vector allow gene
expression to occur much faster, it also has been reported to increase the level of
transgene expression (McCarty, Monahan et al. 2001; Yang, Schmidt et al. 2002). While
these vectors have shown great promise, they are even further limited in their packaging
capacity to around 2.3kb (Wu, Zhao et al. 2007), which may restrict the range of
therapeutic applications.
I.E. Potential Gene Therapy Targets for Epilepsy
Due to the diverse etiology of epilepsy, there are sure to be multiple genes and
mechanisms involved in seizure genesis and epilepsy-induced pathology. While this may
mean that there will not be “one cure to fit them all,” many potential therapeutic targets
can be investigated. Seizure activity is most likely the result of inadequate neuronal
inhibition, excessive neuronal excitation, or a combination of the two states (Engel 1996;
Holmes 1997; Bernard, Hirsch et al. 1999; Ying and Najm 2002). Neuronal inhibition is
14
primarily achieved through GABAergic neurotransmission, and loss of this inhibition has
been implicated in epilepsy, making GABA a good therapeutic target. This is evident by
the fact that many of clinically effective AEDs enhance GABA action either by
increasing the time GABA remains in the synapse (by blocking its intracellular
catabolism or by blocking its reuptake transporter, GAT1), or by acting at GABA
receptors (to increase chloride ion flux into the neuron which hyperpolarizes it and
prevents the firing of new action potentials).
As mentioned before, many patients with epilepsy do not respond to these drugs,
and some patients respond initially, but relapse over time. Perhaps the same problems
will occur with gene therapies directed at GABA. One possible mechanism for decreased
drug efficacy is GABA receptor desensitization, a process whereby overstimulation leads
to the removal of the receptors at and around the synapse by endocytosis (Galpern, Miller
et al. 1990; Qi, Yao et al. 2006), eventually causing a loss of GABA binding, and shifts
the neuronal inhibitory tone toward excitation. Once neurons become hyperexcitable,
they fire action potentials and release glutamate until synchronized excitation can cause
hyperexcitable circuits that kindle seizure genesis. If left untreated, excitotoxicity ensues,
leading to epilepsy-induced pathologies like hippocampal sclerosis, mossy fiber
sprouting, and neuronal death. In fact, administration of a glutamate agonist, kainic acid
is the model we and others use to induce limbic seizures in naïve animals, emphasizing
that a reduction in glutamate action can be a good therapeutic target. There is evidence
from many investigators that epilepsy enhances several other excitatory neurotransmitters
like glycine, aspartate, acetylcholine, N-methyl-D-aspartate (NMDA), α-amino-3-
hydroxy-5-methylisoxazole-4- propionic acid (AMPA), and second messengers like
15
calcium and cyclic GMP (Ure and Perassolo 2000). Even channels for ions such as
sodium, potassium, and calcium regulate neuronal excitability, and their manipulation
could provide in vivo seizure control. However, a liability with many of these targets in a
gene therapy context is that success will depend on the pattern of neuronal transduction,
i.e. the type of neuron and its position within a functional circuit will influence seizure
outcome. For example, reducing excitatory tone of a primary output neuron will reduce
overall excitability, but reducing excitatory tone of an inhibitory interneuron may actually
increase seizure severity.
Perhaps the therapeutic target that holds the greatest potential for treating focal
epilepsy is neuropeptides. Neuropeptides are larger than classical neurotransmitters but
smaller than most proteins, and have less complex tertiary structure. Their main function
is to modulate the release of neurotransmitters, which can influence neuronal excitability.
Neuropeptides diffuse more slowly (100-500ms) but bind tighter to their receptors than
classical neuropeptides (2-5ms), enabling slow long-lasting changes (Hokfelt, Bartfai et
al. 2003). They can also travel to receptors somewhat distant from their release site, a
phenomenon called volume transmission that was first described by Agnati and Fuxe in
1986 (Agnati, Bjelke et al. 1995). Since neuropeptides can influence autoreceptors as
well as the receptors on surrounding neurons, transduction of a few cells within the
seizure focus may result in more wide-spread seizure suppression that is independent of
transduction pattern. To date, the neuropeptides best studied as therapeutics for epilepsy
are neuropeptide Y (NPY) and galanin (see Table 1-2), but others such as dynorphin,
somatostatin, and cholecystokinin have shown some promise.
16
Table 1-2. Potential Therapeutic Targets that Influence Seizure Behavior. Abbreviations are: AAV: adeno-associated virus; ASPA: aspartoacylase; Ad: adenovirus; CB: cytomegalovirus enhancer, chicken beta-actin promoter; CMV: cytomegalovirus promoter; GAD: glutamic acid decarboxylase; GDNF: glial-derived neurotrophic factor; ICP10PK: ICP 10 protein kinase; ih: intrahippocampal; ip: intraperitoneal; NMDA: N-methyl-D-aspartate; NPY: Neuropeptide Y; NSE: neuron- specific enolase; SER: spontaneously epileptic rats; SN: substantia nigra; TET off: tetracycline-off regulatable promoter; WPRE: woodchuck hepatitis virus post-transcriptional element. Target Placement Delivery vector Experimental model Functional effect References Adenosine
Intracerebroventricular
Grafting of fibroblasts or myoblasts
Hippocampal kindling Suppression of generalized
seizures
(Huber, Padrun et al. 2001; Guttinger, Padrun et al. 2005)
Anti NMDA Collicular cortex AAV-CMV vector Focal electrical stimulation Increased seizure threshold (Haberman, Criswell et al. 2002)
AAV-TEToff vector
Decreased seizure threshold
(Haberman, Criswell et al. 2002)
Orogastric tube delivers vector to stomach (oral vaccine)
AAV-CMV
Kainic acid ip
Seizure inhibition, neuroprotection
(During, Symes et al. 2000)
ASPA
Intracerebroventricular
Ad-CB vector
Spontaneous seizures in SER
Transiently reduced incidence of tonic seizures (2 weeks)
(Seki, Matsubayashi et al. 2004)
Cholecystokinin Intracerebroventricular Lipofectin and plasmid Audiogenic seizure-prone rats
Transient seizure inhibition (1 week) (Zhang, Li et al. 1997)
GABAAR Hippocampus AAV-GABAR4 Pilocarpine-induced Status Epilepticus
Transiently reduced spontaneous seizures (4 weeks)
(Raol, Lund et al. 2006)
GAD65 Anterior SN Grafting of neuronal or glial cell lines Entorhinal kindling Delayed rate of kindling
(Thompson, Anantharam et al. 2000)
Posterior SN
Faster rate of kindling (Thompson, Anantharam et al. 2000)
Anterior SN
Grafting neuronal cell line
Pilocarpine-induced Status Epilepticus
Reduced spontaneous seizures
(Thompson and Suchomelova 2004)
Piriform cortex
Grafting neuronal cell line
Amygdala kindling
Increased threshold to seizures
(Gernert, Thompson et al. 2002)
GAD67 SN pars reticulata Grafting of mixed neuronal and glial cell line
Kainic acid ip Delayed seizure behavior (Castillo, Mendoza et al. 2006)
Galanin Collicular cortex AAV-TEToff vector+FIB secretory sequence
Increased seizure threshold (Haberman, Samulski et al. 2003)
Hippocampus
AAV-TEToff vector+FIB secretory sequence
Kainic acid ip
Neuroprotection
(Haberman, Samulski et al. 2003)
AAV-NSE vector+WPRE
Kainic acid ih
Reduced number of seizures
(Lin, Richichi et al. 2003)
Piriform cortex
AAV-CB vector+FIB secretory sequence
Kainic acid ip
Seizure inhibition
(McCown 2006)
Focal electrical stimulation Increased seizure threshold (McCown 2006)
GDNF
Hippocampus
AAV-CB vector+WPRE
Hippocampal kindling
Decreased number of generalized seizures, increased seizure threshold
(Kanter-Schlifke, Georgievska et al. 2007)
Hippocampus
Ad-CMV vector
Kainic acid ip
Delayed seizure behaviors, neuroprotective
(Yoo, Lee et al. 2006)
ICP10PK
Intranasal vaccine
HSV-2∆RR –ICP10 vector
Kainic acid ip
Reduced seizure behaviors, neuroprotection
(Laing, Gober et al. 2006; Laing and Aurelian 2008)
NPY
Hippocampus
AAV-NSE vector+WPRE
Kainic acid ih Delayed seizure behavior
(Richichi, Lin et al. 2004)
Hippocampal kindling
Increased seizure threshold and delayed rate of kindling
(Richichi, Lin et al. 2004)
Piriform cortex
AAV-CB vector+FIB secretory sequence
Kainic acid ip Reduced and delayed seizure behaviors
(Foti, Haberman et al. 2007)
NPY 13-36
Piriform cortex
AAV-CB vector+FIB secretory sequence
Kainic acid ip Reduced and delayed seizure behaviors
(Foti, Haberman et al. 2007)
17
I.F. Ex Vivo Gene Therapy
There are two basic modalities for gene therapy: ex vivo gene therapy which relies
on genetically modifying cells in vitro then implanting them into the target tissue, or in
vivo gene therapy where the transfer of genetic material occurs directly within the host
via viral or non-viral mediated gene delivery (see Figure 1-4). To date, ex vivo gene
therapy experiments in brain have been carried out using fibroblasts (Huber, Padrun et al.
2001), myoblasts (Lisovoski, Wahrmann et al. 1997; Guttinger, Padrun et al. 2005), CNS
progenitor cells (Martinez-Serrano and Bjorklund 1997), immortalized neurons (Gernert,
Thompson et al. 2002; Longhi, Watson et al. 2004), and astrocytes (Lundberg, Horellou
et al. 1996; Ericson, Wictorin et al. 2002). In order for this ex vivo approach to be
successful in treating epilepsy, the implanted cells must be engineered to secrete a
product that will enhance neuronal inhibition. In the Boison lab, genetically engineered
fibroblasts (Huber, Padrun et al. 2001) and myoblasts (Guttinger, Padrun et al. 2005)
were made to secrete the inhibitory neuromodulator adenosine, and then grafted into the
lateral ventricles or hippocampus of rats. While the rate of limbic kindling was
significantly attenuated during the first week post-implantation, by the fourth week there
was a drastic reduction of seizure suppression. The authors determined that the loss of
therapeutic efficacy was due to low viability of the grafted cells. These results point to a
universal problem with ex vivo gene therapy: the loss of graft viability over time. With
regard to epilepsy gene therapy, the gene product must remain functional for a long
period of time, hence the clinical potential of grafting modified cells in the brain is very
low (McCown 2004).
18
Figure 1-4. Ex Vivo versus In Vivo Viral Gene Delivery.
2. Culture Cells
4. Inject Genetically Modified Cells into Rodent
3. Infect Cells with Virus Encoding Therapeutic Gene
1. Harvest Cells from Rodent
A. Ex Vivo Viral Gene Therapy
2. Culture Cells
4. Inject Genetically Modified Cells into Rodent
3. Infect Cells with Virus Encoding Therapeutic Gene
1. Harvest Cells from Rodent
A. Ex Vivo Viral Gene Therapy
4. Inject Genetically Modified Cells into Rodent
3. Infect Cells with Virus Encoding Therapeutic Gene
1. Harvest Cells from Rodent
A. Ex Vivo Viral Gene Therapy
3. Infect Cells with Virus Encoding Therapeutic Gene
1. Harvest Cells from Rodent
A. Ex Vivo Viral Gene Therapy
1. Harvest Cells from Rodent
A. Ex Vivo Viral Gene Therapy
1. Harvest Cells from Rodent
A. Ex Vivo Viral Gene Therapy
B. In Vivo Viral Gene Therapy
1. Inject Virus Encoding Therapeutic Gene Directly into Rodent
B. In Vivo Viral Gene Therapy
1. Inject Virus Encoding Therapeutic Gene Directly into Rodent
19
More recently the Boison lab has used genetically engineered human
mesenchymal stem cells (hMSC) where lentiviral RNAi mediates knockdown of
adenosine kinase (ADK), the gene responsible for adenosine metabolism and influx from
the extracellular space back into cells (Ren, Li et al. 2007). The authors demonstrate
reduced seizure duration and neuronal loss after graft implantation in the hippocampus
followed by intra-amygdaloid injection of kainic acid (Ren, Li et al. 2007). This type of
approach would be compatible with autologous cell grafting in patients, and thus
represents a potential clinical therapy if the grafts of mesenchymal stem cells can remain
viable in the brain over time.
The Boison lab is also currently exploring implantation of genetically modified
embryonic stem cell-derived neural precursors with biallelic genetic disruption of ADK
(Li, Steinbeck et al. 2007). Implanting these grafts into the hippocampus caused
increased levels of extracellular adenosine and suppressed kindling epileptogenesis (Li,
Steinbeck et al. 2007). While the authors only conducted these experiments for 26 days,
they see integration of the grafted cells and neuronal maturation markers expressed in
about half of their grafted cells; leading the authors to speculate that graft viability may
extend beyond the limited time course analyzed.
I.G. In Vivo Viral Vector-Mediated Alterations in Receptor Function
As mentioned before, enhancing inhibitory receptor function may be an effective
target for antiepileptic gene therapy. Thus, an obvious approach would be to increase
GABA-mediated fast neuronal inhibition, which is achieved through GABAA receptors
(GABRs). GABRs are made up of several subunits whose combinations confer a range
of pharmacological function. For example, it has been reported that GABRs rich in
20
GABRα4 and poor in GABRα1 have been found in both humans with temporal lobe
epilepsy and in rodent models of epilepsy, and show reduced GABA-mediated inhibition
in the presence of zinc (Buhl, Otis et al. 1996; Gibbs, Shumate et al. 1997; Brooks-Kayal,
Shumate et al. 1998; Brooks-Kayal, Shumate et al. 1999). In addition, after pilocarpine-
induced status epilepticus, GABRα4 expression increases while GABRα1 expression
decreases in the dentate gyrus of adult rats (Brooks-Kayal, Shumate et al. 1998). While
these adult rats go on to develop spontaneous seizures, postnatal day 10 rats that are
subjected to the same paradigm fail to develop spontaneous seizures (Zhang, Raol et al.
2004). Furthermore, the postnatal day 10 rats exhibit an increase in GABRα1 expression
in dentate gyrus neurons (Zhang, Raol et al. 2004), suggesting that overexpression of
GABRα1 may confer seizure protection if overexpressed in the adults. To investigate
this hypothesis, Raol et al. used an AAV5 vector to express GABRα1 from the GABRα4
promoter in adult rats subsequently injected with pilocarpine (Raol, Lund et al. 2006).
While it was a good idea to use a conditional promoter whose expression is known to be
upregulated by pilocarpine treatment, only transient expression of the transgene GABRα1
was observed (about 4 weeks). A more robust promoter like the constitutively active
cytomegalovirus enhancer, chicken beta-actin promoter (CB) could be used to prevent
this. Even though the effects were transient and not uniform, some seizure protection
was observed; however, about 30% of the rats receiving GABRα1 developed behavioral
phenotypes that were not desirable, including excessive sedation, and anorexia (Raol,
Lund et al. 2006). These results point to a therapeutic liability that must be considered,
and may be a universal risk of directly manipulating fast neuronal inhibition.
21
Another strategy to limit seizure activity would be to reduce or block excitatory
amino-acid receptors, which may help restore inhibitory tone and prevent excitotoxicity.
Since activation of NMDA receptors has been shown to increase seizure sensitivity
(McCown, Givens et al. 1987), it is logical to try to limit their activation. In fact, NMDA
channels are a good target for a gene therapy approach because although they are
comprised of multiple subunits that are coded for by several genes, each channel must
have the required NR1 subunit to be fully functional (Monyer, Sprengel et al. 1992;
Hollmann and Heinemann 1994; Yamakura and Shimoji 1999). Therefore, knockdown
of NMDAR1 protein should result in fewer functional NMDA channels. Haberman et al.
tested this hypothesis using an NMDAR1 antisense construct delivered by an AAV2
vector (Haberman, Criswell et al. 2002). While in vitro NMDAR1 receptor function and
in vivo NMDAR1 protein levels were significantly reduced, the seizure behavior outcome
was complicated by the promoter choice. When the antisense construct was driven by the
CMV promoter, a significant increase in the amount of current was necessary to elicit a
focal seizure (Haberman, Criswell et al. 2002). However, when expression of the same
antisense construct was driven by the tet-off promoter, there was a significant decrease in
the amount of current necessary to elicit a focal seizure. The authors go on to show that
the plausible explanation for the opposing results is that when the CMV promoter was
driving expression of the antisense NMDAR1, a majority of the transduced neurons were
primary output neurons that benefit from a reduction in excitatory input. When the tet-
off promoter was used, the majority of transduced neurons were inhibitory interneurons,
and the resulting reduction in NMDA activation increased the overall excitatory tone and
seizure sensitivity (Haberman, Criswell et al. 2002). This highlights another very
22
important liability when modulating receptors, ion channels, or even neurotransmitters
and transporters: results may be dependant upon the pattern of transduction.
I.H. In Vivo Viral Vector-Mediated Expression of Neuropeptides and Peptide Fragments
Neuropeptides and their receptors are found in areas often associated with seizure
generation, and their physiological effects are predominantly inhibitory suggesting anti-
convulsant action. In particular, both galanin and NPY (and their analogs) have anti-
convulsant effects in several models of experimentally induced seizures (Mazarati, Liu et
al. 1998; Mazarati, Hohmann et al. 2000; Mazarati and Wasterlain 2002; Haberman,
Samulski et al. 2003; Mazarati 2004; McCown 2006; Foti, Haberman et al. 2007).
Experimental evidence suggests that prolonged overstimulation, such as recurring
seizures, depletes galanergic innervation (Mazarati, Liu et al. 1998) and destroys a subset
of GABA-ergic NPY containing interneurons in the hippocampus (Sloviter 1991; Sperk,
Marksteiner et al. 1992; Schwarzer, Williamson et al. 1995). This finding was also
validated in patients with mesial temporal lobe sclerosis (de Lanerolle, Kim et al. 1989).
Taken together, these data suggest that overexpressing galanin and NPY in the ictogenic
brain regions may not only suppress seizures and prevent them from spreading, but might
also compensate for depleted neuropeptides, conferring some neuroprotection.
The use of AAV vectors to express galanin and NPY in the hippocampus and
piriform cortex has been explored with very good results. Haberman et al. first
demonstrated the anti-seizure efficacy of vector-mediated neuropeptide gene expression,
using a novel platform designed to circumvent the potential liabilities of viral vector
tropism (Haberman, Criswell et al. 2002; Haberman, Samulski et al. 2003). An AAV2
vector was constructed in which the coding sequence for the active galanin peptide was
23
preceded by the secretory signal sequence of the laminar protein, fibronectin. Thus, the
active peptide would be constitutively secreted from the transduced cell. The elegance of
this approach hinges on the fact that the newly synthesized neuropeptide will be
packaged in vesicles and continuously released, independent of neuronal activity,
essentially bypassing the traditional neuropeptide regulatory pathways. These studies
showed that the fibronectin secretion signal sequence (FIB) did cause constitutive
secretion of the gene product from transfected cells in vitro and, upon transduction in
vivo, significantly attenuated focal seizure sensitivity and prevented seizure-induced
hippocampal cell damage (Haberman, Samulski et al. 2003).
In a subsequent publication, Lin et al. found that AAV mediated expression of a
human galanin cDNA significantly decreased the number of kainic acid-induced seizure
episodes, but this approach did not alter seizure latency or kainic acid-induced neuronal
damage (Lin, Richichi et al. 2003). McCown then used an AAV2 vector where
expression was driven by the constitutively active CB promoter to deliver galanin to the
piriform cortex in rats (McCown 2006). The vector also contained the FIB sequence to
mediate constitutive secretion of the expressed galanin. This approach not only
prevented electrographic seizures, as measured by electroencephalography (EEG), but
also completely blocked seizure behavior, while delivery of green fluorescent protein
(GFP) had no effect (McCown 2006). Furthermore, in a kindling paradigm, vector-
derived galanin caused a significant increase in the amount of current necessary to elicit
limbic seizures compared to GFP control (McCown 2006). Taken together, these
experiments suggest that the piriform cortex is directly involved in mediating the onset
24
and severity of kainic acid-induced seizure behaviors. Thus, the piriform cortex is a good
place to deliver and test potential therapeutic targets for anti-seizure efficacy.
As previously discussed, there is evidence to suggest that NPY can exhibit anti-
seizure activity in vivo (Woldbye, Larsen et al. 1997; Mazarati and Wasterlain 2002;
Vezzani, Michalkiewicz et al. 2002), by acting primarily through NPY Y1, Y2, and Y5
G protein-coupled receptors in brain (Parker and Herzog 1999; Redrobe, Dumont et al.
1999). Each of these receptors has been reported to affect seizure genesis, but not via the
same mechanism. For example, in the dentate gyrus, activation of Y1 receptors results in
increased neuronal excitation (seizure permissive) (Brooks, Kelly et al. 1987), while
activation of Y2 (Colmers, Klapstein et al. 1991; El Bahh, Cao et al. 2002; Vezzani and
Sperk 2004) and Y5 receptors (Woldbye, Larsen et al. 1997; Marsh, Baraban et al. 1999;
Baraban 2002) mediate inhibitory and anti-convulsant actions. In addition, it has been
demonstrated that in epileptic tissue from patients (Furtinger, Pirker et al. 2001) and
rodents (Kofler, Kirchmair et al. 1997; Schwarzer, Kofler et al. 1998) Y1 receptors are
downregulated, whereas Y2 receptors are upregulated. Even Y5 receptors are transiently
upregulated following kindling and kainic acid-induced seizures (Kopp, Nanobashvili et
al. 1999; Vezzani, Moneta et al. 2000). Taken together, these results suggest that when
designing anti-epileptic therapeutics, it may be advantageous to deliver Y2/Y5-preferring
agonists. In fact, acute intracerebral delivery of the Y2 receptor preferring agonist
NPY13-36 reduces seizure susceptibility following systemic kainic acid administration
(Vezzani, Moneta et al. 2000; Vezzani, Rizzi et al. 2000). These data suggest NPY13-36
would be a good candidate to use in a rAAV vector to treat epilepsy. By using the FIB
secretion strategy, we have the capability of expressing and constitutively secreting
25
peptide fragments, which have demonstrated receptor selectivity. Thus, we infused
AAV2 vectors that mediate expression and constitutive secretion of NPY or the peptide
fragment NPY13-36 into the piriform cortex, and evaluated their ability to suppress
kainic acid-induced limbic seizure behaviors (see Chapter 3).
I.I. Combination Therapy
Delivering individual inhibitory neuropeptides to the brain to prevent seizures has
been well studied (see above), but to date no one has tried to deliver multiple
neuropeptides simultaneously from an AAV vector. In the last 20 years, many different
virus-based strategies were explored to co-express two genes from vectors: 1. internal
promoters, 2. internal initiation (IRES), 3. self-processing peptides (CHYSEL), 4. fusion
proteins, 5. proteolytic processing, and finally 6. a combination of separate vectors where
each carries one transgene (see Figure 1-5). Internal promoter vectors (sometimes
referred to as bicistronic vectors) have two transcriptional units, each with its own open
reading frame that results in the production of two proteins. While these vectors are quite
popular, problems arise because of the uncoupled transcription of both genes. For
example, it has been reported that transcriptional interference and promoter silencing can
result in the transcription of only one gene (Hippenmeyer and Krivi 1991; Nakajima,
Ikenaka et al. 1993; Zaboikin and Schuening 1998).
Internal initiation vectors have two sites of translation initiation and two open
reading frames on a single transcript. The first open reading frame is cap-dependent,
while translation of the second open reading frame depends upon an internal sequence
called an internal ribosomal entry site (IRES). The main concern with IRES vectors is
the reduced expression of the gene downstream of the IRES (typically 20-50% less than
26
Figure 1-5. Strategies to Co-express Gene Products. A) Internal promoter vectors have two transcriptional units, each with its own open reading frame that results in the production of two proteins. B) Internal initiation vectors have a single transcript with two open reading frames and two sites of translation. The first open reading frame is cap-dependent, while translation of the second open reading frame depends upon an internal sequence called an internal ribosomal entry site (IRES). C) Self-processing peptide or cis-acting hydrolase element (CHYSEL) vectors have a single transcriptional unit with a single open reading frame and one site of translation. The CHYSEL sequence is inserted in frame between the two protein sequences, and upon translation, the CHYSEL produces a disruption in translation. D) Proteolytic processing vectors also have a single transcriptional unit with a single open reading frame and one site of translation. The proteolytic cleavage sequence is cloned in frame as a linker, and a chimeric fusion protein is generated that is capable of undergoing post-translational cleavage in the presence of trans proteases. E) Fusion protein vectors have a single transcriptional unit with a single open reading frame and one site of translation. A single chimeric fused protein is generated. (Arrows depict promoters, star depicts cleavage site)
IP Gene 2 polyApolyAGene 1
AAA AAAProtein 1 Protein 2
A
Gene 1 Gene 2 polyAIRES
AAAProtein 1 Protein 2
B
polyA
AAAProtein 1 Protein 2 Post-translational Cleavage
and Removal of SequenceProtein 1 Protein 2
*
Gene 1 Gene 2Cleavage Sequence
D
Gene 2 polyAGene 1
AAAProtein 1 Protein 2
E
Gene 2 polyACHYSEL
AAAProtein 1 CHYSE
Gene 1C
Protein 2L
IP Gene 2 polyApolyAGene 1
AAA AAAProtein 1 Protein 2
AIPIP Gene 2Gene 2 polyApolyApolyApolyAGene 1Gene 1
AAAAAA AAAAAAProtein 1Protein 1 Protein 2Protein 2
A
Gene 1 Gene 2 polyAIRES
AAAProtein 1 Protein 2
BGene 1 Gene 2 polyAIRES
AAAProtein 1 Protein 2
Gene 1 Gene 2 polyAIRES
AAA
Gene 1 Gene 2 polyAIRESGene 1Gene 1 Gene 2Gene 2 polyApolyAIRESIRES
AAAAAAProtein 1Protein 1 Protein 2Protein 2
B
polyA
AAAProtein 1 Protein 2 Post-translational Cleavage
and Removal of SequenceProtein 1 Protein 2
*
Gene 1 Gene 2Cleavage Sequence
DpolyApolyA
AAAProtein 1 Protein 2 Post-translational Cleavage
and Removal of SequenceProtein 1 Protein 2
*AAAAAA
Protein 1Protein 1 Protein 2Protein 2 Post-translational Cleavage
and Removal of SequenceProtein 1Protein 1 Protein 2Protein 2
*
Gene 1Gene 1 Gene 2Gene 2Cleavage SequenceCleavage Sequence
D
Gene 2 polyAGene 1
AAAProtein 1 Protein 2
EGene 2 polyAGene 1
AAAProtein 1 Protein 2
Gene 2Gene 2 polyApolyAGene 1Gene 1
AAAAAAProtein 1 Protein 2Protein 1Protein 1 Protein 2Protein 2
E
Gene 2 polyACHYSEL
AAAProtein 1 CHYSE
Gene 1C
Protein 2L
Gene 2Gene 2 polyApolyACHYSELCHYSEL
AAAAAAProtein 1 CHYSEProtein 1Protein 1 CHYSECHYSE
Gene 1Gene 1C
Protein 2L Protein 2Protein 2LL
27
the expression of the upstream gene) (Mizuguchi, Xu et al. 2000). Perhaps the biggest
drawback of both vector strategies discussed so far (internal promoters and IRES) is that
they contain large DNA sequences (often >0.5kb), which compete with the transgene for
a limited amount of space in an AAV vector (around 4.7kb).
Self-processing peptide or cis-acting hydrolase element (CHYSEL) vectors have a
single transcriptional unit and one site of translation with a single open reading frame.
The CHYSEL sequence is inserted in frame between the two protein sequences, and upon
translation, the CHYSEL produces a disruption in translation. The ribosome then
releases the first protein and continues to translate the second protein, resulting in the
production of two proteins (Donnelly, Luke et al. 2001). While it has been demonstrated
that higher levels of the second protein are achieved when using CHYSEL in contrast to
IRES (Furler, Paterna et al. 2001), a potential problem is that after processing, the
CHYSEL sequence remains fused to the C-terminus of the upstream protein, and an extra
proline residue will be added to the N-terminus of the second protein (de Felipe 2002).
This extra sequence may interfere with protein bioactivity, especially with relatively
small proteins such as neuropeptides that need to bind to receptors to initiate signaling.
Fusion protein vectors are the simplest way to co-express two proteins using a
single transcriptional unit and open reading frame. However, because both proteins
remain attached to each other, there may be a potential loss of function of one or both
proteins, especially if trying to deliver neuropeptides where high affinity receptor binding
is required to initiate signaling cascades. In addition, since fusion proteins are not
cleaved from one another, transport to different subcellular compartments is not feasible.
Therefore, fusion protein strategies could not be used to deliver both secreted proteins
28
and intracellular or membrane bound proteins, further limiting the applicability of this
approach.
There are also major drawbacks with using a co-infection of two separate vectors
where each carries one transgene. Three populations of transduced neurons will result
based on a Poisson distribution: some neurons will be transduced only by the first vector,
some neurons will be transduced only by the second vector, and some neurons will be
transduced by both. This kind of transduction pattern can complicate the analysis of
transgene interactions, because the observed therapeutic effects will not be attributable
solely to the co-transduced neurons. For these reasons we chose to pursue a proteolytic
processing vector strategy where a fusion protein gene product is processed into separate
bioactive proteins. This method will ensure that each transduced neuron expresses both
gene products, and utilizes only a small amount of the AAV vector genome, since
proteolytic cleavage consensus sequences can be as small as two amino acids (Seidah and
Chretien 1999). In addition, we can use this same strategy in the future to express more
than two therapeutic peptides simultaneously, and to manipulate the ratios of expressed
peptides by inserting multiple copies of the same peptide. Furthermore, by using a single
vector we can reduce viral particle number (and viral capsid proteins), which would
likely increase clinical safety.
As mentioned above, neuropeptides are typically synthesized as large pro-proteins
that upon being packaged into vesicles get cleaved into their mature form before
secretion. Furin is one such pro-protein convertase that is active in the constitutive
secretory pathway, and cleaves pro-proteins like neurotrophins, hormones, receptors,
plasma proteins, matrix metalloproteinases, viral envelope glycoproteins, and bacterial
29
endotoxins (Nakayama 1997). Furin cleaves precursors into bioactive proteins by
recognizing and cleaving strings of basic amino acids such as arginines and lysines. In
addition, furin is ubiquitously expressed in neurons and glia throughout the brain (Day,
Schafer et al. 1993), making it an ideal pro-protein convertase to target with our
constitutively secreted neuropeptides platform. The furin pathway has already been used
in a gene therapy context where a furin cleavage site was engineered into the rat
preproinsulin-I gene, which was then packaged into a recombinant retroviral vector and
delivered to the liver (Muzzin, Eisensmith et al. 1997). It was shown that correctly
processed and functional insulin could be secreted from the ectopic organ (Muzzin,
Eisensmith et al. 1997). In a subsequent study, Margaritis et al. used a furin cleavage
consensus sequence in an AAV vector cassette between Factor VIIa subunits, then
delivered the vector into the spleens of hemophiliac mice (Margaritis, Arruda et al. 2004).
Phenotypic correction was achieved using this strategy (Margaritis, Arruda et al. 2004).
While both of these studies demonstrate that engineering a furin cleavage
consensus sequence into a transgene can be successful in a gene therapy context, only
one functional protein was delivered. In a study by Gaken et al., a furin cleavage
consensus sequence was cloned in frame as a linker between two different proteins,
yielding a chimeric protein that can be post-translationally cleaved (Gaken, Jiang et al.
2000). These constructs contained combinations of cytokines or a cytokine and a trans-
membrane protein. They were packaged into retroviral constructs and used to infect
several cell lines where furin cleavage efficiency was evaluated. The authors reported
around 50% cleavage efficiency (Gaken, Jiang et al. 2000).
30
Previous experiments suggest that it is possible to use a proteolytic strategy to
deliver multiple peptides from AAV vectors, but to date no one has tried. We are in a
unique position to conduct these experiments and combine them with our constitutively
secreted neuropeptides platform. We expect improved cleavage efficiencies over
previous attempts because we are using a FIB sequence that will direct our chimeric
protein to the constitutive secretory pathway where furin is most active (Van de Ven,
Creemers et al. 1991). We first proved the concept that the therapeutic gene galanin and
the reporter gene GFP could be correctly cleaved from one another and secreted into the
medium of transfected cells in vitro. Then, we evaluated the in vivo function of each
protein by packaging the cleavable galanin-GFP gene into AAV2 vectors, and infusing
them into the piriform cortex of rats that subsequently received injections of kainic acid
(see Chapter 3). We visualized the GFP protein expression in the piriform cortex
confirming its function, and we evaluated the ability of vector-derived galanin to block
the limbic seizure behaviors elicited by the kainic acid administration. Finally, we
replaced the reporter gene with NPY13-36 and evaluated the combined effects of vector-
derived galanin plus NPY13-36 on kainic acid-induced limbic seizures (see Chapter 3).
Chapter 2
Methods
II.A. rAAV Vectors: Cloning and Construction
II.A.1. Cloning Plasmids for rAAV-CB-FIB-NPY and rAAV-CB-FIB-NPY13-36
The expression cassette backbone is the same for both NPY and NPY13-36
plasmids, where gene expression is driven by the hybrid chicken beta-actin promoter, the
mature coding sequence of the transgene is followed by the SV-40 polyadenylation
sequence (polyA), and the cassette is flanked by AAV2 TRs. The full length NPY active
peptide sequence was amplified by RT-PCR from rat brain RNA using primers directed
to the mature peptide sequence, such that melting and reannealing of two separate PCR
products resulted in 5’ AgeI and 3’ NotI overhangs (Zeng 1998). The 3’ primer included
a stop codon to properly terminate translation. The sequence of the primers used is as
follows: (NPY 5’ long) CCG GTA ATG TAC CCC TCC AAG CCG; (NPY 5’ short) TA
ATG TAC CCC TCC AAG CCG; (NPY 3’short) GC TCA ATA TCT CTG TCT GGT
G; (NPY 3’ long) GGC CGC TCA ATA TCT CTG TCT GGT G. The PCR product was
ligated into the AgeI-NotI sites of the AAV2 backbone plasmid, resulting in the plasmid
pTR-CB-NPY. Next, the fibronectin signal sequence (nucleotides 208–303) was derived
from the rat fibronectin mRNA sequence (GenBank Accession No. X15906) and
oligonucleotides corresponding to both strands were generated (Midland Certified
Reagent Co.). AgeI overhangs were included such that the annealed oligonucleotides
32
could be ligated in front of NPY, resulting in the plasmid pTR-CB-FIB-NPY. For NPY
13-36, the same cloning strategy was used, except the plasmid pTR-CB-FIB-NPY served
as the PCR template and cloning backbone. The primers used to generate NPY13-36
were as follows: (NPY13-36 5’ long) 5’-CCG GTA ATG CCA GCA GAG GAC ATG-
3’, (NPY13-36 5’ short) 5’-TA ATG CCA GCA GAG GAC ATG GC-3’, (NPY13-36 3’
short) 5’-GC TCA ATA TCT CTG TCT GGT G-3’, (NPY13-36 3’ long) 5’-GGC CGC
TCA ATA TCT CTG TCT GGT G-3’. Again, the PCR product was ligated into the
AgeI-NotI site of pTR-CB-FIB-NPY, replacing the “FIB-NPY”. Then the annealed FIB
oligonucleotides were ligated into the AgeI site resulting in the plasmid pTR-CB-FIB-
NPY13-36. All plasmids were sequenced to verify accuracy.
II.A.2. Cloning Plasmids for Multiple Gene Product Delivery Vectors
The plasmids pTR-CB-EGFP, pTR-CB-FIB-EGFP, and pTR-CB-FIB-GAL were
generated as previously described (McCown 2006). Briefly, gene expression is driven by
the hybrid chicken beta-actin promoter, the mature coding sequence of the transgene is
followed by the SV-40 polyA, and the cassette is flanked by AAV2 TRs. The plasmid
EGFP-N1 was purchased from BD-biosciences-clontech, and the GFP was digested out
using AgeI-NotI restriction sites. The fragment was gel purified and ligated into the
AgeI-NotI sites of the AAV2 plasmid backbone, resulting in the plasmid pTR-CB-EGFP.
Then the annealed FIB oligonucleotides were ligated into the AgeI site resulting in the
plasmid pTR-CB-FIB-EGFP. The galanin sequence was amplified by RT-PCR from rat
brain RNA using primers directed to the mature peptide sequence, such that melting and
reannealing of two separate PCR products resulted in 5’ AgeI and 3’ NotI overhangs
(Zeng 1998). The 3’ primer included a stop codon to properly terminate translation. The
33
sequence of the primers used is as follows: (Gal 5’ long) 5’-CCG GTA ATG GGC TGG
ACC CTG AAC-3’, (Gal 5’ short) 5’-TA ATG GGC TGG ACC CTG AAC-3’, (Gal 3’
short) 5’-GC TCA TGT GAG GCC ATG CTT G-3’, (Gal 3’ long) 5’-GGC CGC TCA
TGT GAG GCC ATG CTT G-3’. The PCR product was ligated into the AgeI-NotI sites
of the AAV2 plasmid backbone, resulting in the plasmid pTR-CB-GAL. Then the
annealed FIB oligonucleotides were ligated into the AgeI site resulting in the plasmid
pTR-CB-FIB-GAL. Using pTR-CB-FIB-GAL as a backbone, the other plasmids were
generated by PCR so the furin cleavage consensus sequence arginine-lysine-arginine-
arginine-lysine-arginine (RKRRKR) was cloned in frame between two genes. Briefly,
two pairs of oligonucleotides were designed for each construct, such that a forward and
reverse primer corresponded to each gene plus 3 of the six amino acids that make up the
furin consensus sequence (see Table 2-1). The oligonucleotides included the restriction
sites NheI and NotI that would be used for cloning. Two fragments each containing the
gene of interest plus half of the furin consensus sequence were generated by PCR, then
the products were digested with NheI and NotI, and ligated into the plasmid backbone
using a sticky-blunt-sticky ligation. All plasmids were sequenced to verify accuracy.
II.A.3. DNA Sequencing
All DNA sequencing was performed by the UNC Lineberger Cancer Center DNA
Sequencing Facility. Briefly, 10pmol of primer and 0.7μg of plasmid DNA were diluted
in water to a final volume of 20μL. Samples were further processed by the sequencing
facility.
34
Table 2-1 PCR Primers for Multiple Gene Product Delivery Vectors
Construct Name Primers Used Melting Temperature
TR-CB-FIB-GAL-RKRRKR-EGFP
F1= GTACGGAAGTGTTACTTCTGCTC R1= 5’PTCTCTTTCTTGTGAGGCCATGCTT F2= 5’PAGAAAGAGAATGGTGAGCAAGGGCGAGGAGC R2= CTTATCATGTCTGGATCCCCGCGGCC
55.0°C 56.0°C 66.0°C 64.0°C
TR-CB-FIB-EGFP-RKRRKR-GAL
F1= GTACGGAAGTGTTACTTCTGCTC R1= 5’PTCTCTTTCTCTTGTACAGCTCGTC F2= 5’PAGAAAGAGAGGCTGGACCTGAACAGCG R2= CTTATCATGTCTGGATCCCCGCGGCC
55.0°C 56.0°C 64.0°C 64.0°C
TR-CB-FIB-GAL-EGFP
F1= GTACGGAAGTGTTACTTCTGCTC R1= 5’PTGTGAGGCCATGCTTGTCGCT F2= 5’PATGGTGAGCAAGGGCGAGGAGCTG R2= CTTATCATGTCTGGATCCCCGCGGCC
55.0°C 56.0°C 63.0°C 64.0°C
TR-CB-FIB-GAL-RKRRKR-NPY13-36
F1= GTACGGAAGTGTTACTTCTGCTC R1= 5’PTCTCTTTCTTGTGAGGCCATGCTT F2= 5’P AGAAAGAGACCAGCAGAGGACATGGCC R2= CTTATCATGTCTGGATCCCCGCGGCC
55.0°C 56.0°C 63.0°C 64.0°C
35
II.B. In Vitro Methods
II.B.1. Cell Culture and Transfection
293 cells were cells were maintained at 37°C in a 5% CO2 atmosphere in
Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine
serum (FBS) and penicillin-streptomycin (PS). Cells were plated at a density of 5.5x105
cells per mL medium in 60mm dishes the day prior to transfection. Cells were
transfected using the polyethyleneimine (PEI) technique (Grieger, Choi et al. 2006).
Forty-eight hours after transfection, cells were imaged then medium and lysates were
harvested. Briefly, medium was cleared by centrifugation at 2500 RPM at 4°C for 5
minutes to remove any cellular debris. Cells were scraped and rinsed with 2mL
Dulbecco phosphate buffered saline (DPBS) then lysed using 1mL lysis buffer (50mM
Tris pH8.0, 150mM NaCl, 50mM NaF, 1% NP-40) per 60mm plate. Lysates were
cleared by spinning in centrifuge at maximum speed at 4°C for 5 minutes, supernatant
was transferred to a clean tube.
II.B.2 Immunoprecipitation and Western Blotting
Immunoprecipitation of GFP was performed on cleared medium and lysates.
Briefly, medium and lysates were pre-incubated with 20μL Protein A Sepharose beads
(Amersham Biosciences) and rocked for 1 hour at 4°C to remove non-specific binding.
Supernatant was transferred to a clean tube. For medium 50μL Protein A beads and
10μL Living Colors Full-Length A.V. Polyclonal Antibody (BD) was added. For lysates
25μL Protein A beads and 5μL Living Colors Full-Length A.V. Polyclonal Antibody
(BD) was added. Samples were incubated overnight on a rocker at. Supernatant was
removed by vacuum and beads were washed 3 times using 1mL DPBS. Load dye was
added to beads along with water and betamercaptoethanol, then samples were boiled 5
minutes and cooled to room temperature before loading on 10% Bis-Tris gel (NuPage)
Samples were transferred by western blot to a nitrocellulose membrane and blocked using
10% nonfat milk in tris buffered saline + 0.1% Tween (TBST). Blot was incubated at
4°C overnight in 1:1000 dilution of primary antibody Living Colors A.V. Monoclonal
antibody JL-8 (BD). Blot was rinsed 3 times 5 minutes in TBST then incubated in a
1:3000 dilution of goat-anti-mouse HRP (Pierce) Signal was detected using the West
femto-chemiluminescence kit (Pierce) according to the manufacturer’s instructions.
II.C. rAAV Production, Purification, and Characterization
Recombinant AAV2 was produced and purified as previously described
(Rabinowitz, Rolling et al. 2002) with the following modifications: 293 cells (10 15cm
plates/prep)were transfected with 60μg transgene plasmid, 120μg XX-680, and 100μg
PXR2 via the polyethyleneimine (PEI) technique. Nuclei were isolated and lysed using
sonication. AAV particles were purified by cesium chloride density gradient. Peak
fractions were determined via dot blot hybridization, and extensively dialyzed against
DPBS + 10% (wt/vol) D-sorbitol. Final titer was determined by dot blot hybridization
(modified southern blot) using a probe against the CB promoter sequence so all viruses
could be titered on the same blot (see Figures 2-1 through 2-3) Infectious center assays
(ICA) were performed using C12 cells (293 cells with Rep stably integrated) as
previously described (Grieger, Choi et al. 2006) (see Figures 2-4 through 2-7). AAV
particles were analyzed to determine purity and ratio of empty to full particles by staining
36
37
Figure 2-1. Dot Blot, Standard Curve, and Titer Calculations for AAV2-CB-FIB-NPY and AAV2-CB-FIB-NPY13-36. The top panel shows three dilutions (across) of the viruses performed in duplicate (down). Dilutions of known amounts (nanograms) of one of the plasmids used to make the rAAV vectors were included (pTR-CB-FIB-NPY). Upon hybridization with a radiolabeled probe that was complementary to a fragment of the CB promoter, a standard curve was generated (middle panel). Then a linear regression analysis was done, and the derived equation (displayed in the upper left corner of the standard curve graph) was used to calculate the number of rAAV particles present in each dilution (bottom panel), then the average particles present per mL.
AAV2-CB-FIB-NPY AAV2-CB-FIB-NPY13-36
pTR-CB-FIB-NPY
AAV2-CB-FIB-NPY AAV2-CB-FIB-NPY13-36
pTR-CB-FIB-NPY
1.73E+121.08E+09748630.3
1.89E+122.37E+091595254
1.77E+121.69E+124.23E+092820913
Avg Particles/mLParticles/mLy=Avg*X-BAverage
1.73E+121.08E+09748630.3
1.89E+122.37E+091595254
1.77E+121.69E+124.23E+092820913
Avg Particles/mLParticles/mLy=Avg*X-BAverage
1.54E+129.65E+08674020
1.68E+122.10E+091420987
1.64E+121.71E+124.27E+092845020
1.54E+129.65E+08674020
1.68E+122.10E+091420987
1.64E+121.71E+124.27E+092845020
Standard Curve y = 1520.8x - 6E+07R2 = 0.9974
0.00E+00
5.00E+09
1.00E+10
1.50E+10
0 2000000 4000000 6000000 8000000
Number of Probes
Volu
me
of S
tand
ard
38
Figure 2-2. Dot Blot, Standard Curve, and Titer Calculations for AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP, AAV2-CB-FIB-EGFP, and AAV2-CB-EGFP. The left panel shows three dilutions (across) of the viruses performed in duplicate (down). Dilutions of known amounts (nanograms) of one of the plasmids used to make the rAAV vectors were included (pTR-CB-FIB-GAL-RKRRKR-FIB-EGFP). Upon hybridization with a radiolabeled probe that was complementary to a fragment of the CB promoter, a standard curve was generated (bottom panel). Then a linear regression analysis was done, and the derived equation (displayed in the upper left corner of the standard curve graph) was used to calculate the number of rAAV particles present in each dilution (right panel), then the average particles present per mL.
AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP side and peak fractions
AAV2-CB-FIB-EGFP AAV2-CB-EGFP
50 25 12.5 6.25 3.125
AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP side and peak fractions
AAV2-CB-FIB-EGFP AAV2-CB-EGFP
50 25 12.5 6.25 3.125
5.76271E+117.20E+0838473.47
5.80512E+117.26E+0838756.62
5.47932E+114.87012E+111.22E+0965028.63
Avg particles/mLParticles/mL# * X+BAverage
5.76271E+117.20E+0838473.47
5.80512E+117.26E+0838756.62
5.47932E+114.87012E+111.22E+0965028.63
Avg particles/mLParticles/mL# * X+BAverage
2.16418E+122.71E+09144486.61
2.27565E+122.84E+09151928.995
2.1967E+122.15028E+125.38E+09287117.055
2.16418E+122.71E+09144486.61
2.27565E+122.84E+09151928.995
2.1967E+122.15028E+125.38E+09287117.055
2.51044E+123.14E+09167603.77
2.42044E+123.03E+09161595.12
2.41071E+122.30127E+125.75E+09307278.77
2.51044E+123.14E+09167603.77
2.42044E+123.03E+09161595.12
2.41071E+122.30127E+125.75E+09307278.77
1.88995E+122.36E+09126178.295
1.93021E+122.41E+09128866.455
1.87706E+121.81101E+124.53E+09241815.59
1.88995E+122.36E+09126178.295
1.93021E+122.41E+09128866.455
1.87706E+121.81101E+124.53E+09241815.59
Standard Curve y = 18225x + 1E+09R2 = 0.9899
0
5000000000
10000000000
15000000000
20000000000
25000000000
0 200000 400000 600000 800000 1000000 1200000
Number of Probes
Volu
me
of S
tand
ards
39
Figure 2-3. Dot Blot, Standard Curve, and Titer Calculations for Multiple Gene Product Delivery Vectors. The left panel shows three dilutions (across) of the viruses performed in duplicate (down). The virus order is as follows 1) upper left, AAV2-CB-FIB-GAL-RKRRKR-EGFP, 2) upper right, AAV2-CB-FIB-EGFP-RKRRKR-GAL, 3) lower left, AAV2-CB-FIB-GAL-EGFP, and 4) lower right, AAV2-CB-FIB-GAL-RKRRKR-NPY13-36. Dilutions of known amounts (nanograms) of one of the plasmids used to make the rAAV vectors were included (pTR-CB-FIB-GAL-EGFP). Upon hybridization with a radiolabeled probe that was complementary to a fragment of the CB promoter, a standard curve was generated (bottom panel). Then a linear regression analysis was done, and the derived equation (displayed in the upper left corner of the standard curve graph) was used to calculate the number of rAAV particles present in each dilution (right panel), then the average particles present per mL.
6.44569E+114.03E+08503472.1
1.03451E+121.29E+09909902
1.09097E+121.14743E+122.87E+091629115
Avg Particles/mLParticles/mLAvg * x - BAverage
6.44569E+114.03E+08503472.1
1.03451E+121.29E+09909902
1.09097E+121.14743E+122.87E+091629115
Avg Particles/mLParticles/mLAvg * x - BAverage
5.3877E+113.37E+08473285.3
1.10069E+121.38E+09947668.1
1.16655E+121.2324E+123.08E+091726094
5.3877E+113.37E+08473285.3
1.10069E+121.38E+09947668.1
1.16655E+121.2324E+123.08E+091726094
2.4716E+121.54E+091024766
2.88779E+123.61E+091967467
2.84914E+122.81049E+127.03E+093527150
2.4716E+121.54E+091024766
2.88779E+123.61E+091967467
2.84914E+122.81049E+127.03E+093527150
6.71364E+124.20E+092235117
6.56656E+128.21E+094066745
6.00733E+125.4481E+121.36E+106537437
6.71364E+124.20E+092235117
6.56656E+128.21E+094066745
6.00733E+125.4481E+121.36E+106537437
AAV2-CB-FG-RKRRKR-E AAV2-CB-FE-RKRRKR-G
AAV2-CB-F-G-E AAV2-CB-FG-RKRRKR-NPY13-36
pTR-CB-FIB-GAL-EGFP
AAV2-CB-FG-RKRRKR-E AAV2-CB-FE-RKRRKR-G
AAV2-CB-F-G-E AAV2-CB-FG-RKRRKR-NPY13-36
pTR-CB-FIB-GAL-EGFP
Standard Curve y = 2190.5x - 7E+08R2 = 0.991
-20000000000
2000000000400000000060000000008000000000
100000000001200000000014000000000
0 1000000 2000000 3000000 4000000 5000000 6000000
Number of probes
Volu
me
of S
tand
ards
40
Figure 2-4. Infectious Center Assay of AAV2-CB-FIB-GAL-RKRRKR-EGFP. A) 500 particles, B) 1000 particles, and C) 5000 particles of AAV2-CB-FIB-GAL-RKRRKR-EGFP were co-infected with Ad in C12 cells, blotted on a membrane, and hybridized with radiolabeled probe that was complementary to a fragment of the CB promoter to determine the number of infectious units per mL (IU/mL). This virus is 3.6x109 IU/mL.
AAV2-CB-FIB-GAL-RKRRKR-EGFP
A
B
C
AAV2-CB-FIB-GAL-RKRRKR-EGFP
A
B
C
41
Figure 2-5. Infectious Center Assay of AAV2-CB-FIB-EGFP-RKRRKR-GAL. A) 500 particles, B) 1000 particles, and C) 5000 particles of AAV2-CB-FIB-EGFP-RKRRKR-GAL were co-infected with Ad in C12 cells, blotted on a membrane, and hybridized with radiolabeled probe that was complementary to a fragment of the CB promoter to determine the IU/mL. This virus is 1.0x109 IU/mL.
AAV2-CB-FIB-EGFP-RKRRKR-GAL
A
C
B
AAV2-CB-FIB-EGFP-RKRRKR-GAL
A
C
B
42
Figure 2-6. Infectious Center Assay of AAV2-CB-FIB-GAL-EGFP. A) 500 particles, B) 1000 particles, and C) 5000 particles of AAV2-CB-FIB-GAL-EGFP were co-infected with Ad in C12 cells, blotted on a membrane, and hybridized with radiolabeled probe that was complementary to a fragment of the CB promoter to determine the IU/mL. This virus is 2.0x109 IU/mL.
AAV2-CB-FIB-GAL-EGFP
A
B
C
AAV2-CB-FIB-GAL-EGFP
A
B
C
43
Figure 2-7. Infectious Center Assay of AAV2-CB-FIB-GAL-RKRRKR-NPY13-36. A) 500 particles, B) 1000 particles, and C) 5000 particles of AAV2-CB-FIB-GAL-RKRRKR-NPY13-36 were co-infected with Ad in C12 cells, blotted on a membrane, and hybridized with radiolabeled probe that was complementary to a fragment of the CB promoter to determine the IU/mL. This virus is 9.0x109 IU/mL.
AAV2-CB-FIB-GAL-RKRRKR-NPY13-36
A B C
AAV2-CB-FIB-GAL-RKRRKR-NPY13-36
A B C
44
with 1% aqueous uranyl acetate and visualizing with transmission electron microscopy
(LEO-EM910, accelerating voltage = 80 KV).
II.D. In Vivo Methods
II.D.1. Experimental Animals
All of the animals were pathogen-free male Sprague–Dawley rats obtained from
Charles Rivers. The animals were maintained in a 12 hour light–dark cycle and had free
access to food and water. All care and procedures were in accordance with the Guide for
the Care and Use of Laboratory Animals (DHHS Publication No. [NIH]85-23), and all
procedures received prior approval by the University of North Carolina Institutional
Animal Care and Usage Committee.
II.D.2. rAAV Vector Microinjection
For AAV infusions, rats were first were anesthetized with 50 milligrams per
kilogram, intraperitoneal (mg/kg, ip) pentobarbital and placed into a stereotaxic frame.
Using a 32 gauge stainless steel injector and a Sage infusion pump, the rats received 2 or
3µL of virus (depending on virus titer) over 20 minutes into the piriform cortex (inter-
aural line (IAL) 6.7 millimeters (mm), lateral 6.0mm, vertical 8.4mm, according to the
atlas of Paxinos and Watson, 1998) (see Figure 2-8). The injector was left in place for 3
minutes post-infusion to allow diffusion from the injectors. In all cases, the incisions
were sutured, and the animals were allowed to recover for a minimum of 7 days.
45
Figure 2-8. Coronal Section of the Rat Brain Depicting the Location of Vector Microinjection. rAAV2 vector is stereotaxically injected bilaterally into the piriform cortex, denoted by arrows. The coordinates are inter-aural line (IAL) 6.7 millimeters (mm), lateral 6.0mm, vertical 8.4mm, according to the atlas of Paxinos and Watson (1998).
Interaural 6.70 mm Bregma -2.30 mm
46
II.D.3. In Vivo Detection of AAV-Derived NPY or NPY 13-36
As previously described (Haberman, Samulski et al. 2003; Foti, Haberman et al.
2007) the vector-derived NPY could not be visualized in vivo by immunohistochemistry,
likely due to the fact that the secreted NPY or NPY 13-36 would be rapidly degraded,
especially during the perfusion procedure. Thus, in vivo activity of the vectors AAV-FIB-
NPY or NPY 13-36 was validated by demonstrating the presence of vector-derived
mRNA. Two rats received AAV-FIB-NPY and two received AAV-FIB-NPY 13-36
vector infusions into the piriform cortex as described above. Then, 1 week later, the
animals received an overdose of pentobarbital (100mg/kg, ip) and were subsequently
decapitated. The brain was removed, and the piriform cortex was dissected out. The
tissue was stored in RNAlater (Ambion, Austin, TX, USA) at -80°C. Subsequently, the
RNA was extracted from the tissue (Promega SV-40 total RNA isolation kit; Madison,
WI, USA) and reverse transcribed using AMV reverse transcriptase and oligo(dT)
primers. The subsequent PCR used primers that were designed to span the FIB-NPY (and
thus the FIB-NPY 13-36) sequence, which can only be derived from the rAAV vector:
(FIB, 5’) 5’-CTA GCA GTC CTG TGC CTG-3’, (NPY and NPY13-36, 3’) 5’ –GCT
CAA TAT CTC TGT CTG GTG-3’.
For the multiple gene product delivery vector AAV-CB-FIB-GAL-RKRRKR-
NPY13-36, the same procedure and primers were used with the following modifications:
all seven animals receiving vector infusions in the piriform cortex were sacrificed
immediately after the behavior data was collected from the kainic acid treatment
(described in II.D.5.), and all animals were processed for vector-derived mRNA.
47
II.D.4. In Vivo Detection of AAV-Derived GFP
Although the GFP is likely being secreted in these animals, it is very stable with a
fairly long half-life (around 80 hours (Kamau, Grimm et al. 2001). This allows us to
visualize the pattern of AAV transduction via GFP immunohistochemistry. Ten days
after piriform cortical infusion of AAV vectors containing GFP, the animals received an
overdose of pentobarbital (100mg/kg, ip) and subsequently were perfused transcardially
with ice-cold 100mM sodium phosphate-buffered saline (PBS) (pH 7.4), followed by 4%
paraformaldehyde in 100mM phosphate buffer (pH 7.4). After overnight fixation in the
paraformaldehyde–phosphate buffer, Vibratome sections (40μm thick) were taken and
rinsed in PBS. The sections were mounted and GFP fluorescence was visualized on a
Zeiss 510 confocal microscope.
II.D.5. Kainic Acid Treatment
Seven days after piriform cortical infusion of the AAV vectors, the animals
received a 10mg/kg ip dose of kainic acid (Cayman Chemical Co., Ann Arbor, MI,
USA). Using the limbic seizure scale of Racine (Racine 1972), the latency was recorded
to class I, II, III, IV, and V limbic seizure behaviors. Four hours after kainic acid
treatment, the animals received an overdose of pentobarbital (100mg/kg, ip) and
subsequently were perfused transcardially with ice-cold 100mM sodium phosphate-
buffered saline (PBS) (pH 7.4), followed by 4% paraformaldehyde in 100mM phosphate
buffer (pH 7.4). After overnight fixation in the paraformaldehyde–phosphate buffer,
Vibratome sections (40μm thick) were taken and rinsed in PBS. The sections were
mounted and the infusion placement was determined.
Chapter 3
Results
III.A. rAAV-mediated Expression and Constitutive Secretion of NPY or NPY13-36
Suppresses Seizure Activity In Vivo
A gene therapy approach to suppress seizures is an attractive treatment for focal
epilepsy. Although previous studies validated the effectiveness of gene expression and
constitutive secretion using galanin (McCown 2006), the same approach might not
necessarily prove successful with NPY or the peptide fragment NPY13-36. Therefore,
we used a similar approach with the following modifications: AAV2 vectors were
constructed where the hybrid chicken beta actin promoter drives expression of the FIB-
NPY or FIB-NPY13-36 coding sequences (see Figure 3-1). Then, 2µL of recombinant
AAV-FIB-NPY (3.3 X 1012 viral particles/mL) or AAV-FIB-NPY13-36 (3.0 X 1012 viral
particles/mL) was infused bilaterally into the piriform cortex of rats, as previously
described (McCown 2006). Control rats received no infusion, as we have previously
shown that vectors expressing and secreting reporter genes like GFP (AAV-FIB-EGFP),
as well as vectors expressing peptides that lack secretion sequences (AAV-GAL) have no
effect on seizure sensitivity, seizure behaviors, or seizure-induced cell death (Haberman,
Samulski et al. 2003; McCown 2006). One week later, the rats received a dose of
10mg/kg, i.p. kainic acid to induce seizures. We then recorded the time required to
initiate limbic seizure behaviors.
49
Figure 3-1. AAV Vectors that Express and Constitutively Secrete NPY or NPY13-36 AAV2 vectors were constructed where the hybrid chicken beta actin promoter (CB) drives expression of the FIB-NPY or FIB-NPY13-36 coding sequences.
TR (2)
CB FIB TR (2)
SV-40 Poly-A
NPY13-36
TR (2)
CB FIB NPY SV-40 Poly-A
TR (2)
TR (2)
CB FIB TR (2)
SV-40 Poly-A
NPY13-36
TR (2)
CB FIB TR (2)
SV-40 Poly-A
NPY13-36
TR (2)
CB FIB NPY SV-40 Poly-A
TR (2)
TR (2)
CB FIB NPY SV-40 Poly-A
TR (2)
TR (2)
CB FIB NPY SV-40 Poly-A
TR (2)
50
Figure 3-2. The Effects of AAV-FIB-NPY and AAV-FIB-NPY13-36 Vectors on the Expression of Limbic Seizure Behaviors. The latencies to wet dog shakes and limbic seizure behaviors were determined for 240 min post-kainic acid. The numbers in the columns indicate the number of rats in each group that exhibited any seizure activity, compared to the total number of rats in that treatment group.
51
As seen in Figure 3-2, both AAV treatment groups and the untreated control
group developed wet dog shake behaviors with the same latency.Because the origin of
wet dog shakes appears to be the hippocampus (Frush and McNamara 1986), a local
action of the AAV-FIB-NPY or AAV-FIB-NPY13-36 in the piriform cortex would not be
expected to influence these kainic acid-induced behaviors. However, the findings do
validate the uniformity of kainic acid administration across the different groups. In
marked contrast, however, onset of class III and class IV seizures was significantly
delayed or completely blocked in rats receiving either AAV-FIB-NPY or AAV-FIB-
NPY13-36 compared to control (*t-test; P<0.01). In fact, the majority of rats in both
vector treated groups did not exhibit any seizure behavior at all, whereas all rats in the
untreated group develop class III and class IV seizures within 90 minutes (see Figure 3-
2). Thus, like findings with expression and secretion of galanin, NPY actions in the
piriform cortex significantly attenuated seizure activity induced by the peripheral
administration of the chemical convulsant kainic acid. As previously discussed, the
constitutive secretion of these peptides renders immunohistochemical identification
untenable (Haberman, Samulski et al. 2003), but as previously shown for galanin
(Haberman, Samulski et al. 2003; McCown 2006), the appropriate vector-derived NPY or
NPY13-36 mRNA was present in the area of AAV infusion (see Figure 3-3). Presence of
vector derived mRNA supports the finding that over-expression of NPY or NPY13-36 in
the piriform cortex is responsible for the preventing of seizure behavior.
III.B. A Strategic Approach for Delivering Multiple Gene Products in the Brain
Using a Single AAV Vector
52
Figure 3-3. The In Vivo Presence of FIB-NPY and FIB-NPY13-36 mRNA 1 Week after Vector Infusion into the Piriform Cortex. The appropriate 208bp FIB-NPY (lane A) or 172bp FIB-NPY13-36 (lane C) product is present in the injected piriform cortex, while no product was found in an area slightly distal to the piriform cortex (lane B). Omission of the reverse transcriptase step (lanes D and E) indicated the absence of contaminating viral DNA. The left outside lane contains a 100bp DNA ladder with relevant sizes indicated on the left.
53
III.B.1. In Vitro Characterization of Dual Reporter Gene Product Delivery Vectors
Vectors delivering individual inhibitory neuropeptides to the brain to prevent
seizures has been well studied (Mazarati and Wasterlain 2002; Haberman, Samulski et al.
2003; Richichi, Lin et al. 2004; McCown 2006; Foti, Haberman et al. 2007), but to date
no one has reported the simultaneous delivery of multiple neuropeptides. To achieve
this, we used a proteolytic strategy such that our plasmids contained a single
promoterdriving expression of a single chimeric fusion protein containing the FIB
secretion sequence. Upon being sorted into the constitutive secretion pathway, the fusion
protein should come in contact with the protease furin, and it then should be cleaved into
two separate functional proteins which are then constitutively secreted from the cells.
Using a strategy similar to Margaritis et al., (2004) we engineered our plasmids to
contain a cleavage site for the intracellular protease, furin. The cleavage consensus
sequence arginine-lysine-arginine-arginine-lysine-arginine (RKRRKR) was cloned in
frame as a linker between the coding sequences for the two gene products being
delivered.
As a first proof of concept for the multiple gene product delivery vectors, we used
the two reporter genes luciferase and GFP. Since both gene products are reporter genes
which typically do not undergo secretion from cells, we hypothesized that each gene
would need a secretion sequence in order for the protein to be correctly trafficked within
the cell. Thus, we made constructs that contained either one FIB sequence (in front of the
first gene luciferase) or two FIB sequences. The constructs generated and characterized
by in vitro protein assays were: CB-LUC, CB-FIB-LUC, CB-FIB-LUC-RKRRKR-
EGFP, and CB-FIB-LUC-RKRRKR-FIB-EGFP (see Figure 3-4). In order to determine
54
Figure 3-4. AAV Vectors Characterized in Initial Multiple Gene Product Delivery Studies. (A) CB-LUC is a negative control used in luciferase assays and immunoprecipitation then western blot (IP/WB) experiments to show that luciferase is expressed but not secreted in the absence of a FIB sequence. (B) CB-FIB-LUC is a positive control used in luciferase assays and IP/WB experiments for secretion of luciferase into the medium. (C,D) CB-FIB-LUC-RKRRKR-EGFP and CB-FIB-LUC-RKRRKR-FIB-EGFP are analyzed by IP/WB to determine if the two gene products are made as a fusion protein in the cell then cleaved from one another, and to examine if a single FIB sequence is sufficient for secretion of both gene products into the medium, or would two be necessary.
FIBTR (2)
CB SV-40 Poly-A
TR (2)
B
TR (2)
CB SV-40 Poly-A
TR (2)
ALUC
LUC
TR (2)
CB FIB SV-40 Poly-A
TR (2)
EGFPRKRRKR
CLUC
TR (2)
CB FIB SV-40 Poly-A
TR (2)
EGFPRKRRKR
DLUC FIB
FIBTR (2)
CB SV-40 Poly-A
TR (2)
B
TR (2)
CB SV-40 Poly-A
TR (2)
ALUCTR
(2)CB SV-40
Poly-ATR (2)
ALUC
LUC
TR (2)
CB FIB SV-40 Poly-A
TR (2)
EGFPRKRRKR
CLUCTR
(2)CB FIB SV-40
Poly-ATR (2)
EGFPRKRRKRRKRRKR
CLUC
TR (2)
CB FIB SV-40 Poly-A
TR (2)
EGFPRKRRKR
DLUC FIBTR
(2)CB FIB SV-40
Poly-ATR (2)
EGFPRKRRKRRKRRKR
DLUC FIB
55
if the luciferase was secreted into the medium, we harvested medium and lysates and
performed luciferase assays in the presence of luciferin substrate (see Figure 3-5). To our
surprise, luciferase activity was present in the medium of the non-secreted luciferase
construct (CB-LUC), but is probably due to residual cell lysis and spilling out of the
expressed transgene. In addition, the absence of luciferase activity in the medium of the
secreted constructs (CB-FIB-LUC, and CB-FIB-LUC-RKRRKR-EGFP) suggests either
that the FIB sequence is interfering with the luminescence catalytic domain, or the
luciferase protein is not being expressed. Immunoprecipitation followed by Western
blotting (IP/WB) was performed using antibodies to luciferase and GFP to distinguish
between these possibilities (see Figures 3-6 and 3-7). The luciferase IP/WB revealed that
although there was a complete absence of secretion of luciferase protein into the medium
(Figure 3-6, lanes 2-5), luciferase protein was detected in the all of the cell lysates
(Figure 3-6, lanes 7-10). Thus, luciferase expression is occurring, but that enzymatic
function is abolished when the FIB sequence is included (see Figure 3-5, CB-FIB-LUC).
In addition, the luciferase IP/WB reveals that the fusion protein of luciferase and GFP is
being made in the cells (Figure 3-6, lanes 9-10) even though correctly processed
luciferase is not seen in the lysates or the medium. These results suggest that neither
furin-mediated cleavage nor FIB-mediated secretion are occurring, and are in contrast to
the data obtained with the GFP IP/WB (Figure 3-7).
The GFP IP/WB revealed that some processing of the proteins was occurring, and
that cleaved GFP was secreted into the medium in of CB-FIB-LUC-RKRRKR-FIB-
EGFP transfected cells. CB-FIB-LUC-RKRRKR-EGFP and CB-FIB-LUC-RKRRKR-
FIB-EGFP suggest that two FIB sequences are necessary in order for cleaved GFP to be
56
Luciferase Assay (Avg of Duplicates)
287.5
23730.5
749 980
0
5000
10000
15000
20000
25000
Rel
ativ
e Li
ght U
nits
Mock Transfected
CB-LUC
CB-FIB-LUC
CB-FIB-LUC-RKRRKR-EGFP
Figure 3-5. Luciferase Assay on Concentrated Medium. 293 cells were transfected with the constructs listed above. The medium was harvested 48 hours later, and centrifuged in a concentrating column with a molecular weight cut off membrane of 10kDa. Since firefly luciferase is around 61kDa it will be retained in the supernatant, while salts and smaller proteins flow through. 5mL samples were concentrated into 1mL, then 20μL of each sample was mixed with 100μL of luciferin solution in a 96 well plate and read in the luminometer. Presence of luciferase activity in the medium of the non-secreted luciferase construct (CB-LUC) is probably due to residual cell lysis and spilling out of the expressed transgene. Absence of luciferase activity with the secreted constructs (CB-FIB-LUC, and CB-FIB-LUC-RKRRKR-EGFP) suggests either that the FIB sequence is interfering with the luminescence catalytic domain, or that luciferase protein is not being expressed. IP/WB was performed to distinguish between these possibilities, and to examine protein processing and trafficking.
57
Figure 3-6. Luciferase Antibody Protein Analysis of Luciferase-GFP Multiple Gene Product Delivery Constructs. IP/WB using the same anti-luciferase polyclonal antibody to pull down and blot samples, hence the heavy and light chain antibody bands are visible at around 50kDa and 25kDa respectively. Luciferase is around 61kDa and GFP is around 30kDa. 1 is rainbow marker, 2 is CB-LUC, 3 is CB-FIB-LUC, 4 is CB-FIB-LUC-RKRRKR-EGFP, 5 is CB-FIB-LUC-RKRRKR-FIB-EGFP, 6 is untransfected control, 7 is CB-LUC, 8 is CB-FIB-LUC, 9 is CB-FIB-LUC-RKRRKR-EGFP, 10 is CB-FIB-LUC-RKRRKR-FIB-EGFP. No luciferase is present in the medium following transfection (lanes 2-6), however, luciferase is detected in all of the cell lysates (lanes 7-10). In addition, lanes 9 and 10 show that luciferase-GFP fusion protein is being made in the cells (98kDa), even though processed luciferase (61kDa) is not seen in the lysates or the medium.
Medium 2-6 Lysates 7-10Medium 2-6 Lysates 7-10Luciferase Antibody
58
Figure 3-7. GFP Antibody Protein Analysis of Luciferase-GFP Multiple Gene Product Delivery Constructs. IP/WB using an anti-GFP polyclonal antibody to pull down and an anti-GFP monoclonal antibody to blot. 1 is CB-FIB-LUC-RKRRKR-EGFP, 2 is CB-FIB-LUC-RKRRKR-FIB-EGFP, 3 is CB-FIB-EGFP, 4 is CB-FIB-LUC, 5 is CB-FIB- LUC-RKRRKR-EGFP, 6 is CB-FIB-LUC-RKRRKR-FIB-EGFP, 7 is CB-FIB-EGFP, 8 is CB-FIB-LUC, 9 is untransfected control. CB-FIB-LUC-RKRRKR-EGFP and CB-FIB-LUC-RKRRKR-FIB-EGFP (lanes 1-2) suggest that two FIB sequences are necessary in order for cleaved GFP to be secreted into the medium because it is absent in lane 1 but present in lane 2. CB-FIB-EGFP (lane 3, 7) is a positive control showing GFP is secreted into the medium when FIB is present and is found within the cells. CB-FIB-LUC (lanes 4, 8) is a negative control demonstrating that the GFP antibody does not cross-react with luciferase. Lanes 5 and 6 show that luciferase-GFP fusion protein is made (98kDa), but the multiple bands suggest that it is also being degraded within the cells. Fragments of luciferase-GFP fusion protein may not show up when probing with a luciferase antibody because the luciferase antibody may loose its binding capacity, however, this would not affect GFP antibody binding.
Medium 1-4 Lysates 5-9Medium 1-4 Lysates 5-9GFP Antibody
59
secreted into the medium because GFP was absent in medium of CB-FIB-LUC-
RKRRKR-EGFP transfected cells, but present in the medium of CB-FIB-LUC-
RKRRKR-FIB-EGFP transfected cells (Figure 3-7, lanes 1-2). However, multiple bands
in the lysates for these constructs show that luciferase-GFP fusion protein is made and
then degraded within the cells (Figure 3-7, lanes 5-6). Fragments of luciferase-GFP
fusion protein may not show up when probing with a luciferase antibody because the
luciferase antibody may loose its binding capacity, however, this would not affect GFP
antibody binding. Taken together, we determined that luciferase would not be functional
in these vectors because the protein was expressed but then degraded in the cells, thus it
was not secreted. Therefore we decided to retain the proteolytic vector strategy, but
instead of luciferase, we would use the neuropeptide galanin along with GFP.
III.B.2. In Vitro Characterization of Double FIB Multiple Gene Product Delivery Vectors
Because the initial luciferase data suggested that each gene would need its own
FIB sequence, we made the following additional constructs: CB-FIB-GAL-RKRRKR-
FIB-EGFP and CB-FIB-EGFP-RKRRKR-FIB-GAL (see Figure 3-8). These constructs
were first characterized at the protein level in vitro (see Figures 3-9). Due to the
extremely rapid degradation of galanin in serum (Holst, Bersani et al. 1993), GFP
secretion was assayed. Constructs were made containing GFP and galanin in both
possible orientations, assuring that GFP was correctly processed and cleaved from
galanin whether it was 5’ or 3’ of the cleavage site. The IP/WB revealed that while the
construct CB-FIB-GAL-RKRRKR-FIB-EGFP resulted in secretion of cleaved GFP into
the medium, the construct CB-FIB-EGFP-RKRRKR-FIB-GAL was not processed
60
Figure 3-8. Double FIB Multiple Gene Product Delivery Vectors. (A) CB-EGFP is a negative control used in IP/WB experiments to show that GFP is expressed but not secreted in the absence of a FIB sequence. (B) CB-FIB-EGFP is a positive control used in IP/WB experiments for secretion of GFP into the medium. (C) CB-FIB-GAL-RKRRKR-FIB-EGFP and (D) CB-FIB-EGFP-RKRRKR-FIB-GAL are constructs designed to test cleavage and secretion of both gene products in IP/WB experiments in vitro, and to test the function of both gene products in vivo.
EGFPFIBTR (2)
CB SV-40 Poly-A
TR (2)
TR (2)
CB SV-40 Poly-A
TR (2)
EGFPA
B
CTR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPRKRRKR
FIB
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKR
EGFPD
FIB
EGFPFIBTR (2)
CB SV-40 Poly-A
TR (2)
EGFPFIBTR (2)
CB SV-40 Poly-A
TR (2)
TR (2)
CB SV-40 Poly-A
TR (2)
EGFPA
TR (2)
CB SV-40 Poly-A
TR (2)
EGFPA
B
CTR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPRKRRKR
FIBCTR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPRKRRKR
FIBTR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPRKRRKRRKRRKR
FIB
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKR
EGFPD
FIBTR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKRRKRRKR
EGFPD
FIB
61
Figure 3-9. In Vitro Protein Analysis of Double FIB Multiple Gene Product Delivery Constructs. The following constructs were transfected into 293 cells using PEI 1mg/mL: 1) CB-FIB-EGFP; 2) CB-FIB-GAL-RKRRKR-FIB-EGFP; 3) CB-FIB-EGFP-RKRRKR-FIB-GAL. After 48 hours the lysate and medium were harvested and IP/WB was performed using an anti-GFP polyclonal antibody to pull down and an anti-GFP monoclonal antibody to blot. Lane 1 shows GFP is present in the lysate and is also present in the medium. Lane 2 shows that GFP is present in the lysate and is also in the medium. Note that it appears to be the same size as lane 1 suggesting it has been cleaved from galanin. Lane 3 shows that the GFP is not being processed correctly intracellularly, and intermediate products are present in the lysate. While these intermediates do not seem to be secreted, the amount of correctly processed and secreted protein is reduced. Consequently, we did not use this construct for in vivo experiments.
1 2 3 1 2 3
Lysate Medium
14.3
20.1
30
45
66
97
220
14.3
20.1
30
45
66
97
220
1 2 3 1 2 3
Lysate Medium
14.3
20.1
30
45
66
97
220
14.3
20.1
30
45
66
97
220
correctly within the cells (see Figure 3-8). Thus, we only made virus with CB-FIB-GAL-
RKRRKR-FIB-EGFP, CB-FIB-EGFP and CB-EGFP.
III.B.3. In Vivo Characterization of Double FIB Multiple Gene Delivery Vectors
These viruses were titered by dot blot (see Figure 2-2) and injected into the rat
piriform cortex to analyze the in vivo fluorescence patterns (see Figure 3-10). The in vivo
fluorescence patterns were similar to that of Haberman et al. (2003), such that when the
FIB sequence was present, GFP was localized to the periphery of the neurons, which is
highly suggestive of GFP secretion. However, when the FIB sequence was absent, the
neurons were uniformly fluorescent, suggesting cytoplasmic localization of GFP. Upon
determining that the GFP was functional, we decided to evaluate the function of galanin
from the AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP construct in vivo. In order for
galanin to be functional it must not only be expressed, but also secreted, because the
receptors for galanin are located on the exterior cell membrane. Thus, if infusion of this
construct into the piriform cortex is able to attenuate limbic seizure behavior following
i.p. kainic acid administration, then functional galanin must have been secreted from the
transduced cells. As seen in Figure 3-11, infusion of AAV2-CB-FIB-GAL-RKRRKR-
FIB-EGFP significantly attenuated limbic seizures, and completely blocked seizure
behavior in 3 of 4 animals. While these experiments provided us with an initial proof of
concept, we did not conduct any further experiments with AAV2-CB-FIB-GAL-
RKRRKR-FIB-EGFP, because the reciprocal construct CB-FIB-EGFP-RKRRKR-FIB-
GAL was not processed correctly within the cells. This led us to hypothesize that having
multiple FIB sequences may create sequence-dependent protein sorting and trafficking
62
63
Figure 3-10. In Vivo Fluorescence Pattern in Rat Cortex after Infusion of AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP, AAV2-CB-FIB-EGFP, or AAV2-CB-EGFP. (A) AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP, (B) AAV2-CB-FIB-EGFP, (C) AAV2-CB-EGFP. (A, B) In vivo GFP patterns are highly suggestive of GFP secretion following infusion of AAV-CB-FIB-GAL-RKRRKR-FIB-EGFP and CB-FIB-GFP because there is a subcellular localization of GFP to the perimeter of neurons. (C) Uniform fluorescence suggests cytoplasmic accumulation of GFP but not secretion after infusion of AAV-CB-EGFP. Each image was obtained with a confocal microscope using a 1.4μm thick slice through the middle of the cell.
64
Figure 3-11. The Effects of AAV2-CB-FIB-GAL-RKRRKR-FIB-EGFP on the Expression of Limbic Seizure Behaviors. The latencies to wet dog shakes and limbic seizure behaviors were determined for 240 min post-kainic acid. The numbers in the columns indicate the number of rats in each group that exhibited any seizure activity, compared to the total number of rats in that treatment group. Infusion of AAV-CB-FIB-GAL-RKRRKR-FIB-EGFP in the piriform cortex completely blocks limbic seizure behavior in 3 of 4 animals following i.p. kainic acid administration. Control rats received no infusion, as we have previously shown that vectors expressing and secreting reporter genes like GFP (AAV-FIB-EGFP), as well as vectors expressing peptides that lack secretion sequences (AAV-GAL) have no effect on seizure sensitivity, seizure behaviors, or seizure-induced cell death.
4/4 4/4 1/44/44/4 4/4 1/44/4
problems, thus we decided to explore if a single FIB sequence would be sufficient to
cause secretion of both galanin and GFP in vitro and in vivo.
III.B.4. In Vitro Characterization of Single FIB Multiple Gene Product Delivery Vectors
In order to determine if a single FIB sequence would be sufficient to cause
secretion of both galanin and GFP, we generated and characterized the following
constructs: CB-EGFP, CB-FIB-EGFP, CB-FIB-GAL, CB-FIB-GAL-RKRRKR-EGFP,
CB-FIB-EGFP-RKRRKR-GAL, CB-FIB-GAL-EGFP, and CB-FIB-GAL-RKRRKR-
NPY 13-36 (a construct used only for in vivo experiments and should allow secretion of
two therapeutic neuropeptides) (see Figure 3-12). Again, due to the extremely rapid
degradation of galanin in serum (Holst, Bersani et al. 1993), GFP secretion was assayed.
Constructs were made containing GFP and galanin in both possible orientations, assuring
that GFP was correctly processed and cleaved from galanin whether it was 5’ or 3’ of the
cleavage site. In addition, we made a secreted non-cleavable control of galanin and GFP
to demonstrate the size of a fusion of these two proteins. Figure 3-13 shows that
transfection of our constructs into 293 cells results in similar fluorescence patterns to that
of Haberman et al. (2003) (Figure 3-13, A), and demonstrates that 1) indeed, a single FIB
sequence was sufficient to allow secretion of both gene products (Figure 3-13, B lane 4),
indicating that FIB designates the protein for secretion before furin cleaves it; 2) our
proteins were correctly processed intracellularly regardless of position relative to the
cleavage sequence and secreted into the medium (Figure 3-13, B-C, lanes 3-4); 3) in the
absence of a cleavage sequence the uncleaved fusion protein is secreted into the medium
(Figure 3-13, B lane 5); 4) the absence of fusion protein in the medium suggests that
cleavage between GFP and galanin is 100% efficient (Figure 3-13, B, lanes 3-4).
65
66
Figure 3-12. AAV Vectors Characterized in Final Multiple Gene Product Delivery Studies. (A) CB-EGFP is a negative control used in immunoprecipitation then western blot (IP/WB) experiments to show that GFP is expressed but not secreted in the absence of a FIB sequence. (B) CB-FIB-EGFP is a positive control used in IP/WB experiments for secretion of GFP into the medium. (C) CB-FIB-GAL is a negative control used in IP/WB experiments to show that the GFP antibody does not cross-react to galanin or other non-specific proteins. (D,E) CB-FIB-GAL-RKRRKR-EGFP and CB-FIB-EGFP-RKRRKR-GAL are used both for in vitro cleavage and secretion assay, and used for in vivo studies to test the function of both gene products. (F) CB-FIB-GAL-EGFP is a non-cleavable control for both in vitro and in vivo experiments. (G) CB-FIB-GAL-RKRRKR-NPY 13-36 is a construct used only for in vivo experiments and should allow secretion of two therapeutic neuropeptides.
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPFIBTR (2)
CB SV-40 Poly-A
TR (2)
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPRKRRKR
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKR
EGFP
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFP
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKR
NPY 13-36
TR (2)
CB SV-40 Poly-A
TR (2)
EGFPA
B
C
D
E
F
G
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPFIBTR (2)
CB SV-40 Poly-A
TR (2)
EGFPFIBTR (2)
CB SV-40 Poly-A
TR (2)
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPRKRRKR
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPRKRRKRRKRRKR
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKR
EGFPTR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKRRKRRKR
EGFP
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFPTR (2)
CB FIB GAL SV-40 Poly-A
TR (2)
EGFP
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKR
NPY 13-36
TR (2)
CB FIB GAL SV-40 Poly-A
TR (2)RKRRKRRKRRKR
NPY 13-36
TR (2)
CB SV-40 Poly-A
TR (2)
EGFPTR (2)
CB SV-40 Poly-A
TR (2)
EGFPA
B
C
D
E
F
G
67
Figure 3-13. In Vitro Characterization of Fluorescence Patterns, Cleavage, and Secretion of Proteins Derived from Multiple Gene Product Delivery Vectors. (A) GFP expression patterns in transfected 293 cells at low and high magnification. (B-C) Immunoprecipitation and Western blotting of GFP from transfected cells; either conditioned medium (B) or cell lysate (C) was analyzed. (1) CB-EGFP, GFP is expressed as seen in C lane 1, but is not secreted into the medium (B lane 1). Cells have a bright and uniform distribution of intracellular GFP (A, panel 1). (2) CB-FIB-EGFP, GFP is expressed and secreted into the medium (B-C, lane 2). Fluorescence is concentrated around the perimeter, indicating secretion (A, panel 2). (3) CB-FIB-EGFP-RKRRKR-GAL, GFP is cleaved from galanin and secreted into the medium (B, lane 3). As seen in C lane 3, unprocessed protein is detected in the cell lysate. (4) CB-FIB-GAL-RKRRKR-EGFP, GFP behaves the same as B and C lane 3, so correct processing and secretion occurs whether GFP is 5’ or 3’ of the cleavage sequence. (5) CB-FIB-GAL-EGFP, GFP cannot be cleaved from galanin because this construct lacks a cleavage sequence. The fusion protein is still secreted due to the inclusion of FIB (B lane 5). A small amount of degraded protein is present in the cell lysate, but does not get secreted (C lane 5). Fluorescence patterns are similar with all secreted GFP constructs (A, panels 2-5), although intensity seems to be lower in A, 3, however, the relative amount of GFP detected in the medium is similar (B lanes 2-5). (6) CB-FIB-GAL, GFP is not present in the cells (A, panel 6), and the GFP antibody does not cross-react to galanin (B-C, lane 6). (7) Rainbow marker of known proteins from Amersham, size indicated to the right (B-C, lane 7).
1
2
3
4
5
6
20X 40XA.
Cell LysateC.
Conditioned MediumB.1
2
3
4
5
6
20X 40XA.
Cell LysateC.
Conditioned MediumB.
Cell LysateC. Cell LysateC.
Conditioned MediumB. Conditioned MediumB.
68
III.B.5. In Vivo Functional Test of Single FIB Multiple Gene Product Delivery Vectors
In order to test the in vivo function of these proteins, we packaged our multiple
gene product delivery constructs into AAV2 vectors where the CB promoter drives
expression of the FIB-GAL-RKRRKR-EGFP, FIB-EGFP-RKRRKR-GAL, FIB-GAL-
EGFP, or FIB-GAL-RKRRKR-NPY13 36 coding sequence (see Figure 3-12). Then,
3µL of virus (1.0 X 1012 viral particles/mL) was infused bilaterally into the piriform
cortex of rats, as previously described (McCown 2006) (see Figure 3-14). Control rats
received no infusion, as we have previously shown that vectors expressing and secreting
reporter genes like GFP (AAV-FIB-EGFP), as well as vectors expressing peptides that
lack secretion sequences (AAV-GAL) have no effect on seizure sensitivity, seizure
behaviors, or seizure-induced cell death (Haberman, Samulski et al. 2003; McCown
2006). One week later, the rats received a dose of 10mg/kg, i.p. kainic acid, and
subsequently, the time to limbic seizure behaviors was recorded. As seen in Figure 3-14,
the immunohistochemistry data presented shows that the GFP is functional in both
orientations, but reveals that number of transduced cells is limited. Figure 3-15 confirms
the presence of vector-derived mRNA for the animals injected with AAV2-CB-FIB-
GAL-RKRRKR-NPY13-36. As seen in Figure 3-16, all groups exhibited wet dog shakes
(WDS) with similar latency, validating the efficacy and uniformity of the absorbed dose
of kainic acid. All vector treated animals showed a significant delay in the onset of class
IV seizures (*t-test; P≤0.01). A strong increase in the latency to seizure behavior
demonstrates that the galanin gene product is in fact expressed, secreted, and functional
regardless of whether it is 5’ or 3’ of the cleavage site in our multiple gene product
69
Figure 3-14. Immunofluorescence of Neurons in the Piriform Cortex That Have Been Transduced with GFP-containing Vectors. The shaded area in the coronal section shows the range of placement within the piriform cortex of the AAV vector microinjections. Sections were taken at the level of the needle track and processed for GFP fluorescence. Presence of GFP protein reveals the virus transduction pattern, and confirms that our constructs are functional in both orientations in vivo. The localization of GFP to the periphery of the cells is consistent with GFP secretion.
70
Figure 3-15. The In Vivo Presence of Vector-derived FIB-GAL-RKRRKR-NPY13-36 mRNA 1 Week after Vector Infusion into the Piriform Cortex. To validate viral transfer for the AAV2-CB-FIB-GAL-RKRRKR-NPY13-36 group, we performed RT-PCR on tissue from the piriform cortex. As seen in Figure 3-8, the appropriate vector derived mRNA (252bp) is present in all 7 animals injected with this vector (lanes A-G). The left outside lane contains a 50bp DNA ladder with the size indicated on the left. Omission of the reverse transcriptase step resulted in loss of the product (data not shown). Together, Figures 3-13 and 3-14 confirm successful gene transfer occurred in all of our vector-treated groups.
71
Figure 3-16. The Effects of Multiple Gene Product Delivery Vectors on Limbic Seizure Behavior. The latencies to wet dog shakes and limbic seizure behaviors were determined for 180 min post-kainic acid. All the animals regardless of treatment group developed wet dog shakes with similar latencies, suggesting an equivalent absorbed dose of kainic acid. All AAV vector groups exhibited a significant delay in the time to class IV limbic seizures. (FG-E is AAV2-CB-FIB-GAL-RKRRKR-EGFP, FE-G is AAV2-CB-FIB-EGFP-RKRRKR-GAL, FG-NPY13-36 is AAV2-CB-FIB-GAL-RKRRKR-NPY13-36, FGE is AAV2-CB-FIB-GAL-EGFP, and Control is kainic acid only with no vector; * signifies significance of p≤0.01 compared to Control using two-tailed t-test)
Effects of AAV Vectors on Limbic Seizure Behavior
020406080
100120140160180
FG-E FE-G FG-NPY13-36
FGE Control
Late
ncy
(Min
utes
)
Wet Dog ShakesClass IV
* *
**
72
delivery vectors (CB-FIB-GAL-RKRRKR-EGFP + CB-FIB-EGFP-RKRRKR-GAL,
Figure 3-16). Unexpectedly, galanin was able to decrease seizure activity even when
GFP was not cleaved (CB-FIB-GAL-EGFP, Figure 3-16). While animals receiving
AAV2-CB-FIB-GAL-RKRRKR-NPY13-36 did show a significant delay in the latency to
class IV seizures compared to controls, having two therapeutic neuropeptides did not
confer additional seizure protection over having a single therapeutic neuropeptide (Figure
3-16). Taken together, we can conclude that successful gene transfer occurred in all of
the vector-treated groups, and that the gene products are functional in vivo, providing
proof of concept.
Chapter 4
Discussion
IV.A. rAAV-Mediated Expression and Constitutive Secretion of NPY or NPY13-36 Suppresses Seizure Activity In Vivo
Epilepsy is an attractive target for recombinant Adeno-associated virus (rAAV)
gene therapy, because the temporal lobe structures involved in seizure genesis and
propagation have been shown to be permissive to AAV gene transfer (Freese, Kaplitt et
al. 1997; Vezzani, Michalkiewicz et al. 2002; Haberman, Samulski et al. 2003; Richichi,
Lin et al. 2004; Klugmann, Symes et al. 2005; McCown 2006). Recently, galanin and
NPY have been delivered as transgenes in rAAV vectors, and shown to be effective in
several epilepsy paradigms (Haberman, Samulski et al. 2003; Lin, Richichi et al. 2003;
Richichi, Lin et al. 2004; McCown 2006). While previous approaches utilize pre-pro
cDNA sequences which rely on the cell to modulate release of the gene product,
Haberman et al. (2003) were the first to use a novel secretion strategy whereby the
secretion signal sequence from the constitutively secreted laminar protein fibronectin
(FIB), is combined with the coding sequence for the active therapeutic peptide. Results
from Haberman et al. (2003) and McCown (2006) show that expression and constitutive
secretion of galanin is not only achieved, but also effective in attenuating focal and
limbic seizure activity. In contrast, expression of galanin without the secretory signal or
74
expression and secretion of the reporter gene, GFP had no effect on seizure sensitivity
(Haberman, Samulski et al. 2003; McCown 2006).
In addition to galanin, rAAV delivery of NPY has also been shown to attenuate
limbic seizures (Richichi, Lin et al. 2004). While there is still some question as to which
of the NPY receptors (Y1-Y5) mediates the anti-seizure activity, several studies have
suggested a critical role for the Y2 receptor in mediating anti-epileptic actions (Colmers,
Klapstein et al. 1991; Greber, Schwarzer et al. 1994; Dumont, Cadieux et al. 2000;
Dumont, Cadieux et al. 2000; El Bahh, Cao et al. 2002; El Bahh, Balosso et al. 2005; Lin,
Young et al. 2006). Furthermore, it has been demonstrated that in epileptic tissue from
patients (Furtinger, Pirker et al. 2001) and rodents (Kofler, Kirchmair et al. 1997;
Schwarzer, Kofler et al. 1998) that Y1 receptors are downregulated while Y2 receptors
are upregulated. Taken together, these results suggest that when designing anti-
epileptogenic therapeutics, it may be advantageous to deliver Y2 preferring agonists. In
fact, acute intracerebral delivery of the Y2 receptor preferring agonist NPY13-36 reduces
seizure susceptibility following systemic kainic acid administration (Vezzani, Moneta et
al. 2000; Vezzani, Rizzi et al. 2000). These data along with our current findings suggest
NPY13-36 is a good candidate to use in a rAAV vector to treat epilepsy. By using the
FIB secretion strategy, we have the capability of expressing and constitutively secreting
peptide fragments which have demonstrated receptor selectivity. Thus, we evaluated the
effects of AAV mediated expression and constitutive secretion of NPY and the peptide
fragment NPY13-36 on kainic acid induced limbic seizures. Our data demonstrates that
seizures can be effectively controlled in rats using rAAV to deliver inhibitory
neuropeptides to susceptible neurons.
75
Our approach differed with that of Richichi et al. who used a pre-pro form of
NPY cDNA in their AAV vector (Richichi, Lin et al. 2004). While their approach did
confer some seizure protection, their effects are dependent on three caveats that our
approach circumvents. First, since they use the pre-pro form of NPY, it is likely that only
a subset of transduced neurons will have the ability to correctly process the pre-pro
protein into the active protein, which requires a whole host of proteins including but not
limited to chaperones and pro-protein convertases. Second, even the transduced neurons
that are capable of processing pre-pro NPY (probably neurons that already contain and
release NPY), have a limit to the amount of pre-pro protein that can be correctly
processed and packaged into large core dense vesicles for release. As the authors point
out, pro-NPY could “spill over” into the constitutive pathway, resulting in the formation
of some inactive pro-NPY that cannot be released by exocytosis (Burgess and Kelly
1987), or worse, causes release of inactive protein that could act like a NPY receptor
antagonist. This is a concern especially because Richichi et al. are using a construct with
a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which
dramatically increases the total amount of vector-mediated transgene expression
(Donello, Loeb et al. 1998; Higashimoto, Urbinati et al. 2007). Finally, even the pro-
NPY that is correctly processed into mature NPY is stored in large dense core vesicles
and docked in pools which can only be released in the typical regulated fashion upon
robust neuronal excitation (see Figure 4-1). Usually, only high frequency action
potentials can trigger their release (Hokfelt, Bartfai et al. 2003). Since the NPY receptors
are located on the outside of the neuronal membrane, NPY can only exert function upon
76
Figure 4-1. Regulated versus Constitutive Secretion in Neurons. (1) In the regulated pathway neuropeptides and other proteins are first synthesized as pro-proteins in the endoplasmic reticulum, then they are sorted and processed in the golgi and trans golgi network, finally they are packaged in dense-core vesicles where they can be cleaved into their active forms and tethered in releasable pools until high frequency stimulation (depicted as a lightening bolt) causes exocytosis. (2) In the constitutive secretory pathway proteins are synthesized, sorted, and processed in a similar way, but once they are packaged into secretory vesicles and cleaved into active forms, they are continuously released via exocytosis independent of stimulation.
Continuous Constitutive Secretion Endoplasmic Recticulum
Plasma Membrane Stimulus-induced Regulated
Secretory Vesicles
1
1
2
Golgi Apparatus
2
77
release, so the manner in which the neuropeptide is released is critical to seizure
prevention.
Our approach circumvents all of these issues by using only the active peptide
sequence fused to a constitutive secretion signal. Hence, there is no pro-protein to
process, and release happens in the absence of regulation.While our approach has proven
effective for galanin and NPY, it would probably not work for dynorphin, an endogenous
opioid neuropeptide with anti-seizure activity (Bonhaus, Rigsbee et al. 1987; Mazarati
and Wasterlain 2002; Loacker, Sayyah et al. 2007). Mazarati and Wasterlain (2002)
showed that acute hippocampal delivery of dynorphin A(1-13) had a profound anti-
seizure effect in a rat model of self-sustaining status epilepticus, suggesting that
dynorphin A(1-13) would be a good therapeutic candidate for our constitutive secretion
vectors. However, like most neuropeptides dynorphin A(1-13) is first expressed as a pro-
protein that gets cleaved into active forms of various lengths by a protease that recognizes
basic amino acid residues (for review see (Simonato and Romualdi 1996). After close
examination of the amino acid sequence of dynorphin A(1-13) we discovered that it has a
potential furin cleavage site at residues 6-9 which are R-R-I-R. Hence, based on our
results with the multiple peptide delivery vectors, we predict that designing an AAV
vector where FIB mediates constitutive secretion of dynorphin A(1-13) would result in
undesirable cleavage then secretion of fragments of the expressed protein. This example
illustrates an important concern when delivering gene products as therapeutics: their
sequences must be carefully analyzed and perhaps even optimized for efficient transgene
expression. While our approach might not be effective for every transgene, we
demonstrated that it is effective with NPY and with NPY13-36, confirming that it is now
78
possible to express and secrete active peptide fragments which allow for more selective
receptor targeting. Furthermore, using small peptide fragments in an AAV context could
allow for multiple therapeutic peptides to be expressed from a single vector cassette. It is
likely that combination therapy will be the key to treating epilepsy and many other
complex neurological disorders.
IV.B. A Strategic Approach for Delivering Multiple Gene Products Using a Single AAV Vector
These experiments were conducted primarily to show that it is possible to use a
single AAV vector that upon transduction causes cells to express and secrete two gene
products simultaneously; and also to study the interaction of vector-mediated secretion of
galanin and NPY on limbic seizure behavior. We have shown in vitro and in vivo that
our constructs mediate the expression of a chimeric fusion protein that upon intracellular
processing gets cleaved and constitutively secreted as two functional proteins. These
results expand the utility of engineering a proteolytic cleavage site in an AAV vector
cassette.
While both galanin and NPY (and NPY13-36) have been extensively studied in
seizure models (see IV.A.), combinations of these peptides in such models remain
unexplored. In order for the two neuropeptides to have a synergistic effect, they must
have separate mechanisms of action, or at least divergent down stream pathways.
Mazarati and Wasterlain (2002) concluded that a different pattern of anticonvulsant
action was evident after the acute delivery of NPY versus galanin, suggesting that
delivering both neuropeptides simultaneously could be advantageous. However, it is
clear from our experiments that galanin and NPY13-36 do not potentiate one another in
the piriform cortex because simultaneous delivery did not result in a more complete
79
blockade of limbic seizure behavior. This suggests that galanin and NPY13-36 may have
the same mechanism of action and/or the same intracellular signaling pathways in the
piriform cortex. While unexpected, the data can be explained by carefully examining the
mechanisms of action, receptor subtype distribution, and signaling cascades of both
galanin and NPY in the piriform cortex. Galanin has been shown to reduce glutamate
release from presynaptic nerve terminals in the brain (Zini, Roisin et al. 1993). All three
galanin receptors (GALR1-3) are present in the piriform cortex (O'Donnell, Ahmad et al.
1999; He, Counts et al. 2005; Mazarati, Lundstrom et al. 2006), but GALR2 and GALR3
are the most abundant. GALR1 and GALR3 can signal both by inhibiting adenylyl
cyclase via a pertussis toxin-sensitive G-protein of the Gi/Go family (Parker, Izzarelli et
al. 1995; Smith, Forray et al. 1997), by reducing the concentrations of cAMP (Branchek,
Smith et al. 2000), or by activating G-protein-coupled inwardly rectifying potassium
channels (GIRKs) (Smith, Walker et al. 1998; He, Counts et al. 2005; Mazarati,
Lundstrom et al. 2006). GALR2 stimulates inositol phospholipid turnover and
intracellular calcium mobilization through a pertussis toxin-insensitive Gq/G11-type
mechanism (Fathi, Cunningham et al. 1997; Smith, Forray et al. 1997). This type of
channel activation could increase seizure sensitivity, but this was not observed after
AAV-mediated galanin secretion in the piriform cortex. NPY has several receptors (Y1-
6), but the peptide fragment NPY13-36 has the highest affinity for the Y2 receptor
(Y2R). Y2R are primarily presynaptic, and their activation suppresses glutamate release
(Colmers and Bleakman 1994; Greber, Schwarzer et al. 1994) via suppression of voltage-
dependent calcium influx through neuronal N-type calcium channels in presynaptic nerve
terminals (Toth, Bindokas et al. 1993; Qian, Colmers et al. 1997). Although galanin and
80
NPY receptors appear to have limited overlap in their downstream signaling pathways,
the end result is still the same: both neuropeptides reduce presynaptic glutamate release.
Therefore, a plausible explanation for the observed lack of potentiation in the piriform
cortex is that a maximal effect for inhibition of presynaptic glutamate release is already
achieved with the delivery of galanin.
Another possibility may be that the concentration of furin in the transduced
neurons could be in limited supply such that constitutive expression of our transcript with
the strong CB promoter is overwhelming the cleavage pathway. The consequence of this
might be that a percentage of secreted protein will still be a galanin-NPY13-36 fusion
protein. Furin and other pro-protein convertases are known to be upregulated in neurons
after seizures induced by kainic acid treatment (Meyer, Chretien et al. 1996), but this
upregulation seems to occur primarily in the hippocampus, and is perhaps not as robust in
the piriform cortex. While our in vitro data suggests that cleavage is completely efficient
(because we never detect fusion protein in the medium with galanin-GFP constructs),
perhaps efficiency is related to the size and structure of the cleavable protein. NPY13-36
is very different from GFP in that it is much smaller. In addition, it has been suggested
that Y2R analogs and NPY13-36 may form hairpin or helical structures (Yao, Smith-
White et al. 2002), and this may mask the cleavage site. The predicted results of a
masked cleavage site would be that the majority of the secreted protein would be a
galanin-NPY13-36 fusion protein. The affinity of the fusion protein for the galanin
receptors and/or the NPY receptors might be lower than that of either galanin or NPY
alone, and may explain our observed results. To rule out this possibility two more
control vectors can be made. The first vector can be a non-cleavable cassette with
81
galanin and NPY13-36. If results from this vector are similar to the results from the
cleavable vector, then it is likely that cleavage efficiency in vivo is poor. Another vector
that can be made is a cassette with an IRES sequence in between galanin and NPY13-36.
While levels of translation will be unequal, at least both gene products will be secreted
from the same transduced neuron, which would not happen 100% of the time with co-
infection of separate vectors. If indeed the proteins are not cleaved from one another in
vivo, it points to a potential liability when using a proteolytic strategy: each protein
combination must be tested on a case by case basis to ensure proper folding and
processing of the gene products are occurring. It is likely that this vector strategy might
not be universally suitable for all protein combinations.
Finally, compared to our previous experiments, transduction levels obtained with
the multiple gene product delivery vectors were modest. The limited spread of these
vectors within the piriform cortex probably decreased the sphere of influence of the
secreted neuropeptides. This could explain the lack of complete protection from seizure
behavior that delivering galanin alone in the piriform cortex has previously produced
(McCown 2006). Since the same AAV serotype and promoter was used, the change in
the transduction pattern can not be explained by virus tropism. Other possibilities include
differences in virus infectivity, sequence-dependent problems with second strand
synthesis, or sequence-dependent mRNA transcript instability. The first hypothesis was
investigated by performing an infectious center assay on the AAV vectors used in these
experiments, and confirmed that they were within the normal range (see Figures 2-3, 2-4,
2-5, 2-6). The second hypothesis can be tested by using a self-complementary vector to
bypass rate-limiting second strand synthesis. Since the size of the promoter, transgene,
82
and polyA is around 2.5kb for AAV2-CB-FIB-GAL-RKRRKR-NPY13-36 it should fit
into a double stranded cassette, although efficiency may be slightly lower than a 2kb
construct. Efforts are being made to shorten the CB promoter for more efficient use in
double stranded vectors, and that would restore full infectivity to our construct because
the largest element in our cassette is the CB promoter. The third hypothesis can be tested
by creating a construct that delivers both galanin and NPY13-36 from the same vector but
as separate transcripts by using two promoters and polyA sequences. While the results
are not exactly what we expected, we did demonstrate the concept that an engineered
proteolytic cleavage site can successfully be used in conjunction with a constitutive
secretion signal sequence to allow multiple gene products to be expressed and secreted
simultaneously, using a single cistron cassette in an AAV vector.
Taken together, this body of work describes a gene therapy vector technology
platform to express and constitutively secrete single and multiple proteins from
transduced cells, and to deliver peptide fragments that are capable of selective receptor
targeting. While epilepsy is one model where this technology was applied, it should be
easily transferable for the treatment of other neurological diseases such as Parkinson’s
disease, Alzheimer’s disease, and stroke.
Chapter 5
The Future of AAV-mediated Gene Therapy in the Brain: Obstacles and
Opportunities
V.A. Brain-wide Vector Delivery
One of the most challenging issues in gene therapy is delivering the vector to all
the disease-affected tissues in the patient. Although using gene therapy to treat a
neurological disease limits the area of vector delivery, the complex organization and
specialization of the substructures in the brain currently impedes global brain delivery.
Some attempts have been made to deliver AAV vectors via intracerebroventricular (ICV)
injection (Shenouda, Johns et al. 2006; Qing and Chen 2007) in the hopes that the
cerebrospinal fluid (CSF) would carry the vector throughout the brain, however, no
improvement in virus spread was achieved. It appears that when delivered ICV, AAV
vectors do not spread beyond the third ventricle or median eminence. Some strategies
exist to enhance the spread of AAV in the brain, such as co-administering mannitol
(Bartlett, Samulski et al. 1998), heparin (Nguyen, Sanchez-Pernaute et al. 2001), and
basic fibroblast growth factor (Hadaczek, Mirek et al. 2004). Alternatively, convection-
enhanced delivery (an approach that uses bulk flow--maintaining a pressure gradient over
time--to deliver and distribute molecules to large areas of brain tissue (Hadaczek,
Kohutnicka et al. 2006), or multiple vector injections (McPhee, Janson et al. 2006) can be
used to increase viral spread. A third strategy to increase AAV vector transduction can
84
be achieved by altering critical capsid components of the vector (see the next section for
a more detailed discussion of this point). Until the technical hurdle of global brain
delivery can be overcome, we must be realistic in our expectations of gene therapy to
treat certain neurological diseases. One factor that contributes to limited success is that
AAV2 continues to be the vector of choice for animal experiments and clinical trials,
(because it is the best characterized and most available serotype), however, it is the
poorest of the serotypes at transducing cells beyond the area of injection. While this
quality is beneficial for diseases with a localized nature (like focal epilepsy), it is
detrimental for most neurological diseases that involve multiple brain regions.
While the brain’s complex structure does present a challenge, knowledge of
neuroanatomy and neuronal projection pathways can be employed to design more
efficient delivery paradigms. For example, it has been reported that some but not all
AAV serotypes can be transported in an anterograde or retrograde manner along axonal
projections to a site distant from that of injection (Kaspar, Llado et al. 2003; Passini,
Macauley et al. 2005; Provost, Le Meur et al. 2005). In fact, Cearley and Wolfe (2006)
showed that when the identical vector genome was cross-packaged into AAV7, 8, 9, or
rh10 capsids, there was a difference not only in the transduction patterns of the different
serotypes, but also in the ability of the packaged vector genome to undergo axonal
transport to neuronal cell bodies distal to the injection site (Cearley and Wolfe 2006).
Furthermore, they then used rAAV9 and showed that a single injection into the ventral
tegmental area (VTA) resulted in delivery and transgene expression in several brain
structures known to have reciprocal projections with the VTA (Cearley and Wolfe 2007).
Injection in the VTA dramatically enhanced virus spread and therapeutic efficacy
compared to injection of the same amount of virus in the striatum (Cearley and Wolfe
2007). Another group compared AAV serotype 1, 2, 5, 7, and 8 in a mouse model of the
lysosomal storage disease Niemann-Pick type A (Dodge, Clarke et al. 2005). Niemann-
Pick type A disease is caused by a deficiency in acid sphingomyelinase (ASM) activity.
Thus, the serotypes were injected into the deep cerebellar nuclei (DCN), and evaluated
for the ability to facilitate global distribution of the viral vector and gene product (ASM).
The authors found that AAV1 was the superior serotype in all measures of disease
correction, and that transduction was observed in the DCN and throughout the
cerebellum, brainstem, midbrain, and spinal cord (Dodge, Clarke et al. 2005). By
understanding which brain regions are affected and how they project to one another,
scientists can design more efficient vector delivery strategies that are tailored to each
neurological disease. Successful vector delivery is paramount for gene therapy to
progress beyond clinical trials.
V.B. Vector Safety, Tolerability, and Efficacy
V.B.1. Vector Targeting
The goal of any Phase I clinical trial is to assess if an experimental treatment is
safe and well tolerated by the patients; AAV-mediated gene therapy is no exception. One
way to contribute to the safety and tolerability of a treatment is to minimize off-target
effects. For AAV gene therapy in the brain, targeting subpopulations of cells can be
achieved at two levels; one is by modifying the capsid so it is capable of binding and
mediating viral entry into specific cell types, and the other is by transcriptional control,
where the promoter and its associated cis and trans regulatory elements can be modified
to limit vector-mediated gene expression to specific cell types and conditions.
85
86
where the promoter and its associated cis and trans regulatory elements can be modified
to limit vector-mediated gene expression to specific cell types and conditions.
V.B.1.a. AAV Capsid-mediated Vector Targeting
Capsid modification is one of the most rapidly progressing areas of AAV vector
engineering. Several targeting approaches have been used by various labs including
genome transcapsidation, mixing of different serotypes to create mosaic capsids, domain
swapping from different serotypes, peptide insertion to create chimeric capsids, and
genetically shuffled AAV capsid gene libraries. Each of these approaches might be a
viable way to overcome some rate-limiting barriers in viral entry and post-entry in
neurons, thereby improving gene transfer.
Transcapsidation (sometimes referred to as pseudotyping) is the packaging of an
AAV genome containing the TR from one serotype (usually type 2 because it is the best
characterized and Food and Drug Administration (FDA) approved serotype) into the
capsid of another serotype in order to shift the viral vector tropism (for an in depth review
see (Choi, McCarty et al. 2005). This strategy is being used extensively in the brain to
increase the viral spread beyond the site of injection. For example, rAAV2 vector
genomes psuedotyped with capsids of AAV1 and AAV5 were compared to rAAV2 in
several brain regions. In this case, rAAV1 and rAAV5 had a more extensive area of
transduction than rAAV2 in most regions examined (Burger, Gorbatyuk et al. 2004;
Shevtsova, Malik et al. 2005). As new serotypes are being discovered and characterized
(Gao, Alvira et al. 2002; Gao, Alvira et al. 2003), it will not be long before we have an
arsenal of serotypes that work best for each brain region, and possibly each neurological
disease.
87
Mosaic capsid modification can also expand viral tropism because it involves
mixing capsid subunits from different serotypes to create a hybrid capsid with receptor
binding profiles conferred from each parent serotype (reviewed in (Wu, Asokan et al.
2006). It was shown that mosaic vectors can gain function compared to the parent
serotypes (Rabinowitz, Rolling et al. 2002; Rabinowitz, Bowles et al. 2004). An
AAV1/AAV2 mosaic vector where the capsid input ratio was 1 to 1 was compared to an
AAV2 vector after hippocampal delivery. The AAV2 vector transduced only the hilar
interneurons while the mosaic vector transduced the hilar interneurons and some
pyramidal and granule cells (Richichi, Lin et al. 2004). Although higher transduction
efficiency was achieved by using the mosaic vector, the authors did not include an AAV1
vector in this study. Therefore, it cannot be determined if the mosaic vector would
outperform each parent serotype. In addition, this method of viral production actually
yields non-uniform virions. For example, even though in this case an equal ratio of type
1 and type 2 helper plasmids were used, it is likely that not all particles will contain equal
amounts of type 1 and type 2 subunits. Since standardization of these preps is difficult,
this method of capsid engineering will probably not transition to clinical trials.
Chimeric vectors contain capsid proteins that have been modified by functional
domain or amino acid swapping between different serotypes, or by genetically inserting
peptide ligands into domains of the capsid to change its receptor binding profile
(reviewed in (Wu, Asokan et al. 2006). Bowles et al. applied the marker rescue approach
to AAV capsid engineering so mutations that may help to target vectors but renders them
non-infectious can be rescued by the addition of wild type capsid (Bowles, Rabinowitz et
al. 2003). By adding back wild type capsid with sequence homology and by selecting for
88
infectious particles using specific cell types, infectivity and vector targeting can be
achieved. This rescue may occur by host cell-mediated recombination at regions of
homology between the wild type and mutant capsid subunits (Bowles, Rabinowitz et al.
2003), or by limiting the number of mutation containing subunits that are incorporated
into the capsid (Gigout, Rebollo et al. 2005). Rational domain swapping is similar to
marker rescue, but instead of relying on recombination to make changes in the capsid
subunits, domains are chosen based on their three dimensional location in the assembled
capsid and their potential to impact function while preserving capsid structure (like
variable surface loops). These domains are then swapped into the capsid subunits by
cloning or other techniques. Marker rescue and rational domain swapping are powerful
techniques that when combined with specific cell-type selection can result in the
generation of AAV vectors with novel transduction capabilities (Bowles, Rabinowitz et
al. 2003; Hauck and Xiao 2003).
Chimeric vectors can also be made by inserting peptide ligands into the capsid to
target them to specific cell types expressing the complimentary receptors. Recently, this
approach was used with two peptide ligands that mimic binding domains for cytoplasmic
dynein and NMDA receptors. These peptides were genetically engineered into AAV2
capsid proteins by directed mutagenesis, and it was shown that these chimeric AAV
vectors exhibited significantly enhanced axonal uptake, allowing for more efficient
retrograde transport (Xu, Ma et al. 2005). The authors delivered AAV vectors to the
tongue and analyzed the brain stem for viral DNA as a proof of concept of retrograde
transport, but these vectors could be directly injected into the brain and analyzed for
transduction efficiencies.
89
Fortunately for patients with neurological diseases, most AAV serotypes tested so
far are neuro-tropic, making AAV a potentially ideal vector for treatment. However,
there are certain types of brain cancers like astrocytomas and gliomas, as well as diseases
like multiple sclerosis and other demyelinating diseases that affect oligodendrocytes.
These diseases could be treated with glia-tropic AAV vectors. Actually, one group tried
to use AAV2 vectors where transgene expression was mediated by the oligodendrocyte-
specific myelin basic protein (MBP) promoter, to transduce diseased oligodendrocytes in
a mouse model for the human demyelinating disease globoid cell leukodystrophy (Chen,
McCarty et al. 1999). In this mouse model called twitcher, demyelination occurs as a
result of degenerating oligodendrocytes, the onset of which is around 20 postnatal days
(Suzuki and Taniike 1995). It was reported that transduction of the oligodendrocytes in
the twitcher mice with AAV2 vectors was quite low (Chen, McCarty et al. 1999),
highlighting the need for more effective glia-tropic AAV vectors. While glial cells
cannot be efficiently targeted and transduced in vivo with the serotypes currently
available, engineering the AAV capsid could result in re-targeting the virus so it no
longer primarily infects neurons. DNA shuffling and error-prone PCR are powerful
library-based approaches that direct the evolution of the capsid, and could be used to re-
target AAV vectors to glial cells. DNA shuffling causes recombination to occur between
homologous fragments generated from a pool of related genes. Upon self-priming PCR,
these fragments can reassemble into diverse sequences that are generated in a non-biased
fashion. Error-prone PCR also introduces random mutations that alone or in combination
may confer unique or enhanced function (for review see (Yuan, Kurek et al. 2005). Both
of these techniques in combination with cell type-specific selection and repeated virus
90
cycling can result in lab engineered serotypes that may target and enhance transduction in
refractory cell types such as glia. In addition, these techniques were recently applied to
AAV2 vectors to select for variants that can escape neutralizing antibodies (Maheshri,
Koerber et al. 2006; Perabo, Endell et al. 2006), reinforcing the utility of these
engineering approaches (see section V.B.2. for more detailed discussion).
V.B.1.b. Cell Type-specific Promoter-mediated Vector Targeting
Along with capsid engineering, another way to limit off-target effects is by
restricting the expression of the transgene to the targeted cells by using cell type-specific
promoters. Cell type-specific promoters can only initiate transgene expression when
specific transcription factors or transactivators recognize and bind to regulatory
sequences within the promoter. Expression of these transcription factors and
transactivators is restricted to sub-populations of cells, and thus prevents ubiquitous
transgene expression (for review see (Walther and Stein 1996). Even though the
naturally occurring AAV serotypes primarily transduce neurons, there is evidence to
suggest that using tissue-specific transcription regulatory elements may increase the
strength and persistence of gene expression in vivo (Klein, Meyer et al. 1998; Paterna,
Moccetti et al. 2000; Xu, Janson et al. 2001; Klein, Hamby et al. 2002). Some promising
promoters that restrict transgene expression to neurons are the neuron-specific enolase
(NSE), platelet-derived growth factor b-chain (PDGF-b), and the human synapsin 1 gene
(hSYN) promoters. Transgene expression in neurons was confirmed for these promoters
by immunohistochemistry double labeling experiments, and persisted greater than 9
months in multiple brain regions (Peel, Zolotukhin et al. 1997; Klein, Meyer et al. 1998;
Paterna, Moccetti et al. 2000; Xu, Janson et al. 2001; Shevtsova, Malik et al. 2005).
91
In addition to neurons, the brain contains other cell types including astrocytes,
macrophages, oligodendrocytes, and epithelial cells. In combination with capsid
engineering, it may be possible to direct and restrict AAV-mediated gene expression to
these cell types. Caution must be taken when trying to target AAV transduction to non-
neuronal cells using cell type-specific promoters, because it has been reported that even
in the presence of glial cell-specific promoters, neuronal expression can still occur. For
example, in a study by Xu et al., high levels of neuronal expression and low levels of
glial expression were found when using AAV2 vectors where gene expression was driven
by the glial fibrillary acidic protein (GFAP) promoter (Xu, Janson et al. 2001). The
authors suggest that the observed “leakiness” of expression could be attributed to
promoter-specific effects on enhancer elements within the TRs (Xu, Janson et al. 2001).
This is likely the case because AAV TRs have been shown to posses transcriptional
activity (Flotte, Afione et al. 1993; Haberman, McCown et al. 2000), suggesting that each
cell type-specific promoter must be tested in vivo to confirm that transgene expression is
in fact limited to the targeted cell population. Furthermore, it is known that some
promoters are inactivated over time in various brain regions, causing shutdown of
transgene expression (McCown, Xiao et al. 1996; Klein, Meyer et al. 1998; Raol, Lund et
al. 2006). Thus, longevity of transgene expression should also be evaluated. The clinical
success of treating neurological diseases with AAV-mediated gene therapy will be
greatly improved with the development of cell type-specific promoters that confer
enhanced long-term gene expression.
92
V.B.1.c. Inducible and Conditional Promoters
In addition to cellular targeting, temporal regulation of transgene expression is a
desirable feature that enhances clinical safety of AAV-mediated gene therapy. Precise
control over the level of transgene expression is necessary, particularly when delivering
potent neurotrophic factors and neuropeptides in the brain, because robust over-
expression has been shown to cause deleterious side effects (Kirik, Rosenblad et al. 2000;
Georgievska, Kirik et al. 2002). Regulation of transgene expression can be achieved
through responsive/inducible elements within the promoter sequence of an AAV vector.
Many synthetic and endogenous promoters have been exploited for the purpose of
gene regulation including hypoxia-responsive promoters, metallothionein promoters, heat
shock, and hormone-responsive promoters (Searle, Stuart et al. 1985; Fuqua, Blum-
Salingaros et al. 1989; No, Yao et al. 1996; Semenza, Jiang et al. 1996). Hypoxia-
inducible and similar disease state-inducible systems have been incorporated into AAV
vectors and may be appropriate for use with specific diseases (Phillips, Tang et al. 2002;
Raol, Lund et al. 2006; Shen, Su et al. 2006; Shen, Fan et al. 2008). Perhaps the most
broadly applicable designs for use in an AAV vector are the FK506/rapamycin (Rivera,
Clackson et al. 1996), tetracycline (tet) (Baron and Bujard 2000), and RU486 (Wang,
Tsai et al. 2000) responsive systems (for an in depth review see (Haberman and McCown
2002). These responsive/inducible systems rely on chimeric transcription factors or
transactivators that in the presence of an administered drug (tet, rapamycin, or
mefipristone), bind/detach their promoter and allow/repress expression of the transgene.
Hence, vector-mediated gene expression can be turned on or off by administering drugs,
allowing baselines to be determined in the presence of vector but in the absence of gene
93
product. Additionally, the same animals may be tested before and after drug
administration, allowing for repeated-measures statistical analysis with improved
statistical power.
The rapamycin responsive system has been used successfully in AAV vectors
for gene expression in muscle (Rivera, Ye et al. 1999; Ye, Rivera et al. 1999; Johnston,
Tazelaar et al. 2003), liver (Auricchio, Gao et al. 2002), eye (Auricchio, Rivera et al.
2002), and more recently in the brain (Sanftner, Rivera et al. 2006). But to date, the tet
responsive system has been favored for gene delivery in the brain (Haberman, McCown
et al. 1998; Fitzsimons, McKenzie et al. 2001; Chtarto, Bender et al. 2003; Jiang,
Rampalli et al. 2004; Chtarto, Yang et al. 2007). There have been a few problems
reported with the use of tet responsive elements in AAV vectors, mainly that there is
“leakiness” or incomplete silencing of gene expression in the “off” state (Folliot, Briot et
al. 2003; Chenuaud, Larcher et al. 2004; Gafni, Pelled et al. 2004; Jiang, Rampalli et al.
2004). As mentioned above, AAV TRs have been shown to posses transcriptional
activity (Flotte, Afione et al. 1993; Haberman, McCown et al. 2000), and therefore
optimization and thorough in vivo testing will be needed to resolve this issue. While
some issues still need to be worked out, the combination of inducible/conditional
responsive elements along with cell type-specific promoters represent an attractive means
to target and regulate AAV-mediated gene expression.
V.B.2. Cis-acting Regulatory Elements
Although the serotype and the promoter can each play a role in determining the
transduction efficiency, there are other regulatory elements that when included in AAV
vector genomes can enhance transduction efficiency. The locus control region (LCR)
94
(reviewed in (Kioussis and Festenstein 1997) and the scaffold attachment region (SAR)
are gene regulatory elements that act in cis to ensure that active transcriptional units are
established in cells. LCRs work at the level of chromatin remodeling to keep an open
chromatin configuration, thus allowing transcription of the gene to occur (Festenstein,
Tolaini et al. 1996). These regulatory elements have shown promise in generating
transgenic animals where transgene integration and subsequent expression are required
(Dillon and Grosveld 1993), but limited success has been achieved in using these
elements in viral vectors (Novak, Harris et al. 1990; Plavec, Papayannopoulou et al.
1993). In contrast, inclusion of SAR elements in retroviral vectors have been found to
confer robust and position-independent enhancement of transgene expression (Agarwal,
Austin et al. 1998; Auten, Agarwal et al. 1999). How the SAR elements enhance
transgene expression is not well understood, but one hypothesis is that they act like
insulators to allow local access of transcription factors to the enhancer/promoter
sequences within the domain (Bode, Kohwi et al. 1992; Jenuwein, Forrester et al. 1997).
In fact, companies such as Gene Detect are incorporating SAR elements into their
commercially available AAV vector plasmids (see http://www.genedetect.com/rave.htm).
Perhaps the best known regulatory element used to enhance AAV-mediated
transgene expression is the Woodchuck hepatitis B virus post-transcriptional regulatory
element (WPRE). WPRE has been shown to increase the steady-state level of
mRNA and the efficiency of translation, resulting in higher levels of transgene synthesis
(Loeb, Cordier et al. 1999). Paterna et al. performed the first in vivo quantitative
comparison of AAV vectors containing the PDGF-β promoter in conjunction with a
WPRE in the substantia nigra (Paterna, Moccetti et al. 2000). It was found that a WPRE
95
boosted gene expression 1.8 fold compared to vectors that contained the PDGF-β
promoter and lacked a WPRE (Paterna, Moccetti et al. 2000). Then Xu et al. tested a
variety of promoters with and without a WPRE in four different brain regions (Xu,
Janson et al. 2001). They also found a WPRE to enhance gene expression from every
AAV vector and in every brain region tested (Xu, Janson et al. 2001). Other groups have
also seen improved transduction using AAV constructs with a WPRE in the lung and eye,
(Lipshutz, Titre et al. 2003; Virella-Lowell, Zusman et al. 2005), demonstrating that the
WPRE is definitely effective in an AAV context.
It should be noted that maximum levels of transgene expression are not always
desirable, especially when intracellular processing of the transgene is needed. It is not
hard to envision certain cellular pathways as being rate limiting, and thus overwhelming
them with protein may have deleterious effects. In addition, as previously discussed,
some gene products cause unwanted side effects when robustly expressed (Kirik,
Rosenblad et al. 2000; Georgievska, Kirik et al. 2002). Furthermore, Chrarto et al. show
that incorporating a WPRE in a tet-responsive AAV vector caused increased leakiness
such that the transgene GDNF was expressed at a 2.5 fold greater level in the off state
than baseline levels or a tet construct lacking a WPRE (Chtarto, Yang et al. 2007). Thus,
careful consideration over the inclusion of regulatory elements should be taken when
designing AAV vectors for pre-clinical and clinical trials where tight gene regulation may
be paramount to safety and therapeutic efficacy.
V.B.3. Immune Response
One of the biggest challenges in optimizing viral vectors for gene therapy pertains
to the host-mediated immune response. This response can occur at the cellular level via
96
two pathways: a transient innate inflammatory immune response (i.e. the release of
cytokines, and the recruitment of monocytes and/or macrophages and neutrophils), and
an adaptive immune response where cytotoxic T lymphocytes (CTL) infiltrate the area of
vector injection. In addition, the host-mediated immune response can occur at the
humoral level, where antibodies are generated against the structural components of the
virus or the proteins encoded by the virus. Due to the unique cytoarchitecture of the
brain and the fact that is circumscribed by the blood-brain barrier (BBB), the brain
parenchyma is immune privileged under normal conditions, and has been shown to
exhibit no salient adaptive immune response, and only a minimal innate and humoral
response to viral antigens (Barker and Billingham 1977; Lowenstein 2002). Perhaps this
is because the brain is devoid of lymphoid tissue, and the parenchyma does not normally
contain antigen-presenting dendritic cells, thus an adaptive immune response is not
primed after viral vector delivery (Lowenstein 2002). Consequently, studies examining
the immune response to rAAV vectors in the brain have focused primarily on the humoral
response and its impact on transgene expression.
Earlier studies examined the effects of single and repeat administration of rAAV
into naïve animals with no pre-existing immunity to AAV. Lo et al. delivered AAV2-
LacZ vectors into the mouse brain and reported that serum antibodies to AAV capsid
proteins and β-gal transgene were elicited between 2–4 months post administration, but
did not correlate with transgene expression (Lo, Qu et al. 1999). Furthermore, the
presence of these antibodies did not consistently prevent transgene expression following
re-administration of the same vector 2 months later (Lo, Qu et al. 1999). In another re-
administration study by Mastakov et al., it was found that transgene expression was
97
diminished by approximately 5 to 10 fold in animals where the second injection was done
within a 2-4 week interval, but no reduction was observed if the second injection was
given 3 months the after first injection (Mastakov, Baer et al. 2002). These experiments
suggest that timing between multiple injections may have a critical effect on therapeutic
efficacy of AAV-mediated gene therapy in the brain. Moreover, if rAAV5 was first
injected, then 2 weeks later the same vector cassette was packaged into AAV2 and
injected, no decrease in transgene expression occurred, suggesting that the loss of
transgene expression observed earlier was in fact due to neutralizing antibodies to the
rAAV5 capsid (Mastakov, Baer et al. 2002). Thus, sequential delivery of different rAAV
serotypes into the brain avoids the host immune response. However, this strategy may
create new problems, because (as discussed in section V.A.) the different serotypes
exhibit different transduction patterns in the brain. Perhaps the identification and
mutation of antigenic capsid epitopes that are recognized by neutralizing antibodies can
be achieved without affecting receptor binding and thus the transduction profile. In fact,
Lochrie et al. tried to accomplish this using in silico modeling of antibody-capsid binding
interactions, then mutated residues suspected of being immune epitopes (Lochrie,
Tatsuno et al. 2006). Other approaches include insertion of peptides in or near
neutralizing epitopes (Huttner, Girod et al. 2003), or simultaneous recombination and
mutagenesis of the AAV2 capsid gene followed by selection in the presence of human
antisera (Maheshri, Koerber et al. 2006; Perabo, Endell et al. 2006). Regardless of the
method used to engineer the virus, unless the paradigm that the vector is tested in
accurately reflects a clinical trial scenario, the utility and predictive value of the outcome
will be diminished.
98
More recent experiments examining the humoral immune response have been
conducted in animals that have been pre-immunized via peripheral exposure to AAV.
This model more accurately represents the human population where an estimated 80%
have circulating antibodies to the capsid proteins of wtAAV2, and 30% to 70%
demonstrate the presence of neutralizing anti-capsid antibodies that can bind to the
surface of the virus, thereby preventing attachment to cellular receptors and subsequent
cellular uptake (Chirmule, Propert et al. 1999; Erles, Sebokova et al. 1999; Xiao,
Chirmule et al. 1999; Hildinger, Auricchio et al. 2001; Halbert, Miller et al. 2006). In a
study by Sanftner et al., a peripheral injection of a rAAV2 non-expressing vector (lacking
a promoter) was followed by bilateral intrastriatal injection of rAAV2 encoding a
therapeutic transgene (Sanftner, Suzuki et al. 2004). Neutralizing antibody titers after
rAAV administration in the striatum were highest in the animals pre-immunized with
higher doses of rAAV, and there was a weakly linear and negative relationship between
antibody titer and transgene expression distribution (R2 = 0.30 (Sanftner, Suzuki et al.
2004). No evidence of abnormal morphology, cellular infiltration or inflammatory
markers was found. The authors concluded that rAAV vectors can transduce brain tissue
in the context of pre-existing immunity, but that efficiency of transduction declines
significantly in the presence of very high titers of neutralizing antibodies (Sanftner,
Suzuki et al. 2004). In contrast to these results, another group used a strategy of multiple
pre-immunization injections with wtAAV2 and adjuvant, and found that the resulting
high levels of circulating neutralizing antibodies were sufficient to completely block
striatal transduction (Peden, Burger et al. 2004). It has been suggested that the
conflicting results between these two studies are likely due to differences in the
99
immunization strategies, surgical techniques, BBB integrity, rAAV formulations, and
serological methodologies (McPhee, Janson et al. 2006).
While the Peden study raises some concerns with using rAAV2 as a gene therapy
vector in the brain, a couple of phase I clinical trials have already been completed with
promising results (McPhee, Janson et al. 2006; Kaplitt, Feigin et al. 2007). In the
Canavan disease (CD) trial, an rAAV2 vector encoding a functional copy of the
aspartoacylase gene (ASPA) was injected at 6 sites: bilaterally in the frontal,
periventricular and occipital lobes (McPhee, Janson et al. 2006). It was found that only 1
of 10 subject had circulating neutralizing antibodies to AAV before rAAV
administration, and 3 of 10 had circulating neutralizing antibodies to AAV after rAAV
administration (McPhee, Janson et al. 2006). Fortunately, the subject with pre-existing
neutralizing antibodies had no adverse events, and a physiological response that was
similar to the other subjects (McPhee, Janson et al. 2006). In the Parkinson’s disease
(PD) trial, 2 of 12 subjects had pre-existing neutralizing antibodies, but the levels did not
change in any of the subjects following delivery of an AAV2 vector encoding glutamic
acid decarboxylase (GAD) into the subthalamic nucleus (Kaplitt, Feigin et al. 2007). The
lack of induction of neutralizing antibodies in the PD study compared to the CD study
may be attributed to fewer injection sites and therefore less BBB disruption. Moreover,
in the PD study there was no correlation between pre-existing immunity and clinical
outcome, as measured by improvement in motor function while off medication (Kaplitt,
Feigin et al. 2007). Finally, there was no evidence of pre-existing antibodies to the
transgene, and no induction of such antibodies at any time in any patient over the course
of the 1 year study (Kaplitt, Feigin et al. 2007). While the PD study was only an open
label Phase I study, the results suggest that pre-existing immunity to wtAAV may not
prevent effective AAV-mediated gene transfer in the human brain.
V.C. Conclusions and Progress of Current Gene Therapy Clinical Trials in Brain
Many chronic neurological diseases do not respond to small molecule
therapeutics, and have no effective long-term treatment. In fact, as many as 98% of the
candidate neuro-therapeutic drugs never get developed for clinical use because they have
poor BBB permeability (Pardridge 2001). Gene therapy is a promising alternative to
continuous administration of neuro-therapeutic drugs, because vectors can be locally
delivered to affected brain regions, where they mediate de novo synthesis of therapeutic
compounds. It has been shown that in vivo production of these therapeutic compounds
are more effective than their acute infusion (Frim, Simpson et al. 1993; Frim, Uhler et al.
1993; Venero, Beck et al. 1994; Martinez-Serrano and Bjorklund 1996). In addition,
long-term transgene expression can be achieved in the brain following a single vector
injection. For example, a single injection of an AAV vector into the striatum of primates
resulted in sustained gene expression for more than 6 years (Bankiewicz, Forsayeth et al.
2006). It is likely that the same persistence will be achieved in the human brain because
injections of rAAV in the muscle resulted in sustained gene expression upon biopsy 3.7
years post-delivery (Jiang, Pierce et al. 2006).
Currently, there are 12 open gene therapy clinical trials in the CNS, but most of
them are being conducted with naked DNA as the delivery vector (see table 5-1). Naked
plasmid gene transfer is safe, easy to handle, and cost effective, but has generally been
considered to be less efficient than viral gene transfer (Huang, Hung et al. 1999).
100
101
Table 5-1. Current Open Gene Therapy Trials for Neurological Diseases. This data is current to July 2007. For more details on these clinical trials please see: http://www.wiley.co.uk/wileychi/genmed/clinical/
Trial ID Title Gene Disease Vector Phase UK-129 A multicenter, randomized, double
blind, placebo-controlled study to evaluate the safety, tolerability, and efficacy of BHT-3009 when administered intramuscularly to patients with relapsing remitting multiple sclerosis (Protocol No. BHT-3009-03). EudraCT: 2005-001340-22
Myelin Basic Protein (MBP)
Multiple Sclerosis
Naked/Plasmid DNA
I
UK-132 A Phase II Double Blind, Cross-over Study to Compare the Safety and Efficacy of 125, 250 and 500µg/kg Monarsen (EN101) administered to Patients with Myasthenia Gravis. EudraCT: 2005-002740-26
Antisense Oligodeoxynucleotide against acetylcholinesterase
Myasthenia Gravis
Naked/Plasmid DNA
II
US-180 Phase I Single Dose-ranging Study Of Formulated hIGF-I Plasmid In Subjects With Cubital Tunnel Syndrome
Insulin-like growth factor-1 (IGF-1)
Cubital Tunnel Syndrome
Naked/Plasmid DNA
I
US-632 pVGI.1 (VEGF-2) Gene Transfer for Diabetic Neuropathy
Vascular endothelial growth factor (VEGF)
Diabetic Peripheral Neuropathy
Naked/Plasmid DNA
I/II
US-656 A Phase I/II, Dose-escalation Clinical Trial of SB509 in Subjects with Diabetic Neuropathy
VOP32E VEGF-A Transcription Factor
Diabetic Peripheral Neuropathy
Naked/Plasmid DNA
I/II
US-669 Hippocampal NPY Gene Transfer in Subjects with Intractable Temporal Lobe Epilepsy
Neuropeptide Y (NPY) Epilepsy
Adeno-associated virus
I
US-689 A Phase I, Open-Label Study of CERE-120 (Adeno-Associated Virus Serotype 2 [AAV2]-Neurturin [NTN]) to Assess the Safety and Tolerability of Intrastriatal Delivery to Subjects with Idiopathic Parkinson's Disease
Neurturin (NTN) Parkinson's Disease
Adeno-associated virus
I
US-723 BHT-3009 Immunotherapy in Relapsing Remitting Multiple Sclerosis
Myelin Basic Protein (MBP)
Multiple Sclerosis
Naked/Plasmid DNA
II
US-788 Multicenter, Randomized, Double-blind, Sham Surgery-controlled Study of CERE-120 (Adeno-Associated Virus Serotype 2 [AAV2]-Neurturin [NTN]) to Assess the Efficacy and Safety of Bilateral Intraputamenal (IPu) Delivery in Subjects With Idiopathic Parkinson's Disease
Neurturin (NTN) Parkinson's Disease
Adeno-associated virus
II
US-822 A Phase 2 Repeat Dosing Clinical Trial of SB-509 in Subjects with Diabetic Neuropathy
Zinc finger DNA binding protein (ZPF-TF)
Diabetic Peripheral Neuropathy
Naked/Plasmid DNA
II
US-837 A Phase 2 Repeat Dosing Clinical Trial of SB-509 in Subjects with Moderate to Severe Diabetic Neuropathy and Unmeasurable Nerve Conduction Velocity
Zinc finger DNA binding protein (ZPF-TF)
Diabetic Peripheral Neuropathy
Naked/Plasmid DNA
II
US-851 Cell-Based Gene Therapy Using MRC-MBP for Treatment of Multiple Sclerosis-Phase I/II
Myelin Basic Protein (MBP)
Multiple Sclerosis
Retrovirus I/II
102
Perhaps when the initial AAV gene therapy clinical trials are completed and found to be
well tolerated, more will be approved.
A very exciting clinical trial is being initiated using AAV vectors to delivery NPY
to the hippocampus of patients with intractable TLE. It is the first gene therapy trial to be
conducted in epileptic patients. Pre-clinical studies including those that were presented
in this thesis show that AAV-mediated delivery of NPY to the brain results in seizure
suppression, and therefore has enormous therapeutic potential (Richichi, Lin et al. 2004;
Foti, Haberman et al. 2007). Although this trial will initially be a Phase I study and thus
is not designed to assess transgene efficacy, it is still possible that subjects will obtain
vector-mediated therapeutic benefits.
Currently, the most promising neurological gene therapy trial is the AAV-
mediated delivery of neurturin (NTN) to the striatum of patients with advanced PD.
NTN is a naturally occurring neurotrophic factor that has been shown to be
neuroprotective and regenerative in the damaged dopaminergic neurons that undergo
degeneration in PD (Oiwa, Yoshimura et al. 2002). Loss of dopaminergic innervation of
the striatum as a consequence of substantia nigral degeneration results in the cardinal
symptoms of PD, including tremor, rigidity, and the progressive inability to initiate and
control physical movements (Lang and Lozano 1998). In a recent press release the
company conducting the trial announced that the AAV vector was well tolerated in the
patients; and that the treatment appeared to reduce 8 of the 12 patients’ symptoms by
approximately 52% at 24 months post-treatment (see
http://www.medicalnewstoday.com/articles/91612.php). Furthermore, a multi-center
double-blind, controlled Phase II clinical trial completed enrollment in December of
103
2007, of 58 patients with advanced PD. Two thirds of patients will be given the
treatment and one third will be in a control group. Clinical data from this trial are
expected to be available in late 2008, and if the data are positive, a Phase III trial is
expected to commence in 2009. Thus, this may be the first AAV vector-mediated gene
therapy to undergo Phase III testing.
In conclusion, AAV vectors have demonstrated clinical potential. The most
critical issues that will need to be addressed to ensure safe and effective gene therapy in
the brain are increased gene transfer efficiency, target specificity, and the ability to
regulate gene expression so maximal therapeutic efficacy can be reached with minimal
adverse effects. To this end, substantial progress is being made using recently developed
techniques to engineer the virus, both at the structural level through capsid modification,
and at the gene transcriptional level via promoter choice and regulatory element
inclusion. It is likely that the combination of these developments will facilitate clinical
gene therapy.
104
Appendices
Appendix A. Plasmids Generated and Characterized
Table 6-1. Plasmids Generated and Characterized. Abbreviations are: ELISA: Enzyme-Linked ImmunoSorbent Assay; GAL: galanin; GDNF: glial cell derived neurotrophic factor; IP/WB: Immunoprecipitation and Western blotting; LUC: Luciferase GL3; NA: no assay performed; PCR: polymerase chain reaction; RFP: DsRed2-C1; RKLRKR: arginine-lysine-leucine-arginine-lysine-arginine; RKRRKR: arginine-lysine-arginine-arginine-lysine-arginine; RT-PCR: Reverse transcriptase-polymerase chain reaction; WB: Western blotting. Constructs Generated
Assays Results
ptTak-Dynorphin A 1-13 FIB (has TR2)
WB Protein doesn't stick to membranes and may be cleaved in vivo
ptTAK-NPY13-36 FIB (has TR2)
ELISA NPY13-36 detected in lysates only, not sensitive enough for detection upon secretion into medium
CB-DsRed2-C1-FIB (CB-FIB-RFP) (has TR2)
WB DsRed antibody binds high molecular weight bands and is very poor at detecting proper size
pTR-CB-FIB-EGFP-RKLRKR-FIB-RFP
WB GFP antibody detects fusion protein in lysates
pTR-CB-FIB-EGFP-RKLRKR-RFP
WB GFP antibody detects fusion protein in lysates, and cleaved protein in medium
pTR-CB-FIB-EGFP-RKLGGG-RFP
WB GFP antibody detects fusion protein in lysates
pTR-CB-FIB-LUC (Luciferase GL3)
WB and IP/WB and light assay
Luciferase antibody detects protein in lysates but not medium so luciferase not secreted and not functional in light assay
pTR-CB-FIB-LUC-RKRRKR-EGFP
WB and IP/WB and light assay
Luciferase antibody detects protein in lysates but not medium so luciferase not secreted and not functional in light assay; GFP antibody detects fusion and cleaved protein in lysates
pTR-CB-FIB-LUC-RKRRKR-FIB-EGFP
WB and IP/WB GFP antibody detects cleaved protein in medium and both cleaved and fusion proteins in lysates
pTR-CB-FIB-GAL-RKRRKR-EGFP
IP/WB kainic acid test
GFP antibody detects cleaved protein in medium and both fusion and cleaved protein in lysates; some seizure protection is observed but not to the extent of galanin alone
pTR-CB-FIB-GAL-RKRRKR-FIB-EGFP
IP/WB kainic acid test
GFP antibody detects cleaved protein in medium and both fusion and cleaved protein in lysates; galanin blocks seizures in vivo
pTR-CB-FIB-EGFP-RKRRKR-FIB-GAL
IP/WB Misfolded so does not fluoresce in situ, but can be detected with GFP antibody in IP/WB
pTR-CB-EGFP-RKRRKR-FIB-GAL
Transfection Does fluoresce
pTR-UF-WGDNF (CB-human pre-pro GDNF plus WPRE and TR2)
NA
pTR-UF-GDNF (removed WPRE)
ELISA/ made virus for collaboration
GDNF is detected in medium and lysates; Publishing with this, article in press
105
pBS-SK-huNGF (human pre-pro Nerve Growth Factor )
PCR Worked for cloning
pTR-UF-NGF (cloned human pre-pro Nerve Growth Factor into UF-GDNF plasmid )
ELISA NGF detected in medium
pTR-CB-NGF-RKRRKR-GDNF
ELISA Only GDNF detected in medium and lysates
pTR-CB-FIB-NPY RT-PCR and kainic acid test
Published with this (Foti, Haberman et al. 2007)
pTR-CB-FIB-NPY13-36 RT-PCR and kainic acid test
Published with this (Foti, Haberman et al. 2007)
pCMV-Sport6-hGAL (human galanin)
PCR Worked for cloning
pTR-CB-hGAL NA pTR-CB-FIB-GAL-RKRRKR-FIB-NPY
NA
pTR-CB-FIB-GAL-RKRRKR-FIB-NPY13-36
NA
pTR-CB-FIB-GAL-EGFP IP/WB GFP antibody detects gal-GFP fusion protein in medium, but some degradation product present along with fusion in lysates
pTR-CB-FIB-GAL-EGFP mut (ATG removed from GAL and EGFP)
IP/WB kainic acid test
GFP antibody detects gal-GFP fusion protein in medium, but some degradation product present along with fusion in lysates; some seizure protection is observed but not to the extent of galanin alone
pN1-CB-FIB-EGFP-RKRRKR-GAL (no TR for mutagenesis from EGFP N1 plasmid)
PCR Worked for cloning
pN1-CB-FIB-GAL-RKRRKR-EGFP (no TR for mutagenesis)
PCR Worked for cloning
pN1-CB-FIB-GAL-EGFP mut (no TR for mutagenesis)
PCR Worked for cloning
pTR-CB-KOZ-FIB-GAL-EGFP mut (added Kozak sequence)
IP/WB GFP antibody detects gal-GFP fusion protein in medium, but some degradation product present along with fusion in lysates
pTR-CB-KOZ-FIB-GAL-RKRRKR-EGFP mut (added Kozak sequence)
IP/WB Construct does not behave properly in IP/WB
pTR-CB-KOZ-FIB-EGFP-RKRRKR-GAL mut (added Kozak sequence)
IP/WB GFP antibody detects cleaved protein in medium and fusion proteins in lysates
pTR-CB-KOZ-FIB-GAL-RKRRKR-NPY13-36 (added Kozak sequence)
NA
pTR-CB-FIB-EGFP-RKR Transfection Does fluoresce pTR-CB-FIB-ADNF made virus for
collaboration
pCMV-IL-10 NA pTR-CB-FIB-EGFP-RKRRKR-GAL mut
IP/WB kainic acid test
GFP antibody detects cleaved protein in medium and fusion proteins in lysates; galanin blocks seizures in vivo
106
pTR-CB-FIB-GAL-RKRRKR-NPY13-36 mut
RT-PCR and kainic acid test
Correct vector-derived mRNA product present after rAAV infection in vivo, and some seizure protection is observed but not to the extent of galanin or NPY13-36 alone
pTR-CB-FIB-GAL-RKRRKR-EGFP mut
IP/WB Construct does not behave properly in IP/WB
107
Appendix B. Multiple Gene Product Delivery Vectors
Along with the other in vitro experiments to characterize the secreted proteins
from our multiple gene product delivery vectors, an immunoprecipitation followed by
SDS-PAGE and Coomassie staining for mass spectrometry analysis was performed on
medium harvested from 293 cells transfected with the following constructs: CB-FIB-
EGFP, CB-FIB-GAL-RKRRKR-FIB-EGFP, CB-FIB-EGFP-RKRRKR-FIB-GAL, CB-
FIB-GAL-RKRRKR-EGFP, and CB-EGFP. Upon mass spectrometry analysis, we found
that all samples contained fragments of the FIB sequence and GFP, but not galanin;
except the sample from CB-EGFP, which contained only GFP fragments (see Figure 6-
1). While these results are promising, a positive control such as CB-FIB-GAL-EGFP
must be included next time to assure that galanin can be detected in this assay.
Appendix C. Nerve Growth Factor (NGF) and Glial Cell Derived Neurotrophic Factor (GDNF)
In addition to epilepsy, there are many neurological diseases that can benefit from
AAV-mediated gene therapy. Neurodegeneration is a common pathology of several
diseases (e.g. Huntington’s disease (HD), Parkinson’s disease (PD), Alzheimer’s disease
(AD), and temporal lobe epilepsy (TLE), and if left unchecked causes progressive
decline. Neurotrophic factors have long been recognized to be neuroprotective, and there
is a growing interest in using them as therapeutics. Several studies have shown that NGF,
brain-derived neurotrophic factor (BDNF), and GDNF are each capable of protecting
neurons from death in rat models of HD (Davies and Beardsall 1992; Frim, Short et al.
1993; Galpern, Matthews et al. 1996; Martinez-Serrano and Bjorklund 1996; Perez-
Navarro, Arenas et al. 1996; Araujo and Hilt 1997; Araujo and Hilt 1998; Perez-Navarro,
Arenas et al. 1999; Cruz-Aguado, Turner et al. 2000; Menei, Pean et al. 2000;
108
Figure 6-1. Immunoprecipitation Followed by SDS-PAGE and Coomassie Staining of Multiple Gene Product Delivery Vectors for Mass Spectrometry Analysis. 1, 7) Low molecular weight rainbow marker, 2) CB-FIB-EGFP, 3) CB-FIB-GAL-RKRRKR-FIB-EGFP, 4) CB-FIB-EGFP-RKRRKR-FIB-GAL, 5) CB-FIB-GAL-RKRRKR-EGFP, 6) CB-EGFP. Constructs were transfected into 293 cells using PEI (1mg/mL), and 48 hours later medium was harvested and immunoprecipitated using a GFP polyclonal antibody. After SDS-PAGE, the gel was fixed in 25% isopropanol, 10% acetic acid, and 65% sterile water, and stained overnight with Coomassie 0.01% in 10% acetic acid. Yellow boxes indicate the bands excised from the gel and digested in trypsin for protein laddering matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Samples 2-4 all contained fragments of the FIB signal sequence and GFP, but not galanin. Sample 5 contained only GFP but not FIB or galanin. Sample 6 contained only antibody fragments. While these results are promising, a positive control such as CB-FIB-GAL-EGFP must be included next time to assure that galanin can be detected in this assay.
1 2 3 4 5 6 71 2 3 4 5 6 7
109
Alberch, Perez-Navarro et al. 2002; Maksimovic, Jovanovic et al. 2002; McBride, During
et al. 2003; Kells, Fong et al. 2004). However, individual neurotrophic factors
preferentially spare subpopulations of neurons, and this sparing may be influenced by the
neurotrophic factor’s route of delivery. For example, acute infusions of NGF prior to or
concurrent with quinolinic acid (QA) spares the cholinergic interneurons of the striatum,
but not the striatal GABA-ergic medium spiny neurons (primary neurons vulnerable to
the degenerative cascade seen in HD) or the noncholinergic neurons (containing
somatostatin, or NADPH-diaphorase) (Venero, Beck et al. 1994). In contrast, when NGF
is chronically secreted by genetically modified cells, it potently protects the cholinergic,
GABA-ergic and noncholinergic neurons from QA induced degeneration (Frim, Uhler et
al. 1993; Martinez-Serrano and Bjorklund 1996), and 3-NP induced degeneration (Frim,
Simpson et al. 1993). So, therapeutic gene delivery of neurotrophic factors yields more
comprehensive protection for striatal neurons than acute infusions of the same factors.
Similarly, ex vivo methods of delivering GDNF to the striatum only results in partial
neuroprotection (Perez-Navarro, Arenas et al. 1996; Perez-Navarro, Arenas et al. 1999),
thus, using an in vivo method of neurotrophic factor delivery may overcome this
difficulty. In fact, McBride et al use adeno-associated virus (AAV) as their method of
therapeutic gene delivery to inject a GDNF expressing viral vector into the striatum of
rats before 3-NP challenge (McBride, During et al. 2003). They report significant
reduction in lesion size (assessed by NeuN immunohistochemistry) and increased
survival of striatal GABA-ergic neurons (assessed by a DARPP-32, a marker of GABA-
ergic terminals). In addition, rats receiving GDNF perform better on motor tasks than
controls, suggesting that viral-mediated gene transfer of GDNF into the striatum affords
110
neuroanatomical and behavioral protection from 3-NP. Although striatal neuronal
subtypes were not extensively evaluated in the McBride et al study, Kells et al used AAV
to deliver BDNF or GDNF prior to QA lesioning rats and evaluated subpopulations of
GABA-ergic striatal interneurons. BDNF expression protected NOS-immunopositive
neurons while GDNF protected parvalbumen immunopositive neurons (Kells, Fong et al.
2004). Since neither neurotrophic factor can afford complete protection when
administered singly, a combination of neurotrophic factors should be used. Therefore,
we were interested in using our multiple gene product delivery vectors to deliver both
NGF and GDNF in a rat model of Huntington’s disease. NGF and GDNF are both
synthesized as precursors that must be correctly assembled into homodimers, and cleaved
into their active forms. It has been reported that NGF needs its pro-sequence in order to
fold properly (Rattenholl, Ruoppolo et al. 2001), so we opted not to use the FIB sequence
to mediate secretion, but to use the pre-pro sequences of both NGF and GDNF (each
containing their own secretion sequence) to ensure correct folding and secretion. Using
the same cloning strategy as described in Chapter 2, we constructed the following AAV-
compatible plasmids: TR-CB-EGFP, TR-CB-β-NGF, TR-CB-GDNF, and TR-CB-β-
NGF-RKRRKR-GDNF. To characterize these constructs in vitro, we transfected them
into 293 cells and harvested medium 48 hours post transfection, added leupeptin and
aprotinin protease inhibitors, briefly centrifuged the medium to remove any cellular
debris, and stored the medium at -80oC until use in Enzyme-Linked ImmunoSorbent
Assay (ELISA). We found we could only detect NGF from our TR-CB-β-NGF
transfected samples and our NGF protein spiked samples, but not from our TR-CB-β-
NGF-RKRRKR-GDNF transfected samples (see Figure 6-2). We were however
111
Figure 6-2. NGF and GDNF ELISA. Assay reveals that NGF is not detected in the medium following transfection of CB-β-NGF-RKRRKR-GDNF, but GDNF is detected. Note that the antibodies are specific for each neurotrophin and do not cross-react.
NFG ELISA (Avg of Duplicates)
25
243
17 22
225
23
0
50
100
150
200
250
300
Con
cent
ratio
n (p
g/m
L)
CB-FIB-EGFP
CB-B-NGF
CB-GDNF
CB-B-NGF-RKRRKR-GDNFNGF Protein Spike
Untransfected Control
GDNF ELISA (Avg of Duplicates)
27 36
23562572
2758
260
500
1000
1500
2000
2500
3000
Con
cent
ratio
n (p
g/m
L)
CB-FIB-EGFP
CB-B-NGF
CB-GDNF
CB-B-NGF-RKRRKR-GDNFGDNF Protein Spike
Untransfected Control
112
detecting GDNF from our GDNF protein spiked samples, TR-CB-GDNF transfected
samples, and our TR-CB-β-NGF-RKRRKR-GDNF transfected samples (see Figure 6-2).
This is a puzzling result at first glance because GDNF is the second gene product in our
multi gene product delivery vector, so if we are detecting it, then it is likely that NGF is
also expressed even though we cannot detect it. These results can be explained if the
NGF protein was indeed expressed but misfolded. Misfolded NGF might not show up in
an ELISA if the antibody cannot detect denatured protein. This issue can be resolved by
performing a western blot assay with an antibody designed to detect denatured protein.
However, we felt it was not worth the expense to confirm that the NGF was being
misfolded, because misfolded NGF is not functional and obviously not therapeutic. We
decided to discontinue this project, but we gleaned very critical information from these
experiments about potential protein folding issues when using our multiple gene product
delivery vectors.
Appendix D. Publications
Appendix D.1. Adeno-associated Virus-Mediated Expression and Constitutive Secretion of NPY or NPY13-36 Suppresses Seizure Activity In Vivo.
113
Adeno-associated Virus-Mediated Expression and Constitutive Secretion of NPY or
NPY13-36 Suppresses Seizure Activity In Vivo.
Stacey Foti2, Rebecca P. Haberman5, R. Jude Samulski1,4 and Thomas J. McCown1,3
UNC Gene Therapy Center1, Curriculum in Neurobiology2, Departments of Psychiatry3 and Pharmacology4
University of North Carolina at Chapel Hill
7119 Thurston, CB 7352
University of North Carolina School of Medicine
Chapel Hill, NC, 27599
and
Department of Psychological and Brain Sciences5
Johns Hopkins University
Baltimore, MD
To whom correspondence should be addressed: Thomas J. McCown, Ph.D. UNC Gene Therapy Center 7119 Thurston, CB 7352 Chapel Hill, NC 27599 email:[email protected]
114
Neuropeptide Y (NPY) is a 36–amino acid peptide that attenuates seizure activity
following direct infusion or AAV-mediated expression in the CNS. However, NPY
activates all NPY receptor subtypes, potentially causing unwanted side effects. NPY13-
36 is a C-terminal peptide fragment of NPY that primarily activates the NPY Y2
receptor, thought to mediate the anti-seizure activity. Therefore, we investigated if rAAV
mediated expression and constitutive secretion of NPY or NPY13-36 could alter limbic
seizure sensitivity. Rats received bilateral piriform cortex infusions of AAV vectors that
express and constitutively secrete full length NPY (AAV-FIB-NPY), or NPY13-36
(AAV-FIB-NPY13-36). Control rats received no infusion, as we have previously shown
that vectors expressing and secreting reporter genes like GFP (AAV-FIB-EGFP), as well
as vectors expressing peptides that lack secretion sequences (AAV-GAL) have no effect
on seizures. One week later, all animals received kainic acid (10 mg/kg, i.p.), and the
latencies to wet dog shakes and limbic seizure behaviors were determined. Although
both control and vector treated rats developed wet dog shake behaviors with similar
latencies, the latencies to class III and class IV limbic seizures were significantly
prolonged in both NPY and NPY13-36 treated groups. Thus, AAV-mediated expression
and constitutive secretion of NPY and NPY13-36 is effective in attenuating limbic
seizures, and provides a platform for delivering therapeutic peptide fragments with
increased receptor selectivity.
Key Words: Adeno-associated virus, gene therapy, epilepsy, seizures, kainic acid,
neuropeptide Y, NPY13-36, piriform cortex
115
Epilepsy is an attractive target for recombinant Adeno-associated virus (rAAV)
gene therapy, because the temporal lobe structures involved in seizure genesis and
propagation have been shown to be permissive to AAV gene transfer.1-6 Recently,
galanin and neuropeptide Y (NPY) have been delivered as transgenes in rAAV vectors,
and shown to be effective in several epilepsy paradigms.1,3,6,7 While previous approaches
utilize pre-pro cDNA sequences which rely on the cell to modulate release of the gene
product, Haberman et al.3 were the first to use a novel secretion strategy whereby the
secretion signal sequence from the constitutively secreted laminar protein fibronectin
(FIB), is combined with the coding sequence for the active therapeutic peptide. Results
from Haberman et al.3 and McCown1 show that expression and constitutive secretion of
galanin is not only achieved, but also effective in attenuating focal and limbic seizure
activity. In contrast, expression of galanin without the secretory signal or expression and
secretion of the reporter gene, GFP had no effect on seizure sensitivity.1,3
In addition to galanin, rAAV delivery of NPY has also been shown to attenuate
limbic seizures,6 however, there is still some question as to which of the NPY receptors
(Y1-Y5) mediates the anti-seizure activity. Several studies have suggested a critical role
for the Y2 receptor in mediating anti-epileptic actions.8-14 In addition, it has been
demonstrated that in epileptic tissue from patients15 and rodents16,17 that Y1 receptors are
downregulated while Y2 receptors are upregulated. Taken together, these results suggest
that when designing anti-epileptogenic therapeutics, it may be advantageous to deliver
Y2 preferring agonists. In fact, acute intracerebral delivery of the Y2 receptor preferring
agonist NPY13-36 reduces seizure susceptibility following systemic kainic acid
administration.18,19 These data suggest NPY13-36 would be a good candidate to use in an
116
rAAV vector to treat epilepsy. By using the FIB secretion strategy, we have the
capability of expressing and constitutively secreting peptide fragments which have
demonstrated receptor selectivity. Thus, we evaluated the effects of AAV mediated
expression and constitutive secretion of NPY and the peptide fragment NPY13-36 on
kainic acid induced limbic seizures.
Although previous studies validated the effectiveness of gene expression and
constitutive secretion using galanin, the same approach might not necessarily prove
successful with NPY or the peptide fragment NPY13-36. Therefore, we used a similar
approach with the following modifications: AAV 2 vectors were constructed where the
hybrid chicken beta actin promoter drives expression of the FIB-NPY or FIB-NPY13-36
coding sequences. Then, 2 µl of recombinant AAV-FIB-NPY (3.3 X 1012 viral
particles/ml) or AAV-FIB-NPY13-36 (3.0 X 1012 viral particles/ml) was infused
bilaterally into the piriform cortex of rats, as previously described.1 Control rats received
no infusion, as we have previously shown that vectors expressing and secreting reporter
genes like GFP (AAV-FIB-EGFP), as well as vectors expressing peptides that lack
secretion sequences (AAV-GAL) have no effect on seizure sensitivity, seizure behaviors,
or seizure-induced cell death.1,3 One week later, the rats received a dose of 10 mg/kg, i.p.
kainic acid, and subsequently, the time to limbic seizure behaviors was recorded. As
seen in Figure 1, both AAV treatment groups and the untreated control group developed
wet dog shake behaviors with the same latency. Because the origin of wet dog shakes
appears to be the hippocampus,20 a local action of the AAV-FIB-NPY or AAV-FIB-
NPY13-36 in the piriform cortex would not be expected to influence these kainic acid-
induced behaviors. However, the findings do validate the uniformity of kainic acid
117
administration across the different groups. In marked contrast, the latency to class III or
class IV limbic seizure activity was significantly increased in the AAV-FIB-NPY or
AAV-FIB-NPY13-36 treated groups. In fact, the majority of rats in both vector treated
groups did not exhibit any seizure behavior at all, whereas all rats in the untreated group
develop class III and class IV seizures within 90 minutes (see Figure 1). Thus, like
findings with expression and secretion of galanin, NPY actions in the piriform cortex
significantly attenuated seizure activity induced by the peripheral administration of the
chemical convulsant kainic acid. As previously discussed, the constitutive secretion of
these peptides renders immunohistochemical identification untenable,3 but as previously
shown for galanin,1,3 the appropriate vector-derived NPY or NPY13-36 mRNA was
present in the area of AAV infusion (see Figure 2). Thus, not only does this gene therapy
approach prove effective with NPY, but it is now also possible to express and secrete
active fragments of peptides, allowing for more selective receptor targeting. In addition,
the use of smaller peptide fragments in an AAV context may allow for combinations of
therapeutic peptides to be expressed from a single vector cassette. It is likely that
combination therapy will be the key to treating many complex neurological disorders.
Acknowledgements: We thank Julie Hamra for expert technical assistance. These
studies were supported by NIH grant NINDS NS 35633 to TJM.
118
References:
1 McCown TJ. Adeno-associated virus-mediated expression and constitutive secretion of galanin suppresses limbic seizure activity in vivo. Mol Ther 2006; 14: 63-68.
2 Klugmann M et al. AAV-mediated hippocampal expression of short and long
Homer 1 proteins differentially affect cognition and seizure activity in adult rats. Mol Cell Neurosci 2005; 28: 347-360.
3 Haberman RP, Samulski RJ, McCown TJ. Attenuation of seizures and neuronal
death by adeno-associated virus vector galanin expression and secretion. Nat Med 2003; 9: 1076-1080.
4 Freese A et al. Direct gene transfer into human epileptogenic hippocampal tissue
with an adeno-associated virus vector: implications for a gene therapy approach to epilepsy. Epilepsia 1997; 38: 759-766.
5 Vezzani A et al. Seizure susceptibility and epileptogenesis are decreased in
transgenic rats overexpressing neuropeptide Y. Neuroscience 2002; 110: 237-243. 6 Richichi C et al. Anticonvulsant and antiepileptogenic effects mediated by adeno-
associated virus vector neuropeptide Y expression in the rat hippocampus. J Neurosci 2004; 24: 3051-3059.
7 Lin EJ et al. Recombinant AAV-mediated expression of galanin in rat
hippocampus suppresses seizure development. Eur J Neurosci 2003; 18: 2087-2092.
8 Colmers WF et al. Presynaptic inhibition by neuropeptide Y in rat hippocampal
slice in vitro is mediated by a Y2 receptor. Br J Pharmacol 1991; 102: 41-44. 9 Dumont Y et al. Potent and selective tools to investigate neuropeptide Y receptors
in the central and peripheral nervous systems: BIB03304 (Y1) and CGP71683A (Y5). Can J Physiol Pharmacol 2000; 78: 116-125.
10 Dumont Y et al. BIIE0246, a potent and highly selective non-peptide
neuropeptide Y Y(2) receptor antagonist. Br J Pharmacol 2000; 129: 1075-1088. 11 Greber S, Schwarzer C, Sperk G. Neuropeptide Y inhibits potassium-stimulated
glutamate release through Y2 receptors in rat hippocampal slices in vitro. Br J Pharmacol 1994; 113: 737-740.
12 Lin EJ et al. Differential actions of NPY on seizure modulation via Y1 and Y2
receptors: evidence from receptor knockout mice. Epilepsia 2006; 47: 773-780.
119
13 El Bahh B et al. The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y and not Y receptors. Eur J Neurosci 2005; 22: 1417-1430.
14 El Bahh B, Cao JQ, Beck-Sickinger AG, Colmers WF. Blockade of neuropeptide
Y(2) receptors and suppression of NPY's anti-epileptic actions in the rat hippocampal slice by BIIE0246. Br J Pharmacol 2002; 136: 502-509.
15 Furtinger S et al. Plasticity of Y1 and Y2 receptors and neuropeptide Y fibers in
patients with temporal lobe epilepsy. J Neurosci 2001; 21: 5804-5812. 16 Kofler N, Kirchmair E, Schwarzer C, Sperk G. Altered expression of NPY-Y1
receptors in kainic acid induced epilepsy in rats. Neurosci Lett 1997; 230: 129-132.
17 Schwarzer C, Kofler N, Sperk G. Up-regulation of neuropeptide Y-Y2 receptors
in an animal model of temporal lobe epilepsy. Mol Pharmacol 1998; 53: 6-13. 18 Vezzani A et al. Plastic changes in neuropeptide Y receptor subtypes in
experimental models of limbic seizures. Epilepsia 2000; 41 Suppl 6: S115-121. 19 Vezzani A, Rizzi M, Conti M, Samanin R. Modulatory role of neuropeptides in
seizures induced in rats by stimulation of glutamate receptors. J Nutr 2000; 130: 1046S-1048S.
20 Frush DP, McNamara JO. Evidence implicating dentate granule cells in wet dog
shakes produced by kindling stimulations of entorhinal cortex. Exp Neurol 1986; 92: 102-113.
21 Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor
seizure. Electroencephalogr Clin Neurophysiol 1972; 32: 281-294.
120
FIGURES AND LEGENDS:
Figure 1 – The effects of AAV-FIB-NPY and AAV-FIB-NPY13-36 vectors on the expression of limbic seizure behaviors. Rats received bilateral infusions of AAV-FIB-NPY (2µl/20 minutes; 3.3 X 1012 viral particles/ml) (N=7) or AAV-FIB-NPY13-36 (2µl/20 minutes; 3.0 X 1012 viral particles/ml) (N=7) into the piriform cortex as previously described.1 Seven days later both vector treated groups and an untreated control group (N=8) received an i.p. injection of kainic acid (10 mg/kg). The latencies to limbic seizure behaviors were determined for 240 minutes post-kainic acid. Seizures were scored according to the Racine motor seizure grading scale.21 Briefly, class I seizures were scored when rats exhibited facial twitches and chewing, class II when head nodding, class III when contralateral forelimb clonus was observed, and class IV when bilateral forelimb clonus and rearing was observed. All groups developed wet dog shakes with the same latencies, indicating the uniformity of kainic acid administration across the different groups. In marked contrast, however, onset of class III and class IV seizures was significantly delayed or completely blocked in rats receiving either AAV-FIB-NPY or AAV-FIB-NPY13-36 compared to control. (* t-test, p<0.01). In fact, only 3/7 AAV-FIB-NPY treated rats and 1/7 AAV-FIB-NPY13-36 treated rats developed any class III or class IV seizure behaviors over the entire 240 minute observation period. All 8 rats in the untreated control group developed class III and class IV seizures within 90 minutes.
121
Figure 2 – The in vivo presence of FIB-NPY and FIB-NPY13-36 mRNA one week after vector infusion into the piriform cortex. The appropriate 208 bp FIB-NPY (lane A) or 172 bp FIB-NPY13-36 (lane C) product is present in the injected piriform cortex while no product was found in an area slightly distal to the piriform cortex (lane B). Omission of the RT step (lanes D and E) indicated the absence of contaminating viral DNA. The left outside lane contains a 100 base pair DNA step ladder (Invitrogen) with the size indicated on the left. Rats received bilateral infusions of AAV-FIB-NPY (2µl/20 minutes; 3.3 X 1012 viral particles/ml) (N=2) or AAV-FIB-NPY13-36 (2µl/20 minutes; 3.0 X 1012 viral particles/ml) (N=2) into the piriform cortex as previously described.1 Then, 1 week later, the animals received an overdose of pentobarbital (100 mg/kg, ip) and were subsequently decapitated. The brain was removed, and the piriform cortex was dissected out. The tissue was stored in RNAlater (Ambion, Austin, TX, USA) at -200C. Subsequently, the RNA was extracted from the tissue (Promega SV-40 total RNA isolation kit; Madison, WI, USA) and reverse transcribed using AMV reverse transcriptase and oligo (dT) primers. The subsequent PCR primers were designed to span the FIB-NPY (and thus the FIB-NPY13-36) sequence, which can only be derived from the rAAV vector (FIB, 5’- CTAGCAGTCCTGTGCCTG; NPY and NPY13-36, 3’-GCTCAATATCTCTGTCTGGTG).
122
Appendix D.2. Viral Vector Gene Therapy
Viral Vector Gene Therapy for Epilepsy
Stacey B. Foti1, Shelley J. Russek2, Amy R. Brooks-Kayal3, and
Thomas J. McCown4
Program in Neurobiology, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina1, Department of Pharmacology and Experimental Therapeutics, Boston
University School of Medicine, Boston, Massachusetts2, Division of Neurology, Pediatric
Regional Epilepsy Program, Children’s Hospital of Philadelphia, Philadelphia,
Pennsylvania3, University of North Carolina Gene Therapy Center, University of North
Carolina School of Medicine, Chapel Hill, North Carolina4.
Address Correspondence to :
Thomas J. McCown, Ph.D. UNC Gene Therapy Center 7119 Thurston, CB 7342 University of North Carolina School of Medicine Chapel Hill, NC 27599
123
About 2.1 million people in the United States have epilepsy (Hirtz, Thurman et al.
2007), and with a world wide prevalence of approximately 1 percent (Hauser et al.,
1990), epilepsy represents one of the most common neurological diseases. Not only can
epilepsy be debilitating for patients, it also exacts a significant economic toll. In the
United States the estimated annual economic cost of epilepsy is 12.5 billion dollars, a
figure that includes both the direct cost of treatment and the indirect costs such as lost
productivity and wages (Shafer and Begley 2000). Unfortunately, even with new AEDs
available, the number of drug resistant epilepsies has not decreased (Loscher and Leppik
2002). In fact, 30–40% of all patients with epilepsy have medically intractable seizures,
and only half of these patients are candidates for surgery (Shafer, et al. 1988; Engel et al.
1992; Sander 1993; Siegel 2004). Of those patients that undergo temporal lobe surgery,
20% still have seizures post-op (Spencer et al. 1984), and surgical complications along
with post-operative memory and language deficits can occur (Siegel 2004). For all these
reasons, improving epilepsy treatment remains a priority.
Theoretically, a gene therapy approach to seizure suppression is an attractive
alternative treatment option for focal epilepsy, the most common adult form (Semah et al.
1998). Focal epilepsy arises from a circumscribed, hyperexcitable network of neurons
whose synchronous electrical discharge creates seizure activity. To date, most gene
therapy studies have employed viral vectors based upon adenovirus (AD), adeno-
associated virus (AAV), human immunodeficiency virus (HIV) or herpes simplex virus
(HSV). In all cases, these viral vectors have proven capable of transducing post-mitotic
neurons which provides direct access to the cells responsible for generating and
propagating the seizure activity. Furthermore, these viral vectors have proven capable of
124
influencing brain function on a regional basis, so viral vector gene therapy should have
the ability to influence sites of seizure genesis. However, even with the promising results
obtained using viral vectors (see table 1), a number of factors must be considered when
modeling a gene therapy for the treatment of epilepsy.
Identifying Potential Therapeutic Targets
In general, epilepsy arises from a loss of inhibition, an enhanced state of
excitation or a combination of both processes, so given this wide range of influences, a
wealth of potential therapeutic targets exists. On the most basic level, altering receptor or
ion channel mediated function clearly has been shown to attenuate seizure activity. For
example, antiepileptic drugs (AEDs) exert their actions by (1) increasing GABAergic
inhibition, (2) decreasing excitatory glutamatergic excitation (3) decreasing voltage-
dependant calcium release or (4) decreasing voltage-dependant Na+ conductance (Ure and
Perassolo 2000). Because AD, AAV, HSV-1 and HSV vectors exhibit a clear
neurotropism, any of these targets are readily accessible by these viral vectors. In
addition, basic research has identified a number of other modulatory factors that likewise
can attenuate seizure activity in vivo. Prominent among this class are the neuropeptides,
such as galanin and neuropeptide Y. Although this extensive list of potential targets
would seem to offer many opportunities, the major difficulty is establishing a strategy
that is both effective and non-toxic.
Excitatory Amino Acid Receptors
125
Excitatory amino acid receptors comprise an obvious target for anti-epileptic gene
therapy, and certainly many studies have shown that N-methyl-D-aspartic acid (NMDA)
receptors influence seizure sensitivity. Because a fully functional receptor requires the
NMDA receptor 1 protein (NMDAR1), removal of this subunit protein should
significantly reduce NMDAR1 mediated excitation (Monyer et al., 1992; Hollmann and
Heinmann, 1994). Haberman et al. (2002) showed that AAV vector-mediated delivery of
an antisense RNA specific to the NMDAR1 could indeed reduce NMDA receptor
function in vitro and NMDAR1 protein in vivo. Using a focal seizure model, CMV
promoter driven expression of the NMDAR1 antisense significantly decreased the seizure
susceptibility, proving the principle that viral vector derived antisense could influence
native receptor function in vivo. However, when expression of the same antisense
construct was driven by a tetracycline regulated minimal CMV promoter (Haberman et
al., 1998), the seizure susceptibility actually increased. The fact that NMDA receptor
mediated excitation can drive endogenous GABA inhibition provided a tenable
explanation for these diametrically opposed results. In the case of the CMV promoter,
excitatory principal neurons likely comprised the preponderance of transduced cells, thus
removal of NMDA receptor excitation blunted the seizure susceptibility. In the case of
the regulated minimal CMV promoter, the preponderance of gene expression probably
occurred in inhibitory GABAergic interneurons, so removal of the endogenous excitatory
drive to these interneurons would cause a state of increased seizure sensitivity. The
likelihood of such an occurrence was validated, when equal amounts of each AAV vector
were administered together using different reporter genes. Some neurons supported
126
expression from both promoters, but significant portions exhibited transduction from only
one or the other of the promoters. Thus, a slight change in only the promoter resulted in a
dramatic shift in the final outcome of the gene expression. These surprising findings
greatly complicate the targeting of neurotransmitter receptor proteins or ion channels for
the treatment of epilepsy. Certainly, it is possible that the viral vector tropism and/or
promoter tropism could differ between experimental animals and humans. Thus, without
some detailed a priori knowledge of the transduction pattern in humans, any
manipulation of neurotransmitter receptor proteins or ion channels could lead either to the
desired reduction of seizure susceptibility or a paradoxical heightening of seizure
susceptibility.
Inhibitory GABA Receptors
GABA is the major inhibitory neurotransmitter in the adult brain. Most fast
synaptic inhibition in the mature brain is mediated by GABAA receptors (GABRs),
pentameric anion-selective channels composed of multiple subunit subtypes (α1-6, β1-3,
γ1-3, δ, ε, π, θ and ρ1-3). GABAergic signaling has long been hypothesized to play an
important role in the genesis of epilepsy, and many of the oldest and most commonly
used anticonvulsants such as benzodiazepines and barbiturates act, at least in part,
through augmentation of GABR activity. Mutations in several different GABR subunits
have been identified in families with generalized epilepsies (Macdonald et al., 2004). In
addition, several laboratories have identified changes in GABR function and subunit
composition in human temporal lobe epilepsy (TLE) and in animal models of TLE (Buhl
127
et al., 1996; Gibbs et al., 1997; Brooks-Kayal et al., 1998, 1999). Studies in humans with
temporal lobe epilepsy (TLE) and in adult rodent models of TLE have found reduced
expression of GABR α1 subunit gene and increased expression of GABR α4 subunit
gene in the dentate gyrus (DG)(Brooks-Kayal et al., 1998,1999; Peng et al., 2004; Zhang
et al., 2007). These findings suggest that diminished α1 levels in dentate granule neurons
(DGN) may contribute to epileptogenesis and/or that elevated α1 levels may be
protective. Certainly, reduction of GABR α1 subunit protein using an AAV that
expressed a GABR α1 antisense RNA significantly increased focal seizure susceptibility
(Xiao et al., 1997).
To directly test the hypothesis that the expression of higher α1 subunit levels
inhibits development of epilepsy after SE, an adeno-associated virus gene transfer vector
(AAV) serotype 5 was designed to express a bicistronic RNA that codes for both the
GABR α1 subunit as well as the reporter, enhanced yellow fluorescence protein (eYFP)
(Raol et al., 2006). Expression of this RNA was placed under control of the GABR α4
core promoter region, because it was previously shown to be markedly activated in DG
following SE (Roberts et al., 2005). AAV-vectors containing either the α1/eYFP fused
cDNA (AAV-α1) or the eYFP-reporter only (AAV-eYFP) were injected into DG of adult
rats, and SE was induced two weeks later by intraperitoneal injection of pilocarpine (385
mg/kg). Rats injected with AAV-α1 showed 3-fold higher levels of α1 subunits in DG
by 2 weeks after SE compared to the control groups. AAV-α1 injection resulted in a 3-
fold increase in the mean time to the first spontaneous seizure following SE, and only
39% of AAV-α1 injected rats were observed to develop spontaneous seizures in the first
4 weeks after SE, as compared to 100% of rats receiving sham-injections. Because all
128
groups of rats experienced similar SE after pilocarpine injection, these findings provide
the first direct evidence that increasing the levels of a single GABR subunit in DG can
inhibit the development of spontaneous seizures after SE.
Another unique aspect of this particular study involves the use of a condition-
specific promoter which when upregulated during a disease process, drives expression of
a therapeutic gene. In this case, the upregulation of the GABR α4 promoter drove
expression of the α1 transgene which enhanced α1 expression in DG. Unfortunately, this
vector-derived expression lasted for only the first two weeks after SE. To differentiate
whether the decline in α1 subunit levels by 4 weeks after SE was due to loss of
recombinant or endogenous α1 subunits, mRNA levels for eYFP (produced from the
vector containing the fused α1/eYFP cDNAs) were assayed in AAV-injected rats
sacrificed at 1 or 4 weeks after SE. With presence of eYFP mRNA as a marker for co-
presence of α1, recombinant α1 mRNA levels were found to decline over 6-fold in AAV
injected rats between 1 and 4 weeks after SE. This finding suggests that either the
GABRA4 promoter in the AAV vector was transcriptionally downregulated or that the
recombinant α1/eYFP mRNAs were degraded. A number of studies have demonstrated
robust, long-term gene expression in the hippocampus after AAV transduction (Klein et
al., 1998, Burger et al., 2004), but this stable expression is promoter dependent. For
example, McCown et al. (1996) showed that when gene expression was driven by a CMV
promoter, the initial level of AAV mediated gene expression in the hippocampus declined
over a period of 4 weeks whereas some other areas of brain exhibited no decline in gene
expression. Further studies will be required to define the precise mechanism responsible
for the proposed decrease in activity of the recombinant GABRA4 promoter over time
129
and to rule out whether a change in mRNA stability may also play a role in declining
levels of α1/eYFP transcript.
In addition to the effects on seizures, AAV-mediated elevation of α1 expression
was associated with a behavioral phenotype in a fraction of the AAV-α1 injected rats.
Most of the AAV-α1 injected rats behaved similar to sham-injected rats, but 30%
exhibited abnormal behaviors including excessive sedation, anorexia, and weight loss that
persisted for days to weeks following SE. This effect was not seen after SE in any of the
other groups (including the AAV-eYFP control), suggesting that the effect likely resulted
from elevated α1 levels rather than the effects of AAV injection or SE. Such effects are
not surprising given that α1-containing receptors are the major form of GABRs
mediating sedation in the nervous system (Mohler et al., 2002).
Genetic engineering approaches have also been used to augment other aspects of
GABAergic neurotransmission. Specifically, two groups have transplanted immortalized
neurons genetically engineered to stably express the GABA synthesizing enzyme
glutamic acid decarboxylase (GAD65 or GAD67) into different brain regions and
examined the effects on induced and spontaneous seizures. Transplant of these
conditionally immortalized neurons engineered to produce GABA into either substantia
nigra (SN) or piriform cortex of rats has been found to inhibit kindling (Thompson et al.,
2000; Gernert et al., 2002) and reduce the number and severity of kainic acid-induced
seizures (Castillo et al., 2006). Transplantation of GABA-producing neurons into SN
also has been shown to reduce spontaneous seizures in a TLE model (Thompson and
Suchomelova, 2004). Injection of GABA-producing neurons under the control of
tetracycline injected into SN 45-65 days following lithium-pilocarpine induced status
130
epilepticus reduced the number of spontaneous seizures and epileptiform discharges at 7-
8 days after transplantation compared to animals that received control cells or that
received the GABA-producing cells plus doxycycline to inhibit GABA production
(Thompson and Suchomelova, 2004). Similarly, transplantation of GABA-producing
cells into dentate gyrus raised GABA tissue concentrations, increased the afterdischarge
threshold, shortened the afterdischarge duration and prolonged the latency to expression
of the first stage 5 seizure in a hippocampal kindling model (Thompson, 2005). Although
the engineered cells show evidence of integration, this cell transplantation approach
remains hampered by the limited long-term survival of the transplanted cells (Thompson
and Suchomelova, 2004).
Taken together, it is clear that by controlling GABAergic signaling either through
discrete regulation of its receptor isoforms or through accessibility to its neurotransmitter
GABA, it may be possible in the future to treat a multitude of diseases that stem from an
imbalance between excitatory and inhibitory neurotransmission. The challenge remains
to develop better vehicles and approaches for the selective delivery of gene products over
time in a therapeutically useful manner.
Diverse Unrelated Targets
A number of diverse, unrelated targets also have emerged from basic research that
focused upon neuroprotection, not epilepsy. Glial cell line-derived neurotrophic factor
(GDNF) exerts a potent neuroprotective action in the CNS, especially on dopamine
containing neurons (Choi-Lundberg er al., 1997; Mandel et al., 1997), and when infused
131
intraventricularly, GDNF attenuates kainic acid-induced seizures (Martin et al., 1995).
Using viral vectors, Yoo et al. (2006) subsequently showed that AD mediated GDNF
expression in the hippocampus attenuated behavioral seizures and cell death induced by
peripheral kainic acid administration. Similarly, Kanter-Schlifke et al. (2007A) used an
AAV vector to overexpress glial cell line-derived neurotrophic factor (GDNF) in the
hippocampus and found that this overexpression reduced the seizure severity in both
hippocampal kindling, as well as kindled self-sustained status epilepticus. Thus, viral
vector-mediated expression of GDNF can influence seizure sensitivity. Another anti-
apoptotic agent was identified by Perkins et al., (2003) who reported that the herpes
simplex virus type 2 R1 protein kinase (ICP10 PK) exerted an antiapoptotic action in
hippocampal cultures. Based upon these findings, Laing et al. (2005) showed that nasal
inoculation with a herpes simplex virus type 2 that contains the anti-apoptotic gene
ICP10PK prevented both behavioral seizures and neuronal death elicited by peripheral
kainic acid administration. Thus, it appears that at least in these two instances, gene
products with neuroprotective actions also can significantly influence seizure sensitivity.
A second group of therapeutic targets have emerged from diverse modulators of
synaptic excitation. The protein homer 1a is induced by excessive excitation and is
thought not only to modulate synaptic plasticity, but also to reduce postsynaptic calcium
function (Ango et al., 2001). Using this information, Klugman et al. (2005) found that
AAV-mediated overexpression of Homer 1a attenuated electrographic seizure activity in
a hippocampal status epilepticus model. Another means to reduce excitation would be to
inhibit the efficacy of synaptic transmission. Clostridial toxin light chain digests the
vesicle docking protein, synaptobrevin, which results in impaired synaptic transmission,
132
so Yang et al (2007) used AD vectors to express this protein in rat cortex. AD-mediated
expression of this protein attenuated both electrographic and behavioral seizures elicited
by subsequent penicillin injection into the cortex. Finally, a number of studies have
established that adenosine potently inhibits neuronal activity (Dunwiddie and Masino,
2001), so Huber et al., (2001) engineered fibroblasts to secrete adenosine. When these
cells were implanted into the ventricles of rats, both behavioral and electrographic
seizures were attenuated in electrically kindled rats. Although these studies established
the anti-seizure potency of adenosine in vivo, the effect waned significantly 24 days post-
transplantation. In total, these studies illustrate the wide range of targets by which
various gene products can reduce neuronal excitation by in vivo viral gene therapy.
Neuroactive Peptides
Like other gene therapy studies, previous pharmacological findings with
neuroactive peptides have provided a strong rationale for application in anti-epileptic
gene therapy. Of the many neuropeptides that can modulate neuronal excitability,
galanin and neuropeptide Y have been most thoroughly characterized. For example, the
neuroactive peptides, galanin and neuropeptide Y (NPY), both attenuate seizure activity
in vivo (Woldbye et al.,1997; Mazzarati et al.,1998.). Key to any neuropeptide gene
therapy, though, is the consideration of endogenous neuropeptide function. Most
neuroactive peptides are expressed initially as prepro-peptides, a form that presumably
contains the appropriate information for trafficking to the pre-synaptic terminal and
packaging into vesicles. Thus, if a viral vector expresses a prepro-neuropeptide
133
sequence, one must assume that the transduced cells indeed contain the appropriate
pathways for trafficking and release. Whether determined by the vector or the promoter,
the actual tropism of viral vectors is unknown in the human CNS, so as demonstrated for
NMDA receptor gene therapy, successful neuropeptide gene therapy might require
transduction and gene expression in the appropriate neuronal population. Given these
potential pitfalls, Haberman et al. (2003) devised a novel approach to peptide gene
therapy focusing upon galanin. The laminar protein, fibronectin occurs in most cell types
and normally is secreted in a constitutive manner. This constitutive secretion is
determined by the secretory signal sequence for fibronectin. Using AAV vectors
Haberman et al. (2003) combined the secretory signal sequence for the laminar protein
fibronectin with coding sequence for the active galanin neuropeptide (AAV-FIB-GAL).
By attaching this secretory signal sequence to the coding sequence for the active galanin
peptide, it was reasoned that the viral vector mediated expression would lead to the
constitutive secretion of active galanin. Thus, if the appropriate receptors exist in the
area of transduction, different patterns of neuronal transduction should not adversely
influence the outcome. In fact these investigators showed that indeed AAV-FIB-GAL
supported constitutive secretion of galanin in vitro, and in vivo sufficient galanin was
secreted to significantly attenuate focal seizure activity and prevent kainic acid-induced
neuronal cell death in the hippocampus. If galanin was expressed in the absence of the
FIB, or GFP was constitutively secreted, no effects were found on focal seizure
sensitivity or cell death in the hippocampus. Using this gene therapy approach,
subsequent studies found that bilateral infusion of AAV-FIB-GAL into the piriform
cortex prevented both electrographic and behavioral seizure activity induced by
134
peripheral kainic acid administration and significantly elevated the seizure initiation
threshold in previously kindled rats (McCown, 2006). In contrast, Lin et al. (2003) used
AAV vectors to express human galanin in the hippocampus. Even though these studies
achieved much higher levels of vector derived gene expression in comparison to
Haberman et al. (2003), the effects upon local infusion of kainic acid were restricted to a
decrease in the number of seizures, not the seizure latency. Furthermore, no neuronal
protection was evident. When these investigators further explored the effects of this
approach, they found that galanin overexpression did not alter the course of electrical
hippocampal kindling or modulate short term plasticity of mossy fiber CA3 synapses.
Thus, just because high levels of a peptide are expressed in vivo, such expression does
not necessarily lead to an increase in the peptide’s in vivo actions.
Neuropeptide Y (NPY), like galanin, exhibits anti-seizure activity in vivo
(Woldbye et al., 1997; Mazarati and Wasterlain, 2002), and a recent set of studies by
Richichi et al. (2004) have shown that AAV-mediated expression of prepro-NPY exerts a
range of anti-seizure effects. Expression of a human prepro-NPY gene prevented the
appearance of status epilepticus after intracerebroventricular administration of kainic
acid. Also, hippocampal expression of prepro-NPY increased the electrical seizure
threshold in the hippocampus and significantly retarded the rate of kindling
epileptogenesis. Another important facet of these studies involved the exploitation of
different AAV serotypes. A hybrid AAV1/2 serotype transduced a substantially wider
range of neurons, when compared to the AAV2 serotype, and this increased transduction
135
translated to a greater effect of prepro-NPY expression. Thus, these studies clearly
established the anti-epileptic potential for vector derived prepro-NPY expression in vivo.
A subsequent study by Foti et al. (2007) utilized the constitutive secretion
approach to demonstrate not only the efficacy of vector derived NPY, but also , the
efficacy of the NPY receptor specific fragment NPY(13-36). Although some question
remains as to which of the NPY receptors (Y1-Y5) mediates the anti-seizure activity,
several studies have suggested a critical role for the Y2 receptor in mediating anti-
epileptic actions (Colmers et al., 1991; Greber et al., 1994; El Bahn et al., 2002, 2005;
Lin et al, 2006). Because acute intracerebral delivery of the Y2 receptor preferring
agonist NPY13-36 reduces seizure susceptibility following systemic kainic acid
administration (Vezzani et al., 2000), Foti et al. (2007) used an AAV vector to express
and constitutively secret NPY or NPY(13-36) in the rat piriform. Similar to previous
results with galanin (McCown, 2006), expression of either NPY or NPY(13-36)
significantly suppressed limbic seizures elicited by subsequent peripheral kainic acid
administration. Thus, these studies showed that the constitutive secretory approach can
effectively express and secrete receptor specific peptide fragments in vivo.
Critical Considerations and Future Directions:
The wide range of gene therapy studies amply illustrate the potential of viral
vector gene therapy as an effective treatment modality for epilepsy, but even with these
positive findings, there are a number of considerations that will inevitably impact both
the efficacy and the safety of this therapeutic approach. Although many viral vectors
136
exhibit a neuronal tropism in vivo, one must always take into account how specific
patterns of transduction might alter the outcome of therapeutic gene expression. Viral
vector tropism aside, as shown previously (Haberman et al., 2002) different promoters
can lead to differential gene expression even within the context of the same viral vector.
Because such a change can result in an unwanted outcome, one first must consider the
need to transduce a specific cellular population and the consequences if such specificity
is not achieved. One obvious solution involves the use of cell specific promoters, such as
that used by Raol et al. (2006) where expression would only be achieved in those cells
that contain GABR α4 promoter activity. However, as also seen by Raol et al. (2006)
endogenous promoters may be susceptible to promoter silencing. Secondly, the most
likely clinical population for gene therapy encompasses those individuals with intractable
temporal lobe epilepsy who are surgical candidates. In such a case, these individuals will
have extensive seizure histories and inevitably varying degrees of hippocampal sclerosis.
At present, most viral vector studies have examined the ability to transduce normal tissue.
Thus, a second consideration is whether the viral vectors will support sufficient neuronal
transduction in epileptic tissue with its associated pathology. Finally, it is incumbent to
demonstrate that the proposed gene therapy can prevent spontaneous seizure activity.
Thus, to best model an effective gene therapy, viral vector transduction should be
initiated in animals that exhibit spontaneous seizures. As studies focus upon these
aspects, it is likely that an effective anti-epileptic gene therapy will emerge.
137
References
Ango, R., Prezeau, L., Muller, T., Tu, J.C., Xiao, B., Worley, P.F., Pin, J.P., Bockaert, J., Fagni, L. (2001) Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer. Nature 411:962-965.
Brooks-Kayal, A.R., Shumate, M.D., Jin, H., Rikhter, T.Y., Coulter, D.A. (1998)
Selective changes in single cell GABAA receptor subunit expression and function in temporal lobe epilepsy. Nat. Med. 4:1166-1172.
Brooks-Kayal, A.R., Shumate, M.D., Jin, H., Lin, D.D., Rikhter, T.Y., Holloway, K.L.,
Coulter, D.A. (1999) Human neuronal γ-aminobutyric acidA receptors: coordinated subunit mRNA expression and functional correlates in individual dentate granule cells. J. Neurosci. 19:8312-8318.
Buhl, E., Otis, T., Mody, I. (1996) Zinc-induced collapse of augmented inhibition by
GABA in a temporal lobe epilepsy model. Science 271:369-373. Burger, C., Gorbatyuk, O.S., Velardo, M.J., Peden, C.S., Williams, P., Zolotukhin, S.,
Reir, P.J., Mandel, R.J., Muzyczka, N. (2004) Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2 and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther. 10:302-317.
Castillo, C.G.., Mendoza, S., Freed, W.J., Giordano, M. (2006) Intranigral transplants of
immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat. Behav. Brain Res. 171: 109-115.
Choi-Lundberg, D.L., Lin, Q., Chang, Y.N., Hay, C.M., Mohajeri, H., Davidson, B.L.,
Bohn, M.C. (1997) Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 275:838-841.
Colmers, W.F., Klapstein, G.J., Fournier, A., St-Pierre, S., Treherne, K.A. (1991)
Presynaptic inhibition by neuropeptide Y in rat hippocampal slice in vitro is mediated by a Y2 receptor. Br. J. Pharmacol. 102: 41-44.
Dunwiddie, R.V., Masino, S.A. (2001) The role and regulation of adenosine in the central
nervous system. Annu. Rev. Neurosci. 24:31-55. El Bahh, B., Balosso, S., Hamilton, T., Herzog, H., Beck-Sickinger, A.G., Sperk, G,
Gehlert, D.R., Vezzani, A., Colmers, W.F. (2005) The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y and not Y receptors. Eur. J. Neurosci. 22: 1417-1430.
138
El Bahh B, Cao JQ, Beck-Sickinger AG, Colmers WF. (2002) Blockade of neuropeptide Y(2) receptors and suppression of NPY's anti-epileptic actions in the rat hippocampal slice by BIIE0246. Br. J. Pharmacol. 136: 502-509.
Engel, J., Jr., M. F. Levesque, M.F., Shields, W.D. (1992). Surgical treatment of the
epilepsies: presurgical evaluation. Clin. Neurosurg. 38: 514-34. Foti, S., Haberman, R.P., Samulski, R.J., McCown, T.J. (2007) Adeno-associated virus-
mediated expression and constitutive secretion of NPY or NPY13-36 suppresses seizure activity in vivo. Gene Ther. 14:1534-1536.
Gernert, M., Thompson, K.W., Loscher, W., Tobin, A.J. (2002) Genetically engineered
GABA-producing cells demonstrate anticonvulsant effects and long-term transgene expression when transplanted into the central piriform cortex of rats. Exp. Neurol. 176: 183-192.
Gibbs, J.W. 3rd, Shumate, M.D., Coulter, D.A. (1997) Differential epilepsy-associated
alterations in postsynaptic GABAA receptor function in dentate granule cells and CA1 neurons. J. Neurophysiol. 77:1924-1938.
Greber, S., Schwarzer, C., Sperk, G.. (1994) Neuropeptide Y inhibits potassium-
stimulated glutamate release through Y2 receptors in rat hippocampal slices in vitro. Br. J. Pharmacol. 113: 737-740.
Guttinger, M., Fedele, D., Koch, P., Padrun, V., Pralong, W.F., Brustle, O., Boison, D.
(2005) Suppression of kindled seizures by paracrine adenosine release from stem cell-derived brain implants. Epilepsia 46:1162-1169.
Haberman, R.P., McCown, T.J., Samulski, R.J. (1998) Inducible long-term gene
expression in brain with adeno-associated virus gene transfer. Gene Ther 5:1604-1611.
Haberman, R.P., Criswell, H.E., Snowdy, S., Ming, Z., Breese, G.R., Samulski, R.J.,
McCown, T.J. (2002) Therapeutic Liabilities of In Vivo Viral Vector Tropism: Adeno-associated Virus (AAV) Vectors, NMDAR 1 Antisense and Focal Seizure Sensitivity . Mol Ther 6:495-500.
Haberman, R.P., Samulski, R.J., McCown, T.J. (2003) Attenuation of seizures and
neuronal death by adeno-associated virus (AAV) vector galanin expression and secretion. Nature Med 9:1076-1080.
Hauser, W. A., Hesdorffer, D.C. (1990). Epilepsy frequency, causes, and consequences.
Landover, MD; New York, NY, Demos Publications, New York.
139
Hirtz, D., Thurman, D.J., Gwinn-Hardy, K., Mohamed, M., Chaudhuri, A.R., Zalutsky, R. (2007). "How common are the "common" neurologic disorders?" Neurology 68(5): 326-37.
Hollmann, M., Heinemann, S. (1994) Cloned glutamate receptors. Ann. Rev. Neurosci.
17: 31-108. Huber, A., Padrun, V., Deglon, N., Aebischer, P., Mohler, H., Boison, D. (2001) Grafts
of adenosine-releasing cells suppress seizures in kindling epilepsy. Proc. Nat. Acad. Sci. USA 98:7611-7616.
Kanter-Schlifke, I., Georgievska, B., Kirik, D., Kokais, M. (2007A) Seizure suppression
by GDNF gene therapy in animal models of epilepsy. Mol. Ther. 15:1106-1113,2007.
Kanter-Schlifke, I., Sorensen, A.T., Ledri, M., Kuteeva, E., Hokfelt, R., Kokaia, M.
(2007B) Galanin gene transfer curtails generalized seizures in kindled rats without altering hippocampal synaptic plasticity. Neurosci. 150:984-992.
Klein, R.L., Meyer, E.M., Peel, A.L., Zolotukhin, S., Meyers, C., Muzyczka, N., King,
M.A. (1998) Neuron-specific transduction in the rat septohippocampal or nigrostriatal pathway by recombinant adeno-associated virus vectors. Exp. Neurol. 150:183-194.
Klugman, M., Symes, C.W., Leichtlein, C.B., Klaussner, B.K., Dunning, J., Fong, D.,
Young, D., During, M.J. (2005) AAV-mediated hippocampal expression of short and long Homer 1 proteins differentially affect cognition and seizure activity in adult rats. Mol. Cell. Neurosci. 28:347-360.
Laing, J.M., gober, M.D., Golembewski, E.K., Thompson, S.M., Gyure, K.A., Yarowsky,
P.J., Aurelian, L. (2006) Intranasal administration of the growth-compromised HSV-2 vector DeltaRR prevents kainate-induced seizure and neuronal loss in rats and mice. Mol. Ther. 13:870-881.
Lin, E.D., Richichi, C., Young, D., Baer, K., Vezzani, A., During, M.J. (2003)
Recombinant AAV-mediated expression of galanin in rat hippocampus suppresses seizure development. J. Neurosci. 18:2087-2003.
Lin, E.J., Young, D., Baer, K., Herzog, H., During, M.J. (2006) Differential actions of
NPY on seizure modulation via Y1 and Y2 receptors: evidence from receptor knockout mice. Epilepsia 47: 773-780.
Loscher, W., Leppik, I.E. (2002). Critical re-evaluation of previous preclinical strategies
for the discovery and the development of new antiepileptic drugs. Epilepsy Res. 50:17-20.
140
Macdonald, R.L., Gallagher, M.J., Feng, H.J., Kang, J. (2004) GABA(A) receptor epilepsy mutations. Biochem. Pharmacol. 68:1497-506.
Mandel, R.J., Spratt, S.K., Snyder, R.O., Leff, S.E. (1997) Midbrain injection of
recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson's disease in rats. Proc. Nat. Acad. Sci. 94:14083-14088.
Martin, D., Miller, G., Rosendahl, M., Russell, D.A. (1995) Potent inhibitory effects of
glial derived neurotrophic factor against kainic acid mediated seizures in the rat. Brain Res. 683:172-178.
Mazarati, A.M., Lie, H., Soomets, U., Sankar, R., Shin, D., Katsumori, H., Langel, U.,
Wasterlain, C.G. (1998) Galanin modulation of seizures and seizure modulation of hippocampal galanin in animal models of status epilepticus. J. Neurosci. 18:10070-10077.
McCown, T.J., Xiao, X., Li, J., Breese, G.R., Samulski, R.J. (1996) Differential and
persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector. Brain Res. 713:99-107.
McCown, T. J. (2006) Adeno-associated virus-mediated expression and constitutive
secretion of galanin suppresses limbic seizure activity in vivo. Mol. Ther. 14:63-68.
Mohler, H., Fritschy, J.M., Rudolph, U. (2002) A new benzodiazepine pharmacology. J.
Pharmacol, Exp, Ther, 300:2-8. Monyer, H., Sprengel, R., Schoepfer, R., Herb, A., Higuchi, M., Lomeli, H., Burnashev,
N., Sakmann, B., Seeburg, P.H. (1992) Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256:1217-1221.
Peng, Z., Huang, C.S., Stell, B.M., Mody, I. Houser, C.R. (2004) Altered expression of
the delta subunit of the GABAA receptor in a mouse model of temporal lobe epilepsy. J. Neurosci. 24:10167-75.
Perkins, D., Pereira, E.F., Aurelian, L., (2003) The herpes simplex virus type 2 R1
protein kinase (ICP10 PK) functions as a dominant regulator of apoptosis in hippocampal neurons involving activation of the ERK survival pathway and upregulation of the antiapoptotic protein Bag-1. J. Virol. 77:1292-1305.
Raol, Y.H., Lund, I.V., Bandyopadhyay, S., Zhang, G., Roberts, D.S., Wolfe, J.H.,
Russek, S.J., Brooks-Kayal, A.R. (2006) Enhancing GABA(A) receptor α1 subunit levels in hippocampal dentate gyrus inhibits epilepsy development in an animal model of temporal lobe epilepsy. J. Neurosci. 26:11342-11346.
141
Richichi, C., Lin, E.J., Stefanin, D., Colella, D., Ravizza, T., Grignaschi, G., Veglianese,
P., Sperk, G., During, M.J., Vezzani, A. (2004) Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus. J. Neurosci. 24: 3051-3059.
Roberts, D., Raol, Y.S.H., Budrick, E., Lund, I., Passini, M., Wolfe, J., Brooks-Kayal,
A.R., Russek, S.J. (2005) Egr3 stimulation of GABRA4 promoter activity as a mechanism for seizure-induced up-regulation of GABAA receptor alpha4 subunit expression. Proc. Natl. Acad. Sci. U S A 102:11894-11899.
Sander, J. W. (1993) Some aspects of prognosis in the epilepsies: a review. Epilepsia
34:1007-1016. Seki, T., Matsubayashi, H., Amano, T., Kitada, K., Serkawa, T., Sasa, M., Sakai, N.
(2004) Adenoviral gene transfer of aspartoacylase ameliorates tonic convulsions of spontaneously epileptic rats. Neurochem. Int. 45:171-178.
Semah, F., Picot, M.C., Adan, C., Broglin, D., Arzimanoglou, A., Bazin, B., Cavalcanti,
D., Baulac, M. (1998) Is the underlying cause of epilepsy a major prognostic factor for recurrence? Neurology 51:1256-1262.
Shafer, P. O., Begley, C. (2000). The Human and Economic Burden of Epilepsy.
Epilepsy Behav.1:91-92. Shafer, S. Q., Hauser, W.A., Annegers, J.F., Klass, D.W. (1988). EEG and other early
predictors of epilepsy remission: a community study. Epilepsia 29:590-600. Siegel, A. M. (2004). Presurgical evaluation and surgical treatment of medically
refractory epilepsy. Neurosurg. Rev. 27:1-18. Spencer, D. D., Spencer, S.S., Mattson, R.H., Williamson, P.D., Novelly, R.A. (1984).
Access to the posterior medial temporal lobe structures in the surgical treatment of temporal lobe epilepsy. Neurosurgery 15:667-671.
Thompson, K.W. (2005) Genetically engineered cells with regulatable GABA production
can affect afterdischarges and behavioral seizures after transplantation into the dentate gyrus. Neurosci. 133:1029-1037.
Thompson, K.W., Suchomelova, L.M. (2004) Transplants of cells engineered to produce
GABA suppress spontaneous seizures. Epilepsia 45: 4-12. Thompson, K.W., Anantharam, V., Sehrstock, S., Bongarzone, E., Campagnoni, A.,
Tobin, A.J. (2000) Conditionally immortalized cell lines, engineered to produce and release GABA, modulate the development of behavioral seizures. Exp. Neurol. 161:481-489.
142
Ure, J. A., Perassolo, M. (2000) Update on the pathophysiology of the epilepsies. J. Neurol. Sci. 177:1-17.
Woldbye, D.P.D., Larsen, P.J., Mikkelsen, J.D., Klemp, K., Madsen, T.M., Bolwig, T.G.
(1997) Powerful inhibition of kainic acid seizures by neuropeptide Y via Y5-like receptors. Nat. Med. 3:761-764.
Xiao, X., McCown, T.J., Li, J., Breese, G.R., Morrow, A.L.,Samulski, R.J. (1997)
Adeno-associated virus (AAV) vector antisense gene transfer in vivo decreases GABAA α1 containing receptors and increases collicular seizure sensitivity. Brain Res. 576:76-83.
Yang, J., Teng, Q., Fererici, T., Najm, I., Chabardes, S., Moffitt, M., Alexopoulos, A.,
Riley, J., Boulis, N. (2007) Viral clostridial light chain gene-based control of penicillin-induced neocortical seizures. Mol. Ther. 15:542-551.
Yoo, Y.M., Lee, C.J., Lee, U., Kim, Y.J. (2006) Neuroprotection of adenoviral-vector-
mediated GDNF expression against kainic-acid-induced excitotoxicity in the rat hippocampus. Exp. Neurol. 200:407-417.
Zhang N, Wei W, Mody I, Houser CR (2007). Altered localization of GABA(A) receptor
subunits on dentate granule cell dendrites influences tonic and phasic inhibition in a mouse model of epilepsy. J. Neurosci. 27(28):7520-7531.
Zhang, L.X., Li, X.L., Smith, M.A., Post, R.M., Han, J.S. (1997) Lipofectin-facilitated
transfer of cholecystokinin gene corrects behavioral abnormalities of rats with audiogenic seizures. Neurosci. 77:15-22.
143
Table 1. Potential Therapeutic Targets that Influence Seizure Behavior.
Target Placement Delivery vector
Experimental model
Functional effect
References
Adenosine
Intracerebroventricular
Grafting of fibroblasts or myoblasts
Hippocampal kindling
Suppression of generalized seizures
(Huber et al. ,2001; Guttinger et al., 2005)
Anti NMDA Collicular cortex AAV-CMV vector
Focal electrical stimulation
Increased seizure threshold
(Haberman et al., 2002)
AAV-TEToff vector
Decreased seizure threshold
(Haberman et al., 2002)
ASPA
Intracerebroventricular
Ad-CB vector Spontaneous
seizures in SER
Transiently reduced incidence of tonic seizures (2 weeks)
(Seki et al., 2004)
Cholecystokinin Intracerebroventricular Lipofectin and plasmid
Audiogenic seizure-prone rats
Transient seizure inhibition (1 week)
(Zhang et al., 1997)
GABAAR Hippocampus AAV-GABAR4
Pilocarpine-induced Status Epilepticus
Transiently reduced spontaneous seizures (4 weeks)
(Raol et al., 2006)
GAD65 Anterior SN Grafting of neuronal or glial cell lines
Entorhinal kindling
Delayed rate of kindling
(Thompson et al., 2000)
Posterior SN
Grafting of neuronal or
glial cell lines
Faster rate of kindling
(Thompsonet al., 2000)
Anterior SN
Grafting neuronal cell line
Pilocarpine-induced Status Epilepticus
Reduced spontaneous seizures
(Thompson and Suchomelova 2004)
Piriform cortex
Grafting neuronal cell line
Amygdala kindling
Increased threshold to seizures
(Gernert et al., 2002)
GAD67 SN pars reticulata
Grafting of mixed neuronal and glial cell line
Kainic acid ip Delayed seizure behavior
(Castillo et al., 2006)
Galanin Collicular cortex
AAV-TEToff vector+FIB secretory sequence
Increased seizure threshold
(Haberman et al., 2003)
Hippocampus
AAV-TEToff vector+FIB secretory sequence
Kainic acid ip
Neuroprotection
(Haberman et al., 2003)
144
AAV-NSE vector+WPRE
Kainic acid ih
Reduced number of seizures
(Lin, et al., 2003)
Piriform cortex
AAV-CB vector+FIB secretory sequence
Kainic acid ip
Seizure inhibition
(McCown, 2006)
Focal electrical stimulation
Increased seizure threshold
(McCown, 2006)
GDNF
Hippocampus
AAV-CB vector+WPRE
Hippocampal kindling
Decreased number of generalized seizures, increased seizure threshold
(Kanter-Schlifke et al., 2007)
Hippocampus
Ad-CMV vector
Kainic acid ip
Delayed seizure behaviors, neuroprotective
(Yoo et al., 2006)
ICP10PK
Intranasal vaccine
HSV-2∆RR –ICP10 vector
Kainic acid ip
Reduced seizure behaviors, neuroprotection
(Laing et al., 2006 )
NPY
Hippocampus
AAV-NSE vector+WPRE
Kainic acid ih Delayed seizure behavior
(Richichi et al. ,2004)
Hippocampal kindling
Increased seizure threshold and delayed rate of kindling
(Richichi et al., 2004)
Piriform cortex
AAV-CB vector+FIB secretory sequence
Kainic acid ip
Reduced and delayed seizure behaviors
(Foti, et al., 2007)
NPY 13-36
Piriform cortex
AAV-CB vector+FIB secretory sequence
Kainic acid ip
Reduced and delayed seizure behaviors
(Foti, et al. ,2007)
Abbreviations are: AAV: adeno-associated virus; ASPA: aspartoacylase; Ad: adenoviral; CB: cytomegalovirus enhancer, chicken beta-actin promoter; CMV: cytomegalovirus; GAD: glutamic acid decarboxylase; GDNF: glial-derived neurotrophic factor; ICP10PK: ICP 10 protein kinase; ih: intrahippocampal; ip: intraperitoneal; NMDA: N-methyl-D- aspartate; NPY: Neuropeptide Y; NSE: neuron- specific enolase; SER: spontaneously epileptic rats; SN: substantia nigra; TET off: tetracycline-off regulatable promoter; WPRE: woodchuck virus post-transcriptional regulatory element.
145
References Acland, G. M., G. D. Aguirre, et al. (2001). "Gene therapy restores vision in a canine
model of childhood blindness." Nat Genet 28(1): 92-5. Agarwal, M., T. W. Austin, et al. (1998). "Scaffold attachment region-mediated
enhancement of retroviral vector expression in primary T cells." J Virol 72(5): 3720-8.
Agnati, L. F., B. Bjelke, et al. (1995). "Volume versus wiring transmission in the brain: a
new theoretical frame for neuropsychopharmacology." Med Res Rev 15(1): 33-45.
Alberch, J., E. Perez-Navarro, et al. (2002). "Neuroprotection by neurotrophins and
GDNF family members in the excitotoxic model of Huntington's disease." Brain Res Bull 57(6): 817-22.
Araujo, D. M. and D. C. Hilt (1997). "Glial cell line-derived neurotrophic factor
attenuates the excitotoxin-induced behavioral and neurochemical deficits in a rodent model of Huntington's disease." Neuroscience 81(4): 1099-110.
Araujo, D. M. and D. C. Hilt (1998). "Glial cell line-derived neurotrophic factor
attenuates the locomotor hypofunction and striatonigral neurochemical deficits induced by chronic systemic administration of the mitochondrial toxin 3-nitropropionic acid." Neuroscience 82(1): 117-27.
Auricchio, A., G. P. Gao, et al. (2002). "Constitutive and regulated expression of
processed insulin following in vivo hepatic gene transfer." Gene Ther 9(14): 963-71.
Auricchio, A., V. M. Rivera, et al. (2002). "Pharmacological regulation of protein
expression from adeno-associated viral vectors in the eye." Mol Ther 6(2): 238-42.
Auten, J., M. Agarwal, et al. (1999). "Effect of scaffold attachment region on transgene
expression in retrovirus vector-transduced primary T cells and macrophages." Hum Gene Ther 10(8): 1389-99.
Bankiewicz, K. S., J. Forsayeth, et al. (2006). "Long-term clinical improvement in
MPTP-lesioned primates after gene therapy with AAV-hAADC." Mol Ther 14(4): 564-70.
Baraban, S. C. (2002). "Antiepileptic actions of neuropeptide Y in the mouse
hippocampus require Y5 receptors." Epilepsia 43 Suppl 5: 9-13.
146
Barker, C. F. and R. E. Billingham (1977). "Immunologically privileged sites." Adv Immunol 25: 1-54.
Baron, U. and H. Bujard (2000). "Tet repressor-based system for regulated gene
expression in eukaryotic cells: principles and advances." Methods Enzymol 327: 401-21.
Bartlett, J. S., R. J. Samulski, et al. (1998). "Selective and rapid uptake of adeno-
associated virus type 2 in brain." Hum Gene Ther 9(8): 1181-6. Bauer, H. J. and G. Monreal (1986). "Herpesviruses provide helper functions for avian
adeno-associated parvovirus." J Gen Virol 67 (Pt 1): 181-5. Bernard, C., J. C. Hirsch, et al. (1999). "Excitation and inhibition in temporal lobe
epilepsy: a close encounter." Adv Neurol 79: 821-8. Berns, K. I. and R. M. Linden (1995). "The cryptic life style of adeno-associated virus."
Bioessays 17(3): 237-45. Blacklow, N. R., M. D. Hoggan, et al. (1968). "Epidemiology of adenovirus-associated
virus infection in a nursery population." Am J Epidemiol 88(3): 368-78. Blomer, U., L. Naldini, et al. (1997). "Highly efficient and sustained gene transfer in
adult neurons with a lentivirus vector." J Virol 71(9): 6641-9. Bode, J., Y. Kohwi, et al. (1992). "Biological significance of unwinding capability of
nuclear matrix-associating DNAs." Science 255(5041): 195-7. Bonhaus, D. W., L. C. Rigsbee, et al. (1987). "Intranigral dynorphin-1-13 suppresses
kindled seizures by a naloxone-insensitive mechanism." Brain Res 405(2): 358-63.
Bosch, A., E. Perret, et al. (2000). "Reversal of pathology in the entire brain of
mucopolysaccharidosis type VII mice after lentivirus-mediated gene transfer." Hum Gene Ther 11(8): 1139-50.
Bowles, D. E., J. E. Rabinowitz, et al. (2003). "Marker rescue of adeno-associated virus
(AAV) capsid mutants: a novel approach for chimeric AAV production." J Virol 77(1): 423-32.
Branchek, T. A., K. E. Smith, et al. (2000). "Galanin receptor subtypes." Trends
Pharmacol Sci 21(3): 109-17. Brooks-Kayal, A. R., M. D. Shumate, et al. (1999). "Human neuronal gamma-
aminobutyric acid(A) receptors: coordinated subunit mRNA expression and
147
functional correlates in individual dentate granule cells." J Neurosci 19(19): 8312-8.
Brooks-Kayal, A. R., M. D. Shumate, et al. (1998). "Selective changes in single cell
GABA(A) receptor subunit expression and function in temporal lobe epilepsy." Nat Med 4(10): 1166-72.
Brooks, P. A., J. S. Kelly, et al. (1987). "Direct excitatory effects of neuropeptide Y
(NPY) on rat hippocampal neurones in vitro." Brain Res 408(1-2): 295-8. Buhl, E. H., T. S. Otis, et al. (1996). "Zinc-induced collapse of augmented inhibition by
GABA in a temporal lobe epilepsy model." Science 271(5247): 369-73. Buller, R. M., J. E. Janik, et al. (1981). "Herpes simplex virus types 1 and 2 completely
help adenovirus-associated virus replication." J Virol 40(1): 241-7. Burger, C., O. S. Gorbatyuk, et al. (2004). "Recombinant AAV viral vectors pseudotyped
with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system." Mol Ther 10(2): 302-17.
Burgess, T. L. and R. B. Kelly (1987). "Constitutive and regulated secretion of proteins."
Annu Rev Cell Biol 3: 243-93. Castillo, C. G., S. Mendoza, et al. (2006). "Intranigral transplants of immortalized
GABAergic cells decrease the expression of kainic acid-induced seizures in the rat." Behav Brain Res 171(1): 109-15.
Cearley, C. N. and J. H. Wolfe (2006). "Transduction characteristics of adeno-associated
virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain." Mol Ther 13(3): 528-37.
Cearley, C. N. and J. H. Wolfe (2007). "A single injection of an adeno-associated virus
vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease." J Neurosci 27(37): 9928-40.
Chen, H., D. M. McCarty, et al. (1999). "Oligodendrocyte-specific gene expression in
mouse brain: use of a myelin-forming cell type-specific promoter in an adeno-associated virus." J Neurosci Res 55(4): 504-13.
Chenuaud, P., T. Larcher, et al. (2004). "Optimal design of a single recombinant adeno-
associated virus derived from serotypes 1 and 2 to achieve more tightly regulated transgene expression from nonhuman primate muscle." Mol Ther 9(3): 410-8.
148
Cheung, A. K., M. D. Hoggan, et al. (1980). "Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells." J Virol 33(2): 739-48.
Chirmule, N., K. Propert, et al. (1999). "Immune responses to adenovirus and adeno-
associated virus in humans." Gene Ther 6(9): 1574-83. Choi, V. W., D. M. McCarty, et al. (2005). "AAV hybrid serotypes: improved vectors for
gene delivery." Curr Gene Ther 5(3): 299-310. Chtarto, A., H. U. Bender, et al. (2003). "Tetracycline-inducible transgene expression
mediated by a single AAV vector." Gene Ther 10(1): 84-94. Chtarto, A., X. Yang, et al. (2007). "Controlled delivery of glial cell line-derived
neurotrophic factor by a single tetracycline-inducible AAV vector." Exp Neurol 204(1): 387-99.
Cohen, I., V. Navarro, et al. (2002). "On the origin of interictal activity in human
temporal lobe epilepsy in vitro." Science 298(5597): 1418-21. Colmers, W. F. and D. Bleakman (1994). "Effects of neuropeptide Y on the electrical
properties of neurons." Trends Neurosci 17(9): 373-9. Colmers, W. F., G. J. Klapstein, et al. (1991). "Presynaptic inhibition by neuropeptide Y
in rat hippocampal slice in vitro is mediated by a Y2 receptor." Br J Pharmacol 102(1): 41-4.
Cruz-Aguado, R., L. F. Turner, et al. (2000). "Nerve growth factor and striatal
glutathione metabolism in a rat model of Huntington's disease." Restor Neurol Neurosci 17(4): 217-221.
Davidson, B. L., C. S. Stein, et al. (2000). "Recombinant adeno-associated virus type 2,
4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system." Proc Natl Acad Sci U S A 97(7): 3428-32.
Davies, S. W. and K. Beardsall (1992). "Nerve growth factor selectively prevents
excitotoxin induced degeneration of striatal cholinergic neurones." Neurosci Lett 140(2): 161-4.
Day, R., M. K. Schafer, et al. (1993). "Region specific expression of furin mRNA in the
rat brain." Neurosci Lett 149(1): 27-30. de Felipe, P. (2002). "Polycistronic viral vectors." Curr Gene Ther 2(3): 355-78. de Lanerolle, N. C., J. H. Kim, et al. (1989). "Hippocampal interneuron loss and plasticity
in human temporal lobe epilepsy." Brain Res 495(2): 387-95.
149
Dillon, N. and F. Grosveld (1993). "Transcriptional regulation of multigene loci: multilevel control." Trends Genet 9(4): 134-7.
Dodge, J. C., J. Clarke, et al. (2005). "Gene transfer of human acid sphingomyelinase
corrects neuropathology and motor deficits in a mouse model of Niemann-Pick type A disease." Proc Natl Acad Sci U S A 102(49): 17822-7.
Donello, J. E., J. E. Loeb, et al. (1998). "Woodchuck hepatitis virus contains a tripartite
posttranscriptional regulatory element." J Virol 72(6): 5085-92. Donnelly, M. L., G. Luke, et al. (2001). "Analysis of the aphthovirus 2A/2B polyprotein
'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'." J Gen Virol 82(Pt 5): 1013-25.
Duan, D., P. Sharma, et al. (1998). "Circular intermediates of recombinant adeno-
associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue." J Virol 72(11): 8568-77.
Duan, D., Y. Yue, et al. (2003). "Dual vector expansion of the recombinant AAV
packaging capacity." Methods Mol Biol 219: 29-51. Dumont, Y., A. Cadieux, et al. (2000). "Potent and selective tools to investigate
neuropeptide Y receptors in the central and peripheral nervous systems: BIB03304 (Y1) and CGP71683A (Y5)." Can J Physiol Pharmacol 78(2): 116-25.
Dumont, Y., A. Cadieux, et al. (2000). "BIIE0246, a potent and highly selective non-
peptide neuropeptide Y Y(2) receptor antagonist." Br J Pharmacol 129(6): 1075-88.
During, M. J., R. J. Samulski, et al. (1998). "In vivo expression of therapeutic human
genes for dopamine production in the caudates of MPTP-treated monkeys using an AAV vector." Gene Ther 5(6): 820-7.
During, M. J., C. W. Symes, et al. (2000). "An oral vaccine against NMDAR1 with
efficacy in experimental stroke and epilepsy." Science 287(5457): 1453-60. El Bahh, B., S. Balosso, et al. (2005). "The anti-epileptic actions of neuropeptide Y in the
hippocampus are mediated by Y and not Y receptors." Eur J Neurosci 22(6): 1417-30.
El Bahh, B., J. Q. Cao, et al. (2002). "Blockade of neuropeptide Y(2) receptors and
suppression of NPY's anti-epileptic actions in the rat hippocampal slice by BIIE0246." Br J Pharmacol 136(4): 502-9.
Engel, J., Jr. (1996). "Excitation and inhibition in epilepsy." Can J Neurol Sci 23(3): 167-
74.
150
Engel, J., Jr., M. F. Levesque, et al. (1992). "Surgical treatment of the epilepsies: presurgical evaluation." Clin Neurosurg 38: 514-34.
Ericson, C., K. Wictorin, et al. (2002). "Ex vivo and in vitro studies of transgene
expression in rat astrocytes transduced with lentiviral vectors." Exp Neurol 173(1): 22-30.
Erles, K., P. Sebokova, et al. (1999). "Update on the prevalence of serum antibodies (IgG
and IgM) to adeno-associated virus (AAV)." J Med Virol 59(3): 406-11. Fathi, Z., A. M. Cunningham, et al. (1997). "Cloning, pharmacological characterization
and distribution of a novel galanin receptor." Brain Res Mol Brain Res 51(1-2): 49-59.
Ferrari, F. K., T. Samulski, et al. (1996). "Second-strand synthesis is a rate-limiting step
for efficient transduction by recombinant adeno-associated virus vectors." J Virol 70(5): 3227-34.
Ferrari, F. K., X. Xiao, et al. (1997). "New developments in the generation of Ad-free,
high-titer rAAV gene therapy vectors." Nat Med 3(11): 1295-7. Festenstein, R., M. Tolaini, et al. (1996). "Locus control region function and
heterochromatin-induced position effect variegation." Science 271(5252): 1123-5. Fisher, K. J., G. P. Gao, et al. (1996). "Transduction with recombinant adeno-associated
virus for gene therapy is limited by leading-strand synthesis." J Virol 70(1): 520-32.
Fitzsimons, H. L., J. M. McKenzie, et al. (2001). "Insulators coupled to a minimal
bidirectional tet cassette for tight regulation of rAAV-mediated gene transfer in the mammalian brain." Gene Ther 8(22): 1675-81.
Flotte, T. R. (2001). "Recombinant adeno-associated virus vectors for cystic fibrosis gene
therapy." Curr Opin Mol Ther 3(5): 497-502. Flotte, T. R., S. A. Afione, et al. (1993). "Expression of the cystic fibrosis transmembrane
conductance regulator from a novel adeno-associated virus promoter." J Biol Chem 268(5): 3781-90.
Folliot, S., D. Briot, et al. (2003). "Sustained tetracycline-regulated transgene expression
in vivo in rat retinal ganglion cells using a single type 2 adeno-associated viral vector." J Gene Med 5(6): 493-501.
Foti, S., R. P. Haberman, et al. (2007). "Adeno-associated virus-mediated expression and
constitutive secretion of NPY or NPY13-36 suppresses seizure activity in vivo." Gene Ther 14(21): 1534-6.
151
Freese, A., M. G. Kaplitt, et al. (1997). "Direct gene transfer into human epileptogenic hippocampal tissue with an adeno-associated virus vector: implications for a gene therapy approach to epilepsy." Epilepsia 38(7): 759-66.
Frim, D. M., M. P. Short, et al. (1993). "Local protective effects of nerve growth factor-
secreting fibroblasts against excitotoxic lesions in the rat striatum." J Neurosurg 78(2): 267-73.
Frim, D. M., J. Simpson, et al. (1993). "Striatal degeneration induced by mitochondrial
blockade is prevented by biologically delivered NGF." J Neurosci Res 35(4): 452-8.
Frim, D. M., T. A. Uhler, et al. (1993). "Effects of biologically delivered NGF, BDNF
and bFGF on striatal excitotoxic lesions." Neuroreport 4(4): 367-70. Frush, D. P. and J. O. McNamara (1986). "Evidence implicating dentate granule cells in
wet dog shakes produced by kindling stimulations of entorhinal cortex." Exp Neurol 92(1): 102-13.
Fuqua, S. A., M. Blum-Salingaros, et al. (1989). "Induction of the estrogen-regulated
"24K" protein by heat shock." Cancer Res 49(15): 4126-9. Furler, S., J. C. Paterna, et al. (2001). "Recombinant AAV vectors containing the foot and
mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons." Gene Ther 8(11): 864-73.
Furtinger, S., S. Pirker, et al. (2001). "Plasticity of Y1 and Y2 receptors and neuropeptide
Y fibers in patients with temporal lobe epilepsy." J Neurosci 21(15): 5804-12. Gafni, Y., G. Pelled, et al. (2004). "Gene therapy platform for bone regeneration using an
exogenously regulated, AAV-2-based gene expression system." Mol Ther 9(4): 587-95.
Gaken, J., J. Jiang, et al. (2000). "Fusagene vectors: a novel strategy for the expression of
multiple genes from a single cistron." Gene Ther 7(23): 1979-85. Galpern, W. R., R. T. Matthews, et al. (1996). "NGF attenuates 3-nitrotyrosine formation
in a 3-NP model of Huntington's disease." Neuroreport 7(15-17): 2639-42. Galpern, W. R., L. G. Miller, et al. (1990). "Differential effects of chronic lorazepam and
alprazolam on benzodiazepine binding and GABAA-receptor function." Br J Pharmacol 101(4): 839-42.
Gao, G., M. R. Alvira, et al. (2003). "Adeno-associated viruses undergo substantial
evolution in primates during natural infections." Proc Natl Acad Sci U S A 100(10): 6081-6.
152
Gao, G. P., M. R. Alvira, et al. (2002). "Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy." Proc Natl Acad Sci U S A 99(18): 11854-9.
Georgievska, B., D. Kirik, et al. (2002). "Aberrant sprouting and downregulation of
tyrosine hydroxylase in lesioned nigrostriatal dopamine neurons induced by long-lasting overexpression of glial cell line derived neurotrophic factor in the striatum by lentiviral gene transfer." Exp Neurol 177(2): 461-74.
Gernert, M., K. W. Thompson, et al. (2002). "Genetically engineered GABA-producing
cells demonstrate anticonvulsant effects and long-term transgene expression when transplanted into the central piriform cortex of rats." Exp Neurol 176(1): 183-92.
Gibbs, J. W., 3rd, M. D. Shumate, et al. (1997). "Differential epilepsy-associated
alterations in postsynaptic GABA(A) receptor function in dentate granule and CA1 neurons." J Neurophysiol 77(4): 1924-38.
Gigout, L., P. Rebollo, et al. (2005). "Altering AAV tropism with mosaic viral capsids."
Mol Ther 11(6): 856-65. Greber, S., C. Schwarzer, et al. (1994). "Neuropeptide Y inhibits potassium-stimulated
glutamate release through Y2 receptors in rat hippocampal slices in vitro." Br J Pharmacol 113(3): 737-40.
Grieger, J. C., V. W. Choi, et al. (2006). "Production and characterization of adeno-
associated viral vectors." Nat Protoc 1(3): 1412-28. Grieger, J. C. and R. J. Samulski (2005). "Packaging capacity of adeno-associated virus
serotypes: impact of larger genomes on infectivity and postentry steps." J Virol 79(15): 9933-44.
Guttinger, M., V. Padrun, et al. (2005). "Seizure suppression and lack of adenosine A1
receptor desensitization after focal long-term delivery of adenosine by encapsulated myoblasts." Exp Neurol 193(1): 53-64.
Haberman, R., H. Criswell, et al. (2002). "Therapeutic liabilities of in vivo viral vector
tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity." Mol Ther 6(4): 495-500.
Haberman, R. P. and T. J. McCown (2002). "Regulation of gene expression in adeno-
associated virus vectors in the brain." Methods 28(2): 219-26. Haberman, R. P., T. J. McCown, et al. (1998). "Inducible long-term gene expression in
brain with adeno-associated virus gene transfer." Gene Ther 5(12): 1604-11.
153
Haberman, R. P., T. J. McCown, et al. (2000). "Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element." J Virol 74(18): 8732-9.
Haberman, R. P., R. J. Samulski, et al. (2003). "Attenuation of seizures and neuronal
death by adeno-associated virus vector galanin expression and secretion." Nat Med 9(8): 1076-80.
Hadaczek, P., M. Kohutnicka, et al. (2006). "Convection-enhanced delivery of adeno-
associated virus type 2 (AAV2) into the striatum and transport of AAV2 within monkey brain." Hum Gene Ther 17(3): 291-302.
Hadaczek, P., H. Mirek, et al. (2004). "Basic fibroblast growth factor enhances
transduction, distribution, and axonal transport of adeno-associated virus type 2 vector in rat brain." Hum Gene Ther 15(5): 469-79.
Halbert, C. L., A. D. Miller, et al. (2006). "Prevalence of neutralizing antibodies against
adeno-associated virus (AAV) types 2, 5, and 6 in cystic fibrosis and normal populations: Implications for gene therapy using AAV vectors." Hum Gene Ther 17(4): 440-7.
Harui, A., S. Suzuki, et al. (1999). "Frequency and stability of chromosomal integration
of adenovirus vectors." J Virol 73(7): 6141-6. Hauck, B. and W. Xiao (2003). "Characterization of tissue tropism determinants of
adeno-associated virus type 1." J Virol 77(4): 2768-74. Hauser, W. A., D. C. Hesdorffer, et al. (1990). Epilepsy frequency, causes, and
consequences. Landover, MD; New York, NY, Epilepsy Foundation of America; Demos.
He, B., S. E. Counts, et al. (2005). "Ectopic galanin expression and normal galanin
receptor 2 and galanin receptor 3 mRNA levels in the forebrain of galanin transgenic mice." Neuroscience 133(2): 371-80.
Higashimoto, T., F. Urbinati, et al. (2007). "The woodchuck hepatitis virus post-
transcriptional regulatory element reduces readthrough transcription from retroviral vectors." Gene Ther 14(17): 1298-304.
Hildinger, M., A. Auricchio, et al. (2001). "Hybrid vectors based on adeno-associated
virus serotypes 2 and 5 for muscle-directed gene transfer." J Virol 75(13): 6199-203.
Hippenmeyer, P. J. and G. G. Krivi (1991). "Gene expression from heterologous
promoters in a replication-defective avian retrovirus vector in quail cells." Poult Sci 70(4): 982-92.
154
Hirtz, D., D. J. Thurman, et al. (2007). "How common are the "common" neurologic disorders?" Neurology 68(5): 326-37.
Hokfelt, T., T. Bartfai, et al. (2003). "Neuropeptides: opportunities for drug discovery."
Lancet Neurol 2(8): 463-72. Hollmann, M. and S. Heinemann (1994). "Cloned glutamate receptors." Annu Rev
Neurosci 17: 31-108. Holmes, G. L. (1997). "Epilepsy in the developing brain: lessons from the laboratory and
clinic." Epilepsia 38(1): 12-30. Holst, J. J., M. Bersani, et al. (1993). "On the effects of human galanin in man."
Diabetologia 36(7): 653-7. Huang, L., M.-c. Hung, et al. (1999). Nonviral vectors for gene therapy. San Diego, CA,
Academic Press. Huber, A., V. Padrun, et al. (2001). "Grafts of adenosine-releasing cells suppress seizures
in kindling epilepsy." Proc Natl Acad Sci U S A 98(13): 7611-6. Huttner, N. A., A. Girod, et al. (2003). "Genetic modifications of the adeno-associated
virus type 2 capsid reduce the affinity and the neutralizing effects of human serum antibodies." Gene Ther 10(26): 2139-47.
Jenuwein, T., W. C. Forrester, et al. (1997). "Extension of chromatin accessibility by
nuclear matrix attachment regions." Nature 385(6613): 269-72. Jiang, H., G. F. Pierce, et al. (2006). "Evidence of multiyear factor IX expression by
AAV-mediated gene transfer to skeletal muscle in an individual with severe hemophilia B." Mol Ther 14(3): 452-5.
Jiang, L., S. Rampalli, et al. (2004). "Tight regulation from a single tet-off rAAV vector
as demonstrated by flow cytometry and quantitative, real-time PCR." Gene Ther 11(13): 1057-67.
Johnston, J., J. Tazelaar, et al. (2003). "Regulated expression of erythropoietin from an
AAV vector safely improves the anemia of beta-thalassemia in a mouse model." Mol Ther 7(4): 493-7.
Kamau, S. W., F. Grimm, et al. (2001). "Expression of green fluorescent protein as a
marker for effects of antileishmanial compounds in vitro." Antimicrob Agents Chemother 45(12): 3654-6.
Kanter-Schlifke, I., B. Georgievska, et al. (2007). "Seizure suppression by GDNF gene
therapy in animal models of epilepsy." Mol Ther 15(6): 1106-13.
155
Kaplitt, M. G., A. Feigin, et al. (2007). "Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial." Lancet 369(9579): 2097-105.
Kaspar, B. K., J. Llado, et al. (2003). "Retrograde viral delivery of IGF-1 prolongs
survival in a mouse ALS model." Science 301(5634): 839-42. Kells, A. P., D. M. Fong, et al. (2004). "AAV-mediated gene delivery of BDNF or GDNF
is neuroprotective in a model of Huntington disease." Mol Ther 9(5): 682-8. Kioussis, D. and R. Festenstein (1997). "Locus control regions: overcoming
heterochromatin-induced gene inactivation in mammals." Curr Opin Genet Dev 7(5): 614-9.
Kirik, D., C. Rosenblad, et al. (2000). "Long-term rAAV-mediated gene transfer of
GDNF in the rat Parkinson's model: intrastriatal but not intranigral transduction promotes functional regeneration in the lesioned nigrostriatal system." J Neurosci 20(12): 4686-700.
Klein, R. L., R. D. Dayton, et al. (2006). "Efficient neuronal gene transfer with AAV8
leads to neurotoxic levels of tau or green fluorescent proteins." Mol Ther 13(3): 517-27.
Klein, R. L., R. D. Dayton, et al. (2008). "AAV8, 9, Rh10, Rh43 Vector Gene Transfer in
the Rat Brain: Effects of Serotype, Promoter and Purification Method." Mol Ther 16(1): 89-96.
Klein, R. L., M. E. Hamby, et al. (2002). "Dose and promoter effects of adeno-associated
viral vector for green fluorescent protein expression in the rat brain." Exp Neurol 176(1): 66-74.
Klein, R. L., E. M. Meyer, et al. (1998). "Neuron-specific transduction in the rat
septohippocampal or nigrostriatal pathway by recombinant adeno-associated virus vectors." Exp Neurol 150(2): 183-94.
Klugmann, M., C. W. Symes, et al. (2005). "AAV-mediated hippocampal expression of
short and long Homer 1 proteins differentially affect cognition and seizure activity in adult rats." Mol Cell Neurosci 28(2): 347-60.
Kofler, N., E. Kirchmair, et al. (1997). "Altered expression of NPY-Y1 receptors in
kainic acid induced epilepsy in rats." Neurosci Lett 230(2): 129-32. Kopp, J., A. Nanobashvili, et al. (1999). "Differential regulation of mRNAs for
neuropeptide Y and its receptor subtypes in widespread areas of the rat limbic system during kindling epileptogenesis." Brain Res Mol Brain Res 72(1): 17-29.
156
Laing, J. M. and L. Aurelian (2008). "DeltaRR vaccination protects from KA-induced seizures and neuronal loss through ICP10PK-mediated modulation of the neuronal-microglial axis." Genet Vaccines Ther 6(1): 1.
Laing, J. M., M. D. Gober, et al. (2006). "Intranasal administration of the growth-
compromised HSV-2 vector DeltaRR prevents kainate-induced seizures and neuronal loss in rats and mice." Mol Ther 13(5): 870-81.
Lang, A. E. and A. M. Lozano (1998). "Parkinson's disease. First of two parts." N Engl J
Med 339(15): 1044-53. Li, T., J. A. Steinbeck, et al. (2007). "Suppression of kindling epileptogenesis by
adenosine releasing stem cell-derived brain implants." Brain 130(Pt 5): 1276-88. Lin, E. J., C. Richichi, et al. (2003). "Recombinant AAV-mediated expression of galanin
in rat hippocampus suppresses seizure development." Eur J Neurosci 18(7): 2087-92.
Lin, E. J., D. Young, et al. (2006). "Differential actions of NPY on seizure modulation
via Y1 and Y2 receptors: evidence from receptor knockout mice." Epilepsia 47(4): 773-80.
Lipshutz, G. S., D. Titre, et al. (2003). "Comparison of gene expression after
intraperitoneal delivery of AAV2 or AAV5 in utero." Mol Ther 8(1): 90-8. Lisovoski, F., J. P. Wahrmann, et al. (1997). "Long-term histological follow-up of
genetically modified myoblasts grafted into the brain." Brain Res Mol Brain Res 44(1): 125-33.
Lo, W. D., G. Qu, et al. (1999). "Adeno-associated virus-mediated gene transfer to the
brain: duration and modulation of expression." Hum Gene Ther 10(2): 201-13. Loacker, S., M. Sayyah, et al. (2007). "Endogenous dynorphin in epileptogenesis and
epilepsy: anticonvulsant net effect via kappa opioid receptors." Brain 130(Pt 4): 1017-28.
Lochrie, M. A., G. P. Tatsuno, et al. (2006). "Mutations on the external surfaces of
adeno-associated virus type 2 capsids that affect transduction and neutralization." J Virol 80(2): 821-34.
Loeb, J. E., W. S. Cordier, et al. (1999). "Enhanced expression of transgenes from adeno-
associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy." Hum Gene Ther 10(14): 2295-305.
157
Longhi, L., D. J. Watson, et al. (2004). "Ex vivo gene therapy using targeted engraftment of NGF-expressing human NT2N neurons attenuates cognitive deficits following traumatic brain injury in mice." J Neurotrauma 21(12): 1723-36.
Loscher, W. and I. E. Leppik (2002). "Critical re-evaluation of previous preclinical
strategies for the discovery and the development of new antiepileptic drugs." Epilepsy Res 50(1-2): 17-20.
Lowenstein, P. R. (2002). "Immunology of viral-vector-mediated gene transfer into the
brain: an evolutionary and developmental perspective." Trends Immunol 23(1): 23-30.
Lundberg, C., P. Horellou, et al. (1996). "Generation of DOPA-producing astrocytes by
retroviral transduction of the human tyrosine hydroxylase gene: in vitro characterization and in vivo effects in the rat Parkinson model." Exp Neurol 139(1): 39-53.
Maheshri, N., J. T. Koerber, et al. (2006). "Directed evolution of adeno-associated virus
yields enhanced gene delivery vectors." Nat Biotechnol 24(2): 198-204. Maksimovic, I. D., M. D. Jovanovic, et al. (2002). "Effects of nerve and fibroblast growth
factors on the production of nitric oxide in experimental model of Huntington's disease." Vojnosanit Pregl 59(2): 119-23.
Margaritis, P., V. R. Arruda, et al. (2004). "Novel therapeutic approach for hemophilia
using gene delivery of an engineered secreted activated Factor VII." J Clin Invest 113(7): 1025-31.
Marsh, D. J., S. C. Baraban, et al. (1999). "Role of the Y5 neuropeptide Y receptor in
limbic seizures." Proc Natl Acad Sci U S A 96(23): 13518-23. Martinez-Serrano, A. and A. Bjorklund (1996). "Protection of the neostriatum against
excitotoxic damage by neurotrophin-producing, genetically modified neural stem cells." J Neurosci 16(15): 4604-16.
Martinez-Serrano, A. and A. Bjorklund (1997). "Immortalized neural progenitor cells for
CNS gene transfer and repair." Trends Neurosci 20(11): 530-8. Mastakov, M. Y., K. Baer, et al. (2002). "Immunological aspects of recombinant adeno-
associated virus delivery to the mammalian brain." J Virol 76(16): 8446-54. Mazarati, A., L. Lundstrom, et al. (2006). "Regulation of kindling epileptogenesis by
hippocampal galanin type 1 and type 2 receptors: The effects of subtype-selective agonists and the role of G-protein-mediated signaling." J Pharmacol Exp Ther 318(2): 700-8.
158
Mazarati, A. and C. G. Wasterlain (2002). "Anticonvulsant effects of four neuropeptides in the rat hippocampus during self-sustaining status epilepticus." Neurosci Lett 331(2): 123-7.
Mazarati, A. M. (2004). "Galanin and galanin receptors in epilepsy." Neuropeptides
38(6): 331-43. Mazarati, A. M., J. G. Hohmann, et al. (2000). "Modulation of hippocampal excitability
and seizures by galanin." J Neurosci 20(16): 6276-81. Mazarati, A. M., H. Liu, et al. (1998). "Galanin modulation of seizures and seizure
modulation of hippocampal galanin in animal models of status epilepticus." J Neurosci 18(23): 10070-7.
McBride, J. L., M. J. During, et al. (2003). "Structural and functional neuroprotection in a
rat model of Huntington's disease by viral gene transfer of GDNF." Exp Neurol 181(2): 213-23.
McCarty, D. M., H. Fu, et al. (2003). "Adeno-associated virus terminal repeat (TR)
mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo." Gene Ther 10(26): 2112-8.
McCarty, D. M., P. E. Monahan, et al. (2001). "Self-complementary recombinant adeno-
associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis." Gene Ther 8(16): 1248-54.
McCarty, D. M., S. M. Young, Jr., et al. (2004). "Integration of adeno-associated virus
(AAV) and recombinant AAV vectors." Annu Rev Genet 38: 819-45. McCown, T. J. (2004). "The clinical potential of antiepileptic gene therapy." Expert Opin
Biol Ther 4(11): 1771-6. McCown, T. J. (2006). "Adeno-associated virus-mediated expression and constitutive
secretion of galanin suppresses limbic seizure activity in vivo." Mol Ther 14(1): 63-8.
McCown, T. J., B. S. Givens, et al. (1987). "Amino acid influences on seizures elicited
within the inferior colliculus." J Pharmacol Exp Ther 243(2): 603-8. McCown, T. J., X. Xiao, et al. (1996). "Differential and persistent expression patterns of
CNS gene transfer by an adeno-associated virus (AAV) vector." Brain Res 713(1-2): 99-107.
McMenamin, M. M., T. Lantos, et al. (2004). "Neuropathological consequences of
delivering an adenoviral vector in the rat brain." J Gene Med 6(7): 740-50.
159
McPhee, S. W., C. G. Janson, et al. (2006). "Immune responses to AAV in a phase I study for Canavan disease." J Gene Med 8(5): 577-88.
Menei, P., J. M. Pean, et al. (2000). "Intracerebral implantation of NGF-releasing
biodegradable microspheres protects striatum against excitotoxic damage." Exp Neurol 161(1): 259-72.
Meyer, A., P. Chretien, et al. (1996). "Kainic acid increases the expression of the
prohormone convertases furin and PC1 in the mouse hippocampus." Brain Res 732(1-2): 121-32.
Miller, D. G., L. M. Petek, et al. (2004). "Adeno-associated virus vectors integrate at
chromosome breakage sites." Nat Genet 36(7): 767-73. Miyoshi, H., U. Blomer, et al. (1998). "Development of a self-inactivating lentivirus
vector." J Virol 72(10): 8150-7. Mizuguchi, H., Z. Xu, et al. (2000). "IRES-dependent second gene expression is
significantly lower than cap-dependent first gene expression in a bicistronic vector." Mol Ther 1(4): 376-82.
Monyer, H., R. Sprengel, et al. (1992). "Heteromeric NMDA receptors: molecular and
functional distinction of subtypes." Science 256(5060): 1217-21. Muzyczka, N. (1992). "Use of adeno-associated virus as a general transduction vector for
mammalian cells." Curr Top Microbiol Immunol 158: 97-129. Muzzin, P., R. C. Eisensmith, et al. (1997). "Hepatic insulin gene expression as treatment
for type 1 diabetes mellitus in rats." Mol Endocrinol 11(6): 833-7. Nakajima, K., K. Ikenaka, et al. (1993). "An improved retroviral vector for assaying
promoter activity. Analysis of promoter interference in pIP211 vector." FEBS Lett 315(2): 129-33.
Nakayama, K. (1997). "Furin: a mammalian subtilisin/Kex2p-like endoprotease involved
in processing of a wide variety of precursor proteins." Biochem J 327 (Pt 3): 625-35.
Nguyen, J. B., R. Sanchez-Pernaute, et al. (2001). "Convection-enhanced delivery of
AAV-2 combined with heparin increases TK gene transfer in the rat brain." Neuroreport 12(9): 1961-4.
No, D., T. P. Yao, et al. (1996). "Ecdysone-inducible gene expression in mammalian cells
and transgenic mice." Proc Natl Acad Sci U S A 93(8): 3346-51.
160
Novak, U., E. A. Harris, et al. (1990). "High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells." Proc Natl Acad Sci U S A 87(9): 3386-90.
O'Donnell, D., S. Ahmad, et al. (1999). "Expression of the novel galanin receptor subtype
GALR2 in the adult rat CNS: distinct distribution from GALR1." J Comp Neurol 409(3): 469-81.
Oiwa, Y., R. Yoshimura, et al. (2002). "Dopaminergic neuroprotection and regeneration
by neurturin assessed by using behavioral, biochemical and histochemical measurements in a model of progressive Parkinson's disease." Brain Res 947(2): 271-83.
Pardridge, W. M. (2001). "Crossing the blood-brain barrier: are we getting it right?" Drug
Discov Today 6(1): 1-2. Parker, E. M., D. G. Izzarelli, et al. (1995). "Cloning and characterization of the rat
GALR1 galanin receptor from Rin14B insulinoma cells." Brain Res Mol Brain Res 34(2): 179-89.
Parker, R. M. and H. Herzog (1999). "Regional distribution of Y-receptor subtype
mRNAs in rat brain." Eur J Neurosci 11(4): 1431-48. Passini, M. A., S. L. Macauley, et al. (2005). "AAV vector-mediated correction of brain
pathology in a mouse model of Niemann-Pick A disease." Mol Ther 11(5): 754-62.
Paterna, J. C., T. Moccetti, et al. (2000). "Influence of promoter and WHV post-
transcriptional regulatory element on AAV-mediated transgene expression in the rat brain." Gene Ther 7(15): 1304-11.
Peden, C. S., C. Burger, et al. (2004). "Circulating anti-wild-type adeno-associated virus
type 2 (AAV2) antibodies inhibit recombinant AAV2 (rAAV2)-mediated, but not rAAV5-mediated, gene transfer in the brain." J Virol 78(12): 6344-59.
Peel, A. L., S. Zolotukhin, et al. (1997). "Efficient transduction of green fluorescent
protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters." Gene Ther 4(1): 16-24.
Perabo, L., J. Endell, et al. (2006). "Combinatorial engineering of a gene therapy vector:
directed evolution of adeno-associated virus." J Gene Med 8(2): 155-62. Pereira, D. J., D. M. McCarty, et al. (1997). "The adeno-associated virus (AAV) Rep
protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection." J Virol 71(2): 1079-88.
161
Perez-Navarro, E., E. Arenas, et al. (1999). "Intrastriatal grafting of a GDNF-producing cell line protects striatonigral neurons from quinolinic acid excitotoxicity in vivo." Eur J Neurosci 11(1): 241-9.
Perez-Navarro, E., E. Arenas, et al. (1996). "Glial cell line-derived neurotrophic factor
protects striatal calbindin-immunoreactive neurons from excitotoxic damage." Neuroscience 75(2): 345-52.
Philippe, S., C. Sarkis, et al. (2006). "Lentiviral vectors with a defective integrase allow
efficient and sustained transgene expression in vitro and in vivo." Proc Natl Acad Sci U S A 103(47): 17684-9.
Phillips, M. I., Y. Tang, et al. (2002). "Vigilant vector: heart-specific promoter in an
adeno-associated virus vector for cardioprotection." Hypertension 39(2 Pt 2): 651-5.
Plavec, I., T. Papayannopoulou, et al. (1993). "A human beta-globin gene fused to the
human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells." Blood 81(5): 1384-92.
Provost, N., G. Le Meur, et al. (2005). "Biodistribution of rAAV vectors following
intraocular administration: evidence for the presence and persistence of vector DNA in the optic nerve and in the brain." Mol Ther 11(2): 275-83.
Qi, J. S., J. Yao, et al. (2006). "Downregulation of tonic GABA currents following
epileptogenic stimulation of rat hippocampal cultures." J Physiol 577(Pt 2): 579-90.
Qian, J., W. F. Colmers, et al. (1997). "Inhibition of synaptic transmission by
neuropeptide Y in rat hippocampal area CA1: modulation of presynaptic Ca2+ entry." J Neurosci 17(21): 8169-77.
Qing, K. and Y. Chen (2007). "Central CART gene delivery by recombinant AAV vector
attenuates body weight gain in diet-induced-obese rats." Regul Pept 140(1-2): 21-6.
Rabinowitz, J. E., D. E. Bowles, et al. (2004). "Cross-dressing the virion: the
transcapsidation of adeno-associated virus serotypes functionally defines subgroups." J Virol 78(9): 4421-32.
Rabinowitz, J. E., F. Rolling, et al. (2002). "Cross-packaging of a single adeno-associated
virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity." J Virol 76(2): 791-801.
162
Racine, R. J. (1972). "Modification of seizure activity by electrical stimulation. II. Motor seizure." Electroencephalogr Clin Neurophysiol 32(3): 281-94.
Raol, Y. H., I. V. Lund, et al. (2006). "Enhancing GABA(A) receptor alpha 1 subunit
levels in hippocampal dentate gyrus inhibits epilepsy development in an animal model of temporal lobe epilepsy." J Neurosci 26(44): 11342-6.
Rattenholl, A., M. Ruoppolo, et al. (2001). "Pro-sequence assisted folding and disulfide
bond formation of human nerve growth factor." J Mol Biol 305(3): 523-33. Redrobe, J. P., Y. Dumont, et al. (1999). "Multiple receptors for neuropeptide Y in the
hippocampus: putative roles in seizures and cognition." Brain Res 848(1-2): 153-66.
Ren, G., T. Li, et al. (2007). "Lentiviral RNAi-induced downregulation of adenosine
kinase in human mesenchymal stem cell grafts: a novel perspective for seizure control." Exp Neurol 208(1): 26-37.
Richichi, C., E. J. Lin, et al. (2004). "Anticonvulsant and antiepileptogenic effects
mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus." J Neurosci 24(12): 3051-9.
Rivera, V. M., T. Clackson, et al. (1996). "A humanized system for pharmacologic
control of gene expression." Nat Med 2(9): 1028-32. Rivera, V. M., X. Ye, et al. (1999). "Long-term regulated expression of growth hormone
in mice after intramuscular gene transfer." Proc Natl Acad Sci U S A 96(15): 8657-62.
Rose, J. A., J. V. Maizel, Jr., et al. (1971). "Structural proteins of adenovirus-associated
viruses." J Virol 8(5): 766-70. Samulski, R. J., L. S. Chang, et al. (1987). "A recombinant plasmid from which an
infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication." J Virol 61(10): 3096-101.
Samulski, R. J., L. S. Chang, et al. (1989). "Helper-free stocks of recombinant adeno-
associated viruses: normal integration does not require viral gene expression." J Virol 63(9): 3822-8.
Samulski, R. J., X. Zhu, et al. (1991). "Targeted integration of adeno-associated virus
(AAV) into human chromosome 19." Embo J 10(12): 3941-50. Sander, J. W. (1993). "Some aspects of prognosis in the epilepsies: a review." Epilepsia
34(6): 1007-16.
163
Sanftner, L. M., V. M. Rivera, et al. (2006). "Dimerizer regulation of AADC expression and behavioral response in AAV-transduced 6-OHDA lesioned rats." Mol Ther 13(1): 167-74.
Sanftner, L. M., B. M. Suzuki, et al. (2004). "Striatal delivery of rAAV-hAADC to rats
with preexisting immunity to AAV." Mol Ther 9(3): 403-9. Schiedner, G., N. Morral, et al. (1998). "Genomic DNA transfer with a high-capacity
adenovirus vector results in improved in vivo gene expression and decreased toxicity." Nat Genet 18(2): 180-3.
Schwarzer, C., N. Kofler, et al. (1998). "Up-regulation of neuropeptide Y-Y2 receptors in
an animal model of temporal lobe epilepsy." Mol Pharmacol 53(1): 6-13. Schwarzer, C., J. M. Williamson, et al. (1995). "Somatostatin, neuropeptide Y,
neurokinin B and cholecystokinin immunoreactivity in two chronic models of temporal lobe epilepsy." Neuroscience 69(3): 831-45.
Searle, P. F., G. W. Stuart, et al. (1985). "Building a metal-responsive promoter with
synthetic regulatory elements." Mol Cell Biol 5(6): 1480-9. Seidah, N. G. and M. Chretien (1999). "Proprotein and prohormone convertases: a family
of subtilases generating diverse bioactive polypeptides." Brain Res 848(1-2): 45-62.
Seki, T., H. Matsubayashi, et al. (2004). "Adenoviral gene transfer of aspartoacylase
ameliorates tonic convulsions of spontaneously epileptic rats." Neurochem Int 45(1): 171-8.
Semenza, G. L., B. H. Jiang, et al. (1996). "Hypoxia response elements in the aldolase A,
enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1." J Biol Chem 271(51): 32529-37.
Shafer, P. O. and C. Begley (2000). "The Human and Economic Burden of Epilepsy."
Epilepsy Behav 1(2): 91-92. Shafer, S. Q., W. A. Hauser, et al. (1988). "EEG and other early predictors of epilepsy
remission: a community study." Epilepsia 29(5): 590-600. Shen, F., Y. Fan, et al. (2008). "Adeno-associated viral vector-mediated hypoxia-
regulated VEGF gene transfer promotes angiogenesis following focal cerebral ischemia in mice." Gene Ther 15(1): 30-9.
Shen, F., H. Su, et al. (2006). "Adeno-associated viral-vector-mediated hypoxia-inducible
vascular endothelial growth factor gene expression attenuates ischemic brain injury after focal cerebral ischemia in mice." Stroke 37(10): 2601-6.
164
Shenouda, S. M., C. Johns, et al. (2006). "Long-term inhibition of the central alpha(2B)-adrenergic receptor gene via recombinant AAV-delivered antisense in hypertensive rats." Am J Hypertens 19(11): 1135-43.
Shevtsova, Z., J. M. Malik, et al. (2005). "Promoters and serotypes: targeting of adeno-
associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo." Exp Physiol 90(1): 53-9.
Siegel, A. M. (2004). "Presurgical evaluation and surgical treatment of medically
refractory epilepsy." Neurosurg Rev 27(1): 1-18; discussion 19-21. Simonato, M. and P. Romualdi (1996). "Dynorphin and epilepsy." Prog Neurobiol 50(5-
6): 557-83. Sloviter, R. S. (1991). "Permanently altered hippocampal structure, excitability, and
inhibition after experimental status epilepticus in the rat: the "dormant basket cell" hypothesis and its possible relevance to temporal lobe epilepsy." Hippocampus 1(1): 41-66.
Smith, K. E., C. Forray, et al. (1997). "Expression cloning of a rat hypothalamic galanin
receptor coupled to phosphoinositide turnover." J Biol Chem 272(39): 24612-6. Smith, K. E., M. W. Walker, et al. (1998). "Cloned human and rat galanin GALR3
receptors. Pharmacology and activation of G-protein inwardly rectifying K+ channels." J Biol Chem 273(36): 23321-6.
Song, S., M. Morgan, et al. (1998). "Sustained secretion of human alpha-1-antitrypsin
from murine muscle transduced with adeno-associated virus vectors." Proc Natl Acad Sci U S A 95(24): 14384-8.
Spencer, D. D., S. S. Spencer, et al. (1984). "Access to the posterior medial temporal lobe
structures in the surgical treatment of temporal lobe epilepsy." Neurosurgery 15(5): 667-71.
Sperk, G., J. Marksteiner, et al. (1992). "Functional changes in neuropeptide Y- and
somatostatin-containing neurons induced by limbic seizures in the rat." Neuroscience 50(4): 831-46.
Suzuki, K. and M. Taniike (1995). "Murine model of genetic demyelinating disease: the
twitcher mouse." Microsc Res Tech 32(3): 204-14. Taymans, J. M., L. H. Vandenberghe, et al. (2007). "Comparative analysis of adeno-
associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain." Hum Gene Ther 18(3): 195-206.
165
Thomas, C. E., D. Birkett, et al. (2001). "Acute direct adenoviral vector cytotoxicity and chronic, but not acute, inflammatory responses correlate with decreased vector-mediated transgene expression in the brain." Mol Ther 3(1): 36-46.
Thompson, K., V. Anantharam, et al. (2000). "Conditionally immortalized cell lines,
engineered to produce and release GABA, modulate the development of behavioral seizures." Exp Neurol 161(2): 481-9.
Thompson, K. W. and L. M. Suchomelova (2004). "Transplants of cells engineered to
produce GABA suppress spontaneous seizures." Epilepsia 45(1): 4-12. Tobiasch, E., M. Rabreau, et al. (1994). "Detection of adeno-associated virus DNA in
human genital tissue and in material from spontaneous abortion." J Med Virol 44(2): 215-22.
Toth, P. T., V. P. Bindokas, et al. (1993). "Mechanism of presynaptic inhibition by
neuropeptide Y at sympathetic nerve terminals." Nature 364(6438): 635-9. Ure, J. A. and M. Perassolo (2000). "Update on the pathophysiology of the epilepsies." J
Neurol Sci 177(1): 1-17. Van de Ven, W. J., J. W. Creemers, et al. (1991). "Furin: the prototype mammalian
subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway." Enzyme 45(5-6): 257-70.
Venero, J. L., K. D. Beck, et al. (1994). "Intrastriatal infusion of nerve growth factor after
quinolinic acid prevents reduction of cellular expression of choline acetyltransferase messenger RNA and trkA messenger RNA, but not glutamate decarboxylase messenger RNA." Neuroscience 61(2): 257-68.
Vezzani, A., M. Michalkiewicz, et al. (2002). "Seizure susceptibility and epileptogenesis
are decreased in transgenic rats overexpressing neuropeptide Y." Neuroscience 110(2): 237-43.
Vezzani, A., D. Moneta, et al. (2000). "Plastic changes in neuropeptide Y receptor
subtypes in experimental models of limbic seizures." Epilepsia 41 Suppl 6: S115-21.
Vezzani, A., M. Rizzi, et al. (2000). "Modulatory role of neuropeptides in seizures
induced in rats by stimulation of glutamate receptors." J Nutr 130(4S Suppl): 1046S-8S.
Vezzani, A. and G. Sperk (2004). "Overexpression of NPY and Y2 receptors in epileptic
brain tissue: an endogenous neuroprotective mechanism in temporal lobe epilepsy?" Neuropeptides 38(4): 245-52.
166
Virella-Lowell, I., B. Zusman, et al. (2005). "Enhancing rAAV vector expression in the lung." J Gene Med 7(7): 842-50.
Walther, W. and U. Stein (1996). "Cell type specific and inducible promoters for vectors
in gene therapy as an approach for cell targeting." J Mol Med 74(7): 379-92. Wang, Y., S. Y. Tsai, et al. (2000). "An antiprogestin regulable gene switch for induction
of gene expression in vivo." Adv Pharmacol 47: 343-55. Wang, Z., H. I. Ma, et al. (2003). "Rapid and highly efficient transduction by double-
stranded adeno-associated virus vectors in vitro and in vivo." Gene Ther 10(26): 2105-11.
Woldbye, D. P., P. J. Larsen, et al. (1997). "Powerful inhibition of kainic acid seizures by
neuropeptide Y via Y5-like receptors." Nat Med 3(7): 761-4. Wood, M. J., H. M. Charlton, et al. (1996). "Immune responses to adenovirus vectors in
the nervous system." Trends Neurosci 19(11): 497-501. Wu, J., W. Zhao, et al. (2007). "Self-complementary recombinant adeno-associated viral
vectors: packaging capacity and the role of rep proteins in vector purity." Hum Gene Ther 18(2): 171-82.
Wu, Z., A. Asokan, et al. (2006). "Adeno-associated virus serotypes: vector toolkit for
human gene therapy." Mol Ther 14(3): 316-27. Xiao, W., N. Chirmule, et al. (1999). "Gene therapy vectors based on adeno-associated
virus type 1." J Virol 73(5): 3994-4003. Xiao, X., J. Li, et al. (1998). "Production of high-titer recombinant adeno-associated virus
vectors in the absence of helper adenovirus." J Virol 72(3): 2224-32. Xu, J., C. Ma, et al. (2005). "A combination of mutations enhances the neurotropism of
AAV-2." Virology 341(2): 203-14. Xu, R., C. G. Janson, et al. (2001). "Quantitative comparison of expression with adeno-
associated virus (AAV-2) brain-specific gene cassettes." Gene Ther 8(17): 1323-32.
Yalkinoglu, A. O., R. Heilbronn, et al. (1988). "DNA amplification of adeno-associated
virus as a response to cellular genotoxic stress." Cancer Res 48(11): 3123-9. Yamakura, T. and K. Shimoji (1999). "Subunit- and site-specific pharmacology of the
NMDA receptor channel." Prog Neurobiol 59(3): 279-98.
167
Yanez-Munoz, R. J., K. S. Balaggan, et al. (2006). "Effective gene therapy with nonintegrating lentiviral vectors." Nat Med 12(3): 348-53.
Yang, G. S., M. Schmidt, et al. (2002). "Virus-mediated transduction of murine retina
with adeno-associated virus: effects of viral capsid and genome size." J Virol 76(15): 7651-60.
Yao, S., M. A. Smith-White, et al. (2002). "Stabilization of the helical structure of Y2-
selective analogues of neuropeptide Y by lactam bridges." J Med Chem 45(11): 2310-8.
Ye, X., V. M. Rivera, et al. (1999). "Regulated delivery of therapeutic proteins after in
vivo somatic cell gene transfer." Science 283(5398): 88-91. Ying, Z. and I. M. Najm (2002). "Mechanisms of epileptogenicity in focal malformations
caused by abnormal cortical development." Neurosurg Clin N Am 13(1): 27-33, vii.
Yoo, Y. M., C. J. Lee, et al. (2006). "Neuroprotection of adenoviral-vector-mediated
GDNF expression against kainic-acid-induced excitotoxicity in the rat hippocampus." Exp Neurol 200(2): 407-17.
Yuan, L., I. Kurek, et al. (2005). "Laboratory-directed protein evolution." Microbiol Mol
Biol Rev 69(3): 373-92. Yue, Y. and D. Duan (2003). "Double strand interaction is the predominant pathway for
intermolecular recombination of adeno-associated viral genomes." Virology 313(1): 1-7.
Zaboikin, M. M. and F. G. Schuening (1998). "Poor expression of MDR1 transgene in
HeLa cells by bicistronic Moloney murine leukemia virus-based vector." Hum Gene Ther 9(15): 2263-75.
Zeng, G. (1998). "Sticky-end PCR: new method for subcloning." Biotechniques 25(2):
206-8. Zhang, G., Y. H. Raol, et al. (2004). "Effects of status epilepticus on hippocampal
GABAA receptors are age-dependent." Neuroscience 125(2): 299-303. Zhang, L. X., X. L. Li, et al. (1997). "Lipofectin-facilitated transfer of cholecystokinin
gene corrects behavioral abnormalities of rats with audiogenic seizures." Neuroscience 77(1): 15-22.
Zini, S., M. P. Roisin, et al. (1993). "Galanin reduces release of endogenous excitatory
amino acids in the rat hippocampus." Eur J Pharmacol 245(1): 1-7.