WO2010037143A1

WO2010037143A1 - Vectors and methods of treating brain seizures

Info

Publication number: WO2010037143A1
Application number: PCT/US2009/058899
Authority: WO
Inventors: David J. Poulsen
Original assignee: The University Of Montana
Priority date: 2008-09-29
Filing date: 2009-09-29
Publication date: 2010-04-01

Abstract

Therapeutic tools and methods useful in treating a subject having brain seizures. The methods comprise the step of increasing expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons located in the brain of the subject sufficiently to reduce the severity, frequency, and/or duration of the brain seizures. The therapeutic tools include recombinant adeno-associated viral vector (rAAV) comprises AAV 5' and 3' ITRs flanking a human excitatory amino acid transporter 3 (EAAT3) gene which is operatively linked to a promoter suitable for expression of the EAAT3 in GABAergic neurons of the hippocampus.

Description

VECTORS AND METHODS OF TREATING BRAIN SEIZURES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/101 ,094 filed September 29, 2008, the content of which is expressly incorporated herein in its entirety by reference thereto.

GOVERNMENT SUPPORT

This project was supported in part by NIH Grants P20 RR015583 from the COBRE Program of the National Center for Research Resources and through NIH/NIDCD R21 NS058541 -01 .

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 2,000 byte ASCII (text) file named "Seqjist" created on September 29, 2009.

FIELD OF THE INVENTION

The present invention is directed to methods and therapeutic tools for treating a subject having brain seizures by increasing expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons.

BACKGROUND OF THE INVENTION

Seizures are symptoms of problems in the brain, which result from abrupt, abnormal electrical activity therein. Focal seizures occur in only one part of the brain. Recurring seizures often result from a brain disorder called epilepsy.

Epilepsy is a chronic neurological condition caused by abnormalities in the central nervous system. It is characterized by recurrent seizures originating in the brain and is associated with excessive or abnormal synchronization of neural activity. An estimated 1 % of the population suffers from some form of epilepsy, and more than 2 million people in the US have been diagnosed with epilepsy or experienced a seizure. An epileptic seizure is a brief, excessive surge of electrical activity in the brain that causes a change in consciousness, sensation and behavior. During an epileptic seizure, the regulatory systems that maintain the normal balance between excitation and inhibition of the brain's electrical activity break down. There may be a loss of inhibitory nerve cells or an overproduction of an excitatory neurotransmitter. Groups of abnormal cells are activated synchronously, creating a storm of electrical activity.

Complex focal seizures, and specifically temporal lobe epilepsy (TLE), is one of the most refractory forms of epilepsy. TLE causes both simple partial seizures, wherein the afflicted individual does not lose consciousness, and complex partial seizures, which result in a loss of consciousness and memory impairment. In extreme cases, the latter form of seizure may secondarily generalize. This occurs in about 60% of people with TLE. When the secondary generalization process occurs, seizures spread from the temporal lobe to a wider portion of the brain. The result is a convulsive (grand mal) seizure. Although anticonvulsive drugs may control seizures in some patients, seizures will remain uncontrolled in 40% of patients despite drug therapy. One treatment approach for these patients is to undergo a phased evaluation for consideration of resective surgery.

Resective surgery involves the surgical removal of the portion of the brain responsible for causing seizures. The brain is comprised of four lobes: the frontal, temporal, parietal and occipital. Seizures most often arise from one or both temporal lobes. In the deep front part of the temporal lobes are located the most seizure prone structures in the brain: the hippocampus, and the amygdala. Because of this, temporal lobectomy is one of the most common and one of the most successful types of resective surgery. Typically, one temporal lobe is defined as the site of seizure origin (the epileptogenic region) and the medial temporal lobe including the anterior hippocampus is resected. Patients undergoing the surgery may require up to a 14 day hospitalization. Post-operation headaches and nausea commonly occur, and most doctors will keep a patient on seizure medication for at least several years. In some cases, the medication is necessary for life, albeit at a reduced dosage. Moreover, the part of the brain removed does not grow back. Although antiepileptic drugs (AED) currently exist for treating seizures, patients taking these drugs display a broad spectrum of side-effects, such as liver damage, nausea, vomiting, double vision, loss of coordination, drowsiness, and headache. The widely used drug carbamazepine, for example, exhibits side effects including dizziness, ataxia, drowsiness and reduction of alertness (A. Delcker, Eur. Neuropsychopharmacold 7: 213-8 (1997)). Valproic acid, moreover, may precipitate metabolic disorders, liver disease, gastrointestinal symptomatology, excessive bodyweight gain and alopecia. See, S. J. Wallace, Drug Saf., 15: 378-93 (1996). Barbiturates precipitate metabolic bone disease and rash. (S. J. Wallace, Drug Saf 15: 378-93 (1996)).

As a result, a need exists for improved therapeutic tools and methods of treating a human subject suffering from brain seizures. Ideally, improved treatments would reduce the severity, frequency, and duration of the brain seizures without the side effects or invasive surgical interventions that accompany current methods of treatment.

SUMMARY OF THE INVENTION

The present invention is based on the surprising discovery that brain seizures can be treated with minimal or no side effects by increasing the expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons.

Thus, in one embodiment, the invention is directed to a method of treating a human subject having focal brain seizures. The method comprises the step of increasing expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons located in the brain of the subject where the focal brain seizures occur. The EAAT3 expression is advantageously increased in the GABAergic neurons sufficiently to reduce the severity, frequency, and/or duration of the focal brain seizures to treat the subject having brain seizures.

In a specific embodiment, the method comprises directly administering to the hippocampus of the subject a recombinant adeno-associated viral vector (rAAV). The rAAV used typically comprises an excitatory amino acid transporter 3 (EAAT3) gene operatively linked to a promoter suitable for expression of EAAT3 in GABAergic neurons in the hippocampus, wherein EAAT3 expression is increased sufficiently in the GABAergic neurons to reduce the severity, frequency, and/or duration of the brain seizures in the subject.

Therapeutic tools are also provided herein. The tools are useful in treating humans having chronic brain seizures, e.g. epilepsy. One such tool provided herein is a recombinant adeno-associated viral vector (rAAV) designed specifically to treat brain seizures in human subjects. The rAAV preferably comprises AAV 5' and 3' ITRs flanking a human excitatory amino acid transporter 3 (EAAT3) gene which is operatively linked to a promoter suitable for expression of the EAAT3 in GABAergic neurons of the hippocampus.

Pharmaceutical compositions are also provided. The pharmaceutical compositions comprise the recombinant adeno-associated viral vectors (rAAV) designed to treat brain seizures, as described herein, and a pharmaceutically acceptable carrier. These recombinant adeno-associated viral vector (rAAV) can also be used in the manufacture of a medicament for the treatment of brain seizures.

These and other embodiments of the subject invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1. Increased functional expression of EAAT3. HC2S2 cultures were transduced with AAV1 -CAG-hEAAT3 and levels of EAAT3 expression compared with non transduced control cultures. All three EAAT3 protein bands are shown: non-transduced control HC2S2 cultures (C); cultures transduced with rAAV1 -CAG-EAAT3 (CE3) relative to β-actin normalization.

Figure 2. Functional EAAT3 mediated uptake activity in HC2S2 cultures. [³H]D-aspartic acid uptake activity in HC2S2 cultures was compared between non-transduced HC2S2 control cultures and cultures transduced with rAAV1 - CAG-EAAT3. A 424% increased in [³H]D-aspartic acid was observed in transduced HC2S2 cells over that of control. ^* = p < 0.01 , n=3.

Figure 3. EAAT3 expression regulates intracellular GABA, glutamate and glutamine levels. HPLC analysis of rAAV transduced and control HC2S2 cultures was performed to determine the intracellular concentrations of GABA, glutamine and glutamate. A) GABA levels in HC2S2 cells transduced with rAAV1 -CAG-hEAAT3 were 136% higher than non-transduced controls. B) Glutamate levels in HC2S2 cells transduced with rAAV1 -CAG-hEAAT3 were 56% higher than non-transduced controls. C) Glutamine levels in HC2S2 cells transduced with rAAV1 -CAG-hEAAT3 were 146% higher than non-transduced controls. ^* = p < 0.01 , n=6

Figure 4. rAAV mediated targeted expression to GABAergic neurons. rAAV-GAD65-hrGFP was bilaterally injected into the hippocampaus of adult male Sprague-Dawley rats. After 4 weeks, brains were perfused and fixed with 4% paraformalgehyde and 50μM sections prepared on a vibratome. Sections were stained with anti-GAD65/67 antibody (red). Colocalization of GFP expression (green) with GAD65 staining indicates targeted expression to GABAergic neurons.

Figure 5. Targeted expression of flag tagged rEAAT3 to GABAergic neurons. rAAV-GAD65-flagEAAT3 was bilaterally injected into the hippocampus of adult male Sprague-Dawley rats. After 4 weeks, 50μM vibratome sections were stained with antibody specific for the flag epitope (green) to label recombinant rEAAT3 protein. Sections were dual stained with anti-GAD65/67 antibody (red) to label GABAergic neurons. Colocalization of flag-EAAT3 expression with GAD65 staining indicates targeted expression of the recombinant rEAAT3 to GABAergic neurons.

Figure 6. EAAT3 expression altered seizure susceptibility and rate of progression to SE. The pilocarpine induced seizure model was used to evaluate altered state of seizure susceptibility as a function of EAAT3 expression on inhibitory neuron terminals. Control (v), flag-rEAAT3 (υ), and EAAT3/AS (λ) animals were monitored for progression through distinct behavioral stages based on a modified Racine scale. Time to reach each of the stages was noted, averaged for each treatment group for animals that reached SE. - == p<0.05. One way ANOVA, n=3-7.

Figure 7. Altered EAAT3 expression on inhibitory presynaptic terminals influences neuroprotection. A) Tissue was harvested 48 hours post onset of status epilepticus. Tissue from each treatment group, control, null, EAACI /AS and f!ag-rEAAT3, were stained with Fiuoro-Jade B and imaged to identify the ievel of seizure-mediated neurodamage. B) Total fluorescence intensity of the CA1 region of the hippocampus was calcuiated. One-way ANOVA ^x = p<G.G5, n=3-7.

Figure 8. Similar neuropathology was observed in cortex following piiocarpine-induced seizures. A) Fiuoro-Jade B staining of tissue from each of the treatment groups (control, fiag-rEAAT3 and EAAT3/AS) was performed and images were obtained from similar regions within the cortex, B) Total fluorescence intensity from each of the sampled regions of the cortex revealed no statistical difference in Fiuoro-Jade B levels between each of the treatment groups.

Rgure 9. Stereotaxic coordinates of electrode placement in the hippocampus of the rat. A reference screw near the bregma was used as a ground, and electrodes were placed at 3.6mm from the bregma, 3.5mm to the right of the midline, and at a depth of 3.2mm. KA and virus injections occurred through a port 2mm medial the electrodes and at a depth of 4mm.

Figure 10, Shows a rat with pedestal shown during an EEG a recording

Figure 11. Representative EEG recordings from two rats including their baselines, KA delivery, the 2 weeks after KA delivery, and weeks 1 , 2, and 4 after rAAV delivery. Control rat received AAV8-GAD65-Nu!l and the experimental rat received the AAV8-GAD65-f!ag-rEAAT3, which caused increased expression of rEAAT3 in GABAergic neurons. At two weeks after the injection of virus, there is a marked difference in seizure severities between the two groups. After 4 weeks, the AAV8-GAD65-f!ag-rEAAT3 injected animal showed visibly reduced seizure activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

We demonstrate herein that increasing EAAT3 expression in GABAergic neurons located in the subject's brain can successfully reduce the severity, frequency, and/or duration of the brain seizures. In one specific example described in more detail below, we used recombinant adeno- associated virus (rAAV) vectors to overexpress rat EAAT3 (rEAAT3) in a GABAergic neuronal cell line (HC2S2 cells). We demonstrated, in vitro. increased glutamate uptake activity and intracellular GABA concentrations compared to uninfected controls. We then used rAAV vectors to overexpress and knock down rEAAT3 in GABAergic neurons within the rat hippocampus in vivo. Enhanced expression of rEAAT3 in GABAergic neurons significantly delayed the onset and/or severity of pilocarpine-induced seizures and resulted in reduced neuropathology in animals that experienced status epilepticus. In contrast, knock down of rEAAT3 in GABAergic neurons resulted in greater sensitivity to pilocarpine-induced seizures and enhanced neurodegeneration following seizures. Based on these studies and examples, we provide herein methods and therapeutic tools to treat subject's suffering from brain seizures.

METHODS

The present methods successfully treat human subjects suffering from brain seizures by increasing the expression of excitatory amino acid transporter 3 (EAAT3), e.g., GenBank NM_004170; Accession NM_004170; SLC1 A1 NM 004170.4 Homo sapiens solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), in GABAergic neurons. GABAergic neurons are neurons that convert glutamate to γ-Aminobutyric acid (GABA), typically via the GAD65 and GAD67 enzymes. GABAergic neurons typically also store GABA in synaptic vesicles and release GABA from these vesicles upon stimulation.

In a preferred embodiment, the increased EAAT3 expression is targeted to GABAergic neurons in the target region wherein the seizures occur. Targeted expression means that preferably at least 50%, more preferably at least 85%, and still more preferably at least 95% of the transduced cells with increased expressing of EAAT3 are GABAergic neurons in the target region. In certain preferred embodiments, 98% to 100% of cells with increased expression of EAAT3 are GABAergic neurons.

The methods disclosed herein are designed specifically to treat brain seizures, e.g. epileptic seizures and focal brain seizures. A focal seizure is localized (partial or focal onset seizures). Focal seizures can be further divided on the extent to which consciousness is affected (simple focal seizures and complex focal seizures). If consciousness is unaffected, then it is a simple focal seizure; otherwise it is a complex focal seizure. A focal seizure may spread within the brain — a process known as secondary generalization. An epileptic seizure is a transient symptom of excessive or synchronous neuronal activity in the brain. The medical syndrome of recurrent, unprovoked seizures is typically termed epilepsy.

In one embodiment, the invention is directed to a method of treating a human subject having focal brain seizures. The method preferably comprises the step of increasing expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons located in the brain of the subject where the focal brain seizures occur. The EAAT3 expression is advantageously increased in the GABAergic neurons sufficiently to reduce the severity, frequency, and/or duration of the focal brain seizures to treat the subject having brain seizures. Increased expression of EAAT3 is compared to the GABAergic neuronal cells prior to transduction. One specific and preferred method of increasing expression of EAAT3 in GABAergic neurons is by directly administering, to a region of the brain where the focal brain seizures occur, a recombinant adeno-associated virus (rAAV) vector having a human EAAT3 gene, preferably by stereotaxic injection, described more fully below.

Another preferred and specific method comprises directly administering to the hippocampus of the subject a recombinant adeno-associated viral vector (rAAV). The rAAV used typically comprises an excitatory amino acid transporter 3 (EAAT3) gene operatively linked to a promoter suitable for expression of EAAT3 in GABAergic neurons in the hippocampus, wherein EAAT3 expression is increased sufficiently in the GABAergic neurons to reduce the severity, frequency, and/or duration of the brain seizures in the subject.

THERAPEUTIC TOOLS

The invention also provides therapeutic tools useful in treating a subject having brain seizures, such as epilepsy. One such tool is a recombinant adeno-associated viral vector (rAAV) designed specifically to treat human brain seizures. The rAAV preferably comprises AAV 5' and 3' ITRs flanking a human excitatory amino acid transporter 3 (EAAT3) gene which is operatively linked to a promoter suitable for expression of the EAAT3 in GABAergic neurons of the hippocampus. In a specific preferred embodiment, the promoter is glutamic acid decarboxylase 65 (GAD65). Preferably the vector further comprises the 3' Woodchuck hepatitis virus post-transcriptional regulation element (WPRE).

AAV: At least 13 different AAV serotypes have been identified based on amino acid sequence differences in their respective capsid proteins. The AAV used in the present invention can be any AAV serotype. In a preferred embodiment, however, the serotype comprises serotype 1 , 2, 8 or a mixture of two or more serotypes.

The AAV used in the present invention is a derivative of the adeno- associated virus, into which exogenous DNA has been introduced. The construction of infectious recombinant AAV and methods of purification are well known in the art. See, e.g., U.S. Patent Nos. 5,173,414; 5,139,941 ; 5,741 ,683; 6,458,587; 6,475,769; 6,503,888; 6,783,972; 6,943,019; 7,015,026; 7,037,713; and 7,282,199; Zolotukhin, Gene Ther 6:973-985 (1999); and Grimm, Hum Gene Ther 9(18): 2745-60 (1998) and Hum Gene Ther 10(15): 2445-50 (1999), all of which are incorporated herein by reference.

The AAV genome is composed of a linear, single-stranded DNA molecule that contains 4681 bases. The genome includes inverted terminal repeats (ITRs) at each end that function in cis as origins of DNA replication and as packaging signals for the virus. The ITRs are approximately 145 bp in length. The internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV rep and cap regions, respectively. These regions code for the viral proteins that provide AAV helper functions, i.e., the proteins involved in replication and packaging of the virion. Specifically, a family of at least four viral proteins is synthesized from the AAV rep region, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. The rAAV cap region encodes at least three proteins, VP1 , VP2 and VP3. For a detailed description of the AAV genome, see, e.g., Muzyczka, Current Topics in Microbiol, and Immunol 158:97-129 (1992) and U.S. Patent No. 7,282,199. rAAV vectors can be engineered to carry an exogenous nucleotide sequence of interest, preferably EAAT3, more preferably human EAAT3, by deleting, in whole or in part, the internal portion of the rAAV genome and inserting the DNA sequence of interest between the ITRs. The ITRs remain functional in such vectors allowing replication and packaging of the rAAV containing the heterologous nucleotide sequence of interest. The heterologous nucleotide sequence is also typically linked to a promoter sequence capable of driving gene expression in the patient's target cells under the certain conditions. Termination signals, such as polyadenylation sites, can also be included in the vector.

For propagation of the vector in vitro, susceptible cells are co- transfected with the rAAV-derived vector and a suitable rAAV-derived helper virus or plasmid. Preferably, the vector retains from rAAV essentially only the recognition signals for replication and packaging.

The rAAV-derived sequences do not necessarily have to correspond exactly with their wild-type prototypes. For example, the rAAV vectors of the present invention may feature mutated inverted terminal repeats, etc., provided that the vector can still be replicated and packaged and still infect GABAergic neuronal cells.

In one embodiment the rAAV is produced using the plasmid-based methods and the virus is purified on iodixanol gradients as described in Zolotukhin, Methods 28: 158-167 (2002).

Viruses are titered by the number of genomic particles per ml. Titers of the rAAV can vary, particularly depending upon the target cell, but preferably the rAAV used is a high-titer virus of at least 10⁹ gp/μl and more preferably, at least 10¹⁰ gp/μl or alternatively at least 10¹¹ gp/μl. Methods of producing high- titer viruses are also well known in the art. See, e.g., U.S. Patent Nos. 6,632,670 (teach methods of generating high-titer, contaminant free, recombinant rAAV vectors in large quantities); 7,015,026.

DNA: By "exogenous DNA" is meant any heterologous DNA, i.e., not normally found in wild-type rAAV that can be inserted into the rAAV for transfer into the target cell, e.g. EAAT3. For the DNA gene to be expressible, the coding sequence must be "operably linked" to a promoter sequence functional in the target cell. By "operatively linked" is meant that the promoter can drive expression of the exogenous DNA, as is known in the art, and can include the appropriate orientation of the promoter relative to the exogenous DNA. Furthermore, the exogenous DNA preferably has all appropriate sequences for expression. The DNA can include, for example, expression control sequences, such as an enhancer, and necessary information processing sites. Typically, because of the packaging limitations of rAAV, the exogenous DNA will have a length of about up to 5,000 bases. Preferably, the DNA is 4,500 bases.

Promoter: The promoter can be any desired promoter, selected by known considerations, such as the level of expression of the DNA operatively linked to the promoter and the GABAergic neuronal cells in which the DNA is to be expressed. Promoters can be an exogenous or an endogenous promoter.

In a preferred embodiment, the promoter is designed for expression in GABAergic neuronal cells, e.g., glutamic acid decarboxylase 65 (GAD65) (e.g., GenBank NT_008705.16, Bases 26444235-26445738; Accession NT_008705.16, bases 26555235-26445738; GenBank AF090195.1 , Bases 1233-2733; Accession AF090195.1 Bases 1233-2733). Thus, in one example, the rAAV is directly delivered to a specific region of the brain and the promoter induces expression of EAAT3 in GABAergic neuronal cells.

Preferably the promoter is one that will not induce expression in other cells located near the site of administration. Thus, in this way, the promoter can be said to be specific to have GABAergic neuronal cell specific expression. Thus, the promoter will not induce expression in other cells that might be transduced. In general, to find a tissue-specific promoter, one identifies a protein which is expressed only (or primarily) in that tissue, and then isolates the gene encoding that protein. (The gene may be a normal cellular gene, or a viral gene of a virus, which infects that cell). The promoter of that gene is likely to retain the desired tissue-specific activity when linked to another gene.

The tissue specificity of a promoter may be associated with a particular genetic element, which may be modified, or transferred into a second promoter.

Delivery: Delivery can be accomplished by any standard means for administering rAAV. For example, by simply contacting the rAAV, optionally contained in a desired liquid such as tissue culture medium, or a buffered saline solution, with the target cell. The rAAV can be allowed to remain in contact with the target cell for any desired length of time, and typically the rAAV is administered and allowed to remain for a time sufficient to effectively transduce the target cell.

For in vivo delivery, the rAAV may be delivered by any suitable method. Preferably delivery is via direct injection of the virus. In one embodiment the rAAV is directly administered to a region of the brain of the subject where the focal brain seizures occur. In another embodiment, the rAAV is directly administered into the hippocampus.

A "therapeutically effective amount" will fall in a relatively broad range that can be determined through clinical trials. For example, for in vivo injection. More importantly is the number of genomic particles (gp) administered to the hippocampus or region where the focal seizures occur. Preferably the rAAV is administered such that 10⁹ to 10¹⁴ gp, more preferably 10¹⁰ to 10¹² gp, and most preferably around 10¹¹ gp are administered to the region.

To deliver the rAAV specifically to a particular region of subject's brain, the rAAV is preferably administered by stereotaxic microinjection. For example, on the day of surgery, patients will have the stereotactic frame base fixed in place (screwed into the skull). The brain with stereotactic frame base (MRI-compatible with fiducial markings) will be imaged using high resolution MRI. The MRI images will then be transferred to a computer which runs stereotactic software. A series of coronal, sagittal and axial images will be used to determine the target (site of rAAV vector injection) and trajectory. The software directly translates the trajectory into 3 dimensional coordinates appropriate for the stereotactic frame. Burr holes are drilled above the entry site and the stereotactic apparatus positioned with the needle implanted at the given depth. The rAAV vector will then be injected at the target sites. Since the rAAV vector will integrate into the target cells, rather than producing viral particles, the subsequent spread of the vector will be minor, and mainly a function of passive diffusion from the site of injection, prior to integration. The degree of diffusion may be controlled by adjusting the ratio of vector to fluid carrier.

Preferably the rAAV vectors are designed to target GABAergic neurons in the subject's brain, more particularly in the region of the brain where the seizures are occurring. In certain non-limiting embodiments, the rAAV can be associated with a homing agent that binds specifically to a surface receptor of the cell. Thus, the vector may be conjugated to a ligand which is specific to GABAergic neurons cells. The conjugation may be covalent, e.g., a crosslinking agent such as glutaraldehyde, or noncovalent, e.g., the binding of an avidinated ligand to a biotinylated vector. Another form of covalent conjugation is provided by engineering the helper virus used to prepare the vector stock so that one of the encoded coat proteins is a chimera of a native AAV coat protein and a peptide or protein ligand, such that the ligand is exposed on the surface.

Whatever the form of conjugation, it is necessary that it not substantially interfere either with the integration of the AAV vector, or with the binding of the ligand to the cellular receptor.

The term "transduction" denotes the delivery of a DNA molecule to a recipient cell either in vivo or in vitro, via rAAV, preferably, as used herein, delivery of EAAT3 to GABAergic neuronal cells.

PHARMACEUTICAL COMPOSITIONS

The pharmaceutical compositions preferably comprise the recombinant adeno-associated viral vector (rAAV) designed to treat brain seizures, as described herein, and a pharmaceutically acceptable carrier.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a human. In one specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which an active ingredient is administered. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991 ).

Preferred excipients confer a protective effect on the rAAV such that loss of rAAV, as well as the loss of transduceability resulting from formulation procedures, packaging, storage, transport, and the like, is minimized.

Examples of the invention are provided, and are understood to be exemplary only, and do not limit the scope of the invention or the appended claims. A person of ordinary skill in the art will appreciate the invention can be practiced in any forms according to the claims and disclosure here.

EXAMPLES Methods Plasmid Design and Virus Preparation cDNA was produced from rat cortical brain tissue using the Cells-to cDNA Il kit (Ambion, Austin, TX) and used as template for PCR amplification. Primers were designed for amplification of the rat GAD65 promoter sequence (Skak, Gene 236:231 -241 (1999)). The upstream primer contained a Kpnl restriction site: δ'-GGTACCGGCGCTCCGCAG-S' (SEQ ID NO:1 ) and the downstream primer containing a BcM restriction site: 5'- TGATCAGGGTTCTGCTAGTCTGG-3' (SEQ ID NO:3). Amplified products were cloned into the TOPO Blunt PCR cloning plasmid (Invitrogen, Carlsbad, CA) and fragments confirmed by double stranded sequence analysis. The GAD65 promoter sequence was subcloned into the Kpnl and BamHI sites of the pAM-hrGFP-WPRE vector. Two additional GAD65 plasmids were designed with the hrGFP sequence replaced by either the rEAAT3 sequence tagged with a flag epitope (DYKDDDK) at the 5' end (flag-rEAAT3), or the rEAAT3 in the antisense orientation (rEAAT3/AS). Rat cDNA was used to PCR amplify the flag-rEAAT3 sequence with an upstream primer containing an Xhol restriction site and the flag epitope:5'- CTCGAGATGGATTATAAAGATGACGATGACAAATGTATGGGGAAG CCCACGAG-3' (SEQ ID NO:3) and a downstream primer containing a Hindlll restriction site: δ'-AAGCTTAGGCATCTAAGGCCAGGC-S' (SEQ ID NO:4). All constructs contained the 3' Woodchuck hepatitis virus post-transcriptional regulation element (WPRE). The WPRE construct has been shown to enhance transgene expression (Loeb, Hum Gene Ther 10:2295-2305 (1999); Paterna, Gene Ther 7:1304-131 1 (2000); Hlavaty, Virology 341 :1 -1 1 (2005)). The GAD65 promoter and expression cassettes are flanked by rAAV serotype 2 (rAAV2) inverted terminal repeats. Prior studies have shown chimeric rAAV at select serotype ratios increases overall rAAV mediated transduction (Rabinowitz, J Virol 78:4421 -4432 (2004)). For this study, we used a 3:1 ratio of rAAV serotype 1 to serotype 2.

The three pAM-GAD65 plasmids, containing the hrGFP, rEAAT3/AS and flag-rEAAT3 expression cassettes, and one cassette containing the CAG promoter (pAM-CAG-hEAAT3) were used in the production of recombinant rAAV. HEK293T cells cultures were transfected with the pAM-GAD65 and pAM-CAG constructs using Polyfect Transfection Reagent (Qiagen, Valencia, CA) (Selkirk, Eur J Neurosci 21 : 2291 -2296 (2005)). For the production of the rAAV1 -CAG-hEAAT3-WPRE virus, the plasmids used in the transfection were pFΔ6 (adenoviral helper plasmid), pH21 (cap gene for rAAV serotype 1 and rep gene for serotype 2) and the rAAV plasmid containing the CAG promoter upstream from the hEAAT3 sequence. For the production of the chimeric rAAV1/2-GAD65 viruses, four plasmids were used in the transfection were pFΔ6' pH21 , pRVI (cap and rep genes for rAAV serotype 2), and the rAAV plasmid containing the GAD65 promoter upstream from the flag-rEAAT3, rEAAT3/AS, or hrGFP gene sequence, flanked by rAAV2 inverted terminal repeats.

Virus was isolated from HEK293T cells through repeated freeze-thaw cycles, incubation for 30 minutes at 37⁰C with 5OU benzonase (Novagen, Madison, Wl) and 0.5% sodium deoxycholate, briefly sonicated and further purified by iodixonol density gradient centrifugation as previously reported (Zolotukhin, Gene Ther 6:973-985 (1999)). The titer (genomic particles/ml (gp/ml)) of the final virus isolate was determined by quantitative real time- polymerase chain reaction (RT-PCR) using an ABI Prism 7700 with primer and probe sets specific for the cis-acting WPRE enhancer sequence.

HC2S2 Cell Line

HC2S2 cells are an immortalized rat hippocampal GABAergic cell line (kindly provided by Dr. Fred H. Gage, SaIk Institute, La JoIIa, CA) and were cultured as previously reported (Hoshimaru, Proc Natl Acad Sci USA 93:1518- 1523 (1996); Asahi, J Neurosci Res 52:699-708 (1998)). Briefly, cells were maintained in growth media (D M E IWF- 12 (50:50) containing glutamine, N2 supplement (Invitrogen, Carlsbad, CA), bFGF at 20ng/ml, and amphotercin B in flasks coated with poly-L-ornithine and mouse laminin (Invitrogen, Carlsbad, CA). To initiate differentiation, cells were cultured on coated 12 well plates at 1 .0 x 10⁴ cells/well in differentiation media (D M E IWF- 12 (50:50) containing glutamine, N2 supplement (Invitrogen, Carlsbad, CA), bFGF at 2ng/ml, amphotercin B and 20 μg/ml tetracyline). Tetracycline was added daily to cell culture media at 20μg/ml. After 3 days, cells stopped dividing and began to send out neuronal processes. HC2S2 cells were transduced with rAAV1 -CAG-hEAAT3 on day 3 in culture with 5.0 x 10⁵ gp/cell. Cells were maintained in culture media, which was changed every other day for 7 days.

Glutamate Uptake

[³H]D-Aspartic-acid (PerkinElmer, Boston, MA) uptake was used to assess functional hEAAT3 activity. Seven days post transduction with rAAV1 - CAG-hEAAT3, differentiation media was removed and HC2S2 cells were gently rinsed with pre-warmed sodium-containing HEPES buffer (2OmM HEPES, 120 mM NaCI, 5mM KCI, 1 mM KH₂PO₄, 2mM CaCI₂, 1 OmM D- glucosθ, pH 7.4) or a sodium-free HEPES Buffer (2OmM HEPES, 120 mM choline, 5mM KCI, 1 mM KH₂PO₄, 2mM CaCI₂, 1 OmM D-glucose, pH 7.4). Media was replaced with HEPES +/- sodium containing 10OnM [³H]D-aspartic acid and incubated for 4 minutes at 37⁰C. Uptake was terminated by the removal of [³H]D-aspartic acid containing media, rinsed twice in ice cold sodium-free HEPES and cells were lysed in 0.1 N NAOH, 0.01 % SDS. Aliquots of the lysate were collected, incubated in liquid scintillation cocktail and counted for measure of [³H]D-aspartic acid on a Beckman LS 6500 Scintillation System. Specific hEAAT3 mediated uptake of [³H]-D-aspartic acid was calculated as total sodium-dependent uptake less the uptake in the absence of sodium and reported as pmol/mg protein/min. Protein concentration was determined on sister wells using the Bio-Rad DC protein assay (Hercules, CA).

Western Blot Analysis

Western blot analysis of the HC2S2 cell cultures were performed to evaluate total EAAT3 expression levels in control cells and cells transduced with rAAV1 -CAG-hEAAT3. Cells were removed from 12 wells plates and homogenized in lysis solution (2.5% sodium deoxycholate, 0.1 % protease inhibitor cocktail set III (Calbiochem, San Diego, CA), and 0.05% benzonase (EMD Biosciences, San Diego, CA) in PBS. Protein concentrations of lysate samples were determined using the Bio-Rad DC protein assay (Hercules, CA). Aliquots of homogenized HC2S2 cells (30μg) were loaded onto a NuPAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA). Proteins were transferred to Immuno-Blot PVDF membrane (Bio-Rad, Hercules, CA) and blocked in Tris buffer containing Tween-20 and 0.5% non-fat milk. Membranes were probed with antibodies to EAAT3 (1 :1000, ADI, San Antonio, TX) and actin (1 :1000, Sigma, St. Louis, MO). Proteins were visualized using ECL (Pierce, Rockford, IL) with species-specific HRP conjugated secondary antibodies. Blots were imaged on a Kodak Image Station 440 CF. Dosimetry analysis of all three bands, representing different multimers of EAAT3, were used to calculate fold change in EAAT3 protein expression corrected to β-actin controls. High-Performance Liquid Chromotography (HPLC) analysis

HPLC analysis was performed to evaluate the total intracellular concentrations of specific amino acids in control or rAAV1 -CAG-hEAAT3 transduced HC2S2 cultures. Cells were lysed in 0.1 N perchloric acid solution, centrifuged at 14,00Og for 10 minutes at 4⁰C and supernatant was retained. Protein concentrations were determined using the Bio-Rad DC microplate protein assay (Hercules, CA). Samples (10μl_) were derivatized with o-phtaldialdehyde (OPA) solution (90μl_) and 40μl of the derivatized sample was loaded for HPLC analysis.

Detection of amino acid content was performed by HPLC using an Amersham Bioscience AKTA purifier system (GE Healthcare, Piscataway, NJ) equipped with a Spherisord 5μM ODS1 reverse phase column (4.6x250mm, Waters Corporation, Tokyo Japan). Amino acid levels were quantified by fluorescence detection at 440 nm (emission) and 330nm (excitation). Mobile phase A was composed of 0.03M sodium acetate and 1% tetrahydrofuran. Mobile phase B was compose of 0.02M sodium acetate in 80% acetonitrile. Amino acids were separated using a linear increase in concentration of mobile phase B from 0% to 40% over 40 minutes at a flow rate of 0.8 ml/min. Retention time for GABA, glutamate and glutamine (Sigma, St. Louis, MO) were 29, 8, and 23 minutes respectively as confirmed by amino acid standards. Specific concentrations were determined through analysis of amino acid standards over an applicable range of concentrations.

Stereotaxic Injections

Eight-week old male Sprague-Dawley rats were anesthetized by brief isoflurane inhalation (Abbott Laboratories, North Chicago, IL) and intramuscular injection of ketamine (100mg/kg of body weight, Vetus Animal Health, Westbury, NY) then placed into a stereotaxic frame (Stoelting, Wood Dale, IL). Animals were maintained on 1 .0 LPM of oxygen and 2% isoflurane. Injections were performed from the following coordinates beginning at midline of the bregma: -3.3mm dorsal from midline of the bregma, +/- 1 .8mm lateral and -3.0mm depth from surface of the skull. The three rAAV1/2-GAD65p vectors (hrGFP, rEAAT3/AS, and flag-rEAAT3, 1 x 10¹³ gp/ml) were diluted 1 :1 with 25% mannitol (American Regent Inc., Shirley, NY) and 8 μl was delivered to each hippocampus at a rate of 0.5 μl/min.

Seizure Study

Four weeks post rAAV bilateral stereotaxic injections, animals were evaluated for seizure susceptibility. The pilocarpine (100mg/ml, BioChemika, Buchs, Switzerland) and atropine (100mg/ml, Sigma, St. Louis, MO) were prepared in a 0.9% saline solution, pH adjusted to 7.4 and filter sterilized. Thirty minutes prior to pilocarpine treatment, each animal received 100mg/kg atropine intraparitoneal (IP) to limit peripheral effects of pilocarpine. Pilocarpine solution was delivered I. P. at a concentration of 300mg/kg body weight. Animals were monitored and seizure-like behavior determined based on a modified Racine scale (Racine, Electroencephalogr Clin Neurophysiol 32:281 -294 (1972); Sperk, Prog Neurobiol 42:1 -32 (1994)). The behaviors were broken down into the following stages: 1 ) chewing, salivation, walking backwards, 2) head bobbing, tremors, wet-dog shakes, 3) forearm clonus, rearing/falling, 4) 1 class 5 seizure 5) 3+ class 5 seizures and 6) general tonic- clonic behavior, status epilepticus. Time to reach each stage was recorded for each animal and times were averaged between animals within the same treatment group. Sixty minutes post-onset of status epilepticus animals received 10mg/kg body weight of Diazepam (Abbott Laboratories, North Chicago, IL) to stop seizures.

lmmunohistochemistry

Brain tissues were harvested from animals injected with rAAV at 4 weeks post surgery and from pilocarpene injected animals that progressed to status epilepticus in the seizure study 48 hours after onset of status epilepticus. Animals were anesthetized with isoflurane and perfused with 4% paraformaldehyde, decapitated and brain tissue removed. Tissue was incubated post harvest for 24 hours in 4% PFA, and stored at 4⁰C until sectioning. Tissue was sectioned on a vibratome into 50μm slices, placed on charged slides, briefly dehydrated and stored at 4⁰C until used.

Cell type specific expression of GFP was evaluated 4 weeks following in vivo injection of rAAV1/2-GAD65p-hrGFP and rAAV1 /2-GAD65p-flag- rEAAT3 in brain slices from non-seizure animals. Slices were incubated in blocking buffer (1 .5% normal goat serum, 0.3% Triton-X 100) for 1 hour at room temperature (RT). After initial blocking, slices were stained with antibodies to GAD65 (1 :300) or the flag epitope (DYKDDDK) (1 :1000) (Chemicon, Temecula, CA) for 1 hour at RT, rinsed in blocking buffer and incubated in species-specific conjugated secondary antibody (1 :200, Molecular Probes, Eugene, OR). Slices were coverslipped with Fluorosave (Calbiochem, San Diego, CA). Images were obtained on a Bio-Rad Radiance 2000 MP laser scanning confocal microscope (Hercules, CA).

A marker for neurodegradation, Fluoro-Jade B (Histo-Chem, Inc., Jefferson, AR), was used to evaluate the level of seizure mediated damage. Tissues were processed as previously published (Wang, J Spinal Cord Med 23:31 -39 (2000)). Briefly, cryosectioned tissue was incubated in 1 % sodium hydroxide and 80% ethanol solution for 5 minutes, rinsed in 70% ethanol for 2 minutes, and rinsed in distilled water for an additional 2 minutes. Tissue was then incubated with gentle agitation in 0.06% potassium permanganate for 10 minutes, rinsed with distilled water and incubated in 0.0004% FluoroJade B working solution for 20 minutes. Tissues were rinsed with distilled water and allowed to dry in a 37⁰C incubator over-night. Slides were immersed in xylene for 1 minute and coverslipped with DPX. Tissue was imaged using an Olympus IMT-2 inverted fluorescent microscope attached to an Olympus digital camera (Olympus, Melville, NY) and captured using Magnafire SP imaging software package (Optronics, Goleta, CA). Total fluorescent intensity per region of interest was determined using Image Pro Plus software (MediaCybernetics, Silver Springs, MD).

Data Analysis

One-way ANOVA was performed to determine statistical significance of western blot dosimetry analysis, functional [³H]D-aspartic acid uptake, rates of seizure-like behavior and level of Fluoro-Jade B staining between the control tissue and transduced tissue. Statistical analysis was performed using Prism Software (GraphPad Software, Inc., San Diego, CA). Results

Overexpression of EAAT3 increases intracellular GABA levels

To determine if functional EAAT3 expression and activity could be increased, we first used an in vitro model in which the recombinant adeno- associated virus (rAAV) vector (rAAV1 -CAG-hEAAT3) was used to transduce cultures of HC2S2 cells with the hEAAT3 gene under control of the CAG promoter. HC2S2 cells are transformed rat hippocampal neurons under the control of the tet operon. Under normal culture conditions the cells grow and exhibit characteristics of a transformed cell line. However, in the presence of tetracycline, HC2S2 cells stop dividing and exhibit characteristics of differentiated adult rat hippocampal GABAergic neurons.

Western blot analysis indicated a 565% increase in EAAT3 expression in HC2S2 cultures transduced with rAAV1 -CAG-hEAAT3 compared to non- transduced control cultures (Figure 1 ). We further wanted to determine if the exogenous hEAAT3 was functional. Therefore, we compared [³H]D-aspartic acid uptake in nontransduced control and rAAV1 -CAG-hEAAT3 transduced HC2S2 cultures. A 424% increase in transporter activity was observed in transduced cultures compared to non-transduced controls, indicating that both EAAT3 expression and functional activity could be enhanced within GABAegic neurons (Figure 2).

To further test the hypothesis that EAAT3 activity influences GABA synthesis, we used HPLC analysis to measure the intracellular amino acid content of HC2S2 cells. Intracellular GABA levels were 136% higher in rAAV1 -CAG-hEAAT3 transduced cultures compared to controls (Figure 3A). Additionally, increasing EAAT3 expression resulted in a 56% greater intracellular concentration of glutamate (Figure 3B) as well as a 146% increase in intracellular glutamine (Figure 3C).

Targeted expression of EAAT3 to GABAergic neurons in vivo

To test our hypothesis in vivo required the ability to selectively target and modulate expression of the rat EAAT3 (rEAAT3) gene within GABAergic neurons of the rat hippocampus. Therefore, we first generated a rAAV vector (rAAV1/2-GAD65-hrGFP) that carried an expression clone, in which the GAD65 promoter was used to selectively drive expression of the GFP marker gene in GABAergic neurons. Adult, male Sprague-Dawley rats were sterotactically injected with rAAV1/2-GAD65-hrGFP bilaterally into the hippocampus. Four weeks post injection, tissue was harvested and sections were stained with an anti-GAD65 antibody to label GABAergic neurons and confirm cell type specificity of transgene expression. GAD65 is localized to GABAergic. A robust patter of GFP expression was observed throughout the CA1 -CA3 and hilus regions, which showed strong colocalization with GAD65 (Figure 4).

The rEAAT3 gene sequence was subcloned into this same rAAV vector in both the sense and antisense orientation to facilitate the overexpression and knock down of rEAAT3 in GABAergic neurons. rEAAT3 is normally expressed on inhibitory presynaptic terminals and co-localizes with GAD65 (Conti, Cereb Cortex 8:108-1 16 (1998)). Adult male Sprague-Dawley rats were injected with rAAV vectors carrying either the rEAAT3 gene in the antisense orientation (rEAAT3/AS) or in the sense orientation fused with a flag epitope tag at the N-terminus (flag-rEAAT3). Four weeks post injection tissue was harvested and sectioned. Brain tissue transduced with rAAV1 /2-GAD65- Flag-rEAAT3 was dual stained with antibodies against GAD65 and the flag epitope. Again, robust expression of flag-rEAAT3 was detected throughout the CA1 -CA3 region with strong colocalization with GAD65 staining (Figure 5). Images taken of tissue transduced with rAAV1/2-GAD65-rEAAT3/AS did not reveal detectable differences in rEAAT3 expression (data not shown). Due to the limited sensitivity of standard immunohistochemical techniques, knock down of this protein could not be efficiently detected. This may be due to the antisense construct knocking down, but not completely knocking out rEAAT3 expression within inhibitory neurons. In addition, rEAAT3 present in postsynaptic terminals of glutamatergic pyramidal neurons would not be altered and would be detected in immunohistochemical staining.

Modulation of rEAAT3 expression in GABAergic neurons influences sensitivity to pilocarpine-induced seizures.

We used the rat model of pilocarpine-induced seizures to further test the hypotheses that modulation of rEAAT3 expression on GABAergic neurons would alter GABA production and inhibitory synaptic strength. Age-matched adult male Sprague-Dawley rats were injected with rAAV1/2-GAD65- rEAAT3/AS (rEAAT3/AS), or rAAV1/2-GAD65-flag-rEAAT3 (Flag-rEAAT3). Virus injected groups and non-transduced controls were monitored for progression through distinct behavioral stages leading to status epilepticus (SE) after receiving IP injections of pilocarpine (300mg/kg) (Racine, Electroencephalogr Clin Neurophysiol 32:295-299 (1972a); Racine, Electroencephalogr Clin Neurophysiol 32:281 -294 (1972b); Sperk, Prog Neurobiol 42:1 -32 (1994)).

Overall, 40% of control and 50% of flag-rEAAT3 injected rats progressed to status epilepticus (SE) (Figure 6). In contrast, 100% of rEAAT3/AS injected rats progressed to SE. Significant differences were observed in the rate at which animals progressed to SE. The average time required for the untreated control group to reach SE was 16.58 +/- 0.55 minutes (Figure 6). In contrast, the rate at which rEAAT3/AS treated animals progressed to SE was significantly reduced to 1 1.28 +/- 0.65 minutes. Most notably, animals that overexpressed rEAAT3 progressed to SE 2.5 times slower than untreated controls. Significant differences were also noted between stages 4-6. Control animals typically experienced 3-4 seizures prior to entering SE. In contrast, it was not unusual for animals within the flag- rEAAT3 overexpressing group to experience as many as 7-9 seizures prior to reaching SE. However, the rEAAT3/AS treatment group frequently progressed from stage 4 (1 class 5 seizure) directly into tonic-clonic seizures, skipping stage 5 altogether.

Modulation of rEAAT3 expression in GABAergic neurons influences neuodegeneration following pilocarpine-induced seizures

We further wanted to determine if the modulation of rEAAT3 in GABAergic neurons could alter neuropathology following SE. Therefore, brain sections of animal from each treatment group that had undergone SE were harvested 48 hours after the onset of SE and stained with Fluoro-Jade B. Observations were made from similar regions of the hippocampus and cortex. Significantly more seizure-mediated neuropathology was observed in the antisense treated animals compared to the untreated controls. In contrast, significantly less damage was seen in the rEAAT3 overexpressing animals compared to controls (Figure 7A and 7B). The differences in neuroprotection/neuropathology were most striking within the CA1 region of the hippocampus, the targeted region of viral vector delivery. In addition, all three groups showed similar levels of neuropathology within the cortex, where no virus was injected (Figure 8A and 8B).

Summary

By altering the expression of EAAT3 we were able to change the threshold of seizure susceptibility, severity, duration, frequency and the rate of progression to SE. Animals transduced with rAAV1/2-GAD65-flag-rEAAT3 progressed to SE at a significantly slower rate. This may be attributed to increased capacity to withstand pilocarpine mediated excitatory stimulus due to elevated GABA supply and increased inhibitory postsynaptic strength. Increased GABA synthesis may enable extended inhibitory signaling during cyclic epileptic discharge.

ADDITIONAL EXAMPLES

In vivo model of increased expression of EAAT3 in GABAergic neurons

Glutamate transport from extracellular to intracellular spaces is one of the primary mechanisms for preventing excitotoxicity and reducing neuronal damage caused by epilepsy. While various types of glutamate transporters are found in both neuronal and glial synaptic terminals, only excitatory amino acid transporter 3 (EAAT3) is uniquely located in the presynaptic terminals of inhibitory GABAergic neurons. Rats with significantly reduced EAAT3 activity have reduced GABA synthesis, develop EEG seizures correlated with behavioral freezing episodes, limbic hyperexcitability and decreased tonic inhibition (Sepkuty, J. Neurosci, 22:6372-6379 (2002)). It has also been shown that reducing rEAAT3 activity reduces the strength of the inhibitory post-synaptic potential (IPSP), potentially contributing to the increased hyperexcitability of the system (Mathews J. Neuosci 23:2040-2048 (2003).

Methods:

A bipolar depth probe composed of two twisted wires and a guide cannula port (Plastics One, Roanoke, VA) were implanted in the hippocampus of male Sprague Dawley rats (300-45Og) (Fig. 9). A surface electrode placed in the skull and in contact with the undisrupted dura served as a ground. All three electrodes were collected into a 363 Plastics One pedestal and cemented to the skull of the animal. 300ng of kanic acid (KA) was delivered directly into the hippocampus to establish spontaneous but clinically unapparent seizures. Rats then underwent weekly 24-hour EEG recordings that allowed unrestricted movement while recording (Fig. 10). Once spontaneous EEG seizures were observed (typically 2 weeks after KA injection), an AAV vector was directly delivered into the hippocampus. The vector included the GAD65 promoter that ensured selective expression of either an EAAT3 expression cassette or a null expression cassette in only GABAergic neurons. Weekly 24-hr recordings were continued for four weeks after delivery of the virus (Fig. 1 1 ).

Summary of Results:

Increased expression of EAAT3 in GABAergic neurons resulted in a reduction in frequency and severity of spontaneous seizures in the rat hippocampus.

The present invention is not to be limited in scope by the specific embodiments described herein. Various modification of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the claims. It is also understood that all values are approximate, and are provided for description purposes only.

All patents, patent application, publications, product descriptions, and protocols cited throughout this application are incorporated herein by reference in their entireties for all purposes.

Claims

CLAIMSWhat is claimed is:

1 . A method of treating a human subject having focal brain seizures, the method comprising increasing expression of excitatory amino acid transporter 3 (EAAT3) in GABAergic neurons located in the brain of the subject where the focal brain seizures occur, wherein EAAT3 expression is increased in the GABAergic neurons sufficiently to reduce the severity, frequency, and/or duration of the focal brain seizures, thereby treating the subject.

2. The method of claim 1 , wherein the subject has chronic epilepsy and the method reduces the severity of the focal brain seizures in the subject.

3. The method of claim 1 , wherein the step of increasing expression of EAAT3 comprises directly administering, to a region of the brain where the focal brain seizures occur, a recombinant adeno-associated virus (rAAV) vector having a human EAAT3 gene.

4. The method of claim 3, wherein the rAAV vectors are administered directly to the hippocampus of the subject.

5. The method of claim 4, wherein the rAAV vectors are administered by stereotaxic injection.

6. The method of claim 1 , wherein the rAAV vector is rAAV-GAD65- hEAAT3.

7. A method of treating a human subject having brain seizures, the method comprising directly administering to the hippocampus of the subject a recombinant adeno-associated viral vector (rAAV) comprising an excitatory amino acid transporter 3 (EAAT3) gene which is operatively linked to a promoter suitable for expression of EAAT3 in GABAergic neurons in the hippocampus, wherein EAAT3 expression is increased sufficiently in the GABAergic neurons to reduce the severity, frequency, and/or duration of the brain seizures, thereby treating the subject.

8. The method of claim 7, wherein said promoter sequence is a glutamic acid decarboxylase 65 (GAD65) promoter.

9. The method of claim 8, wherein the rAAV further comprises the 3' Woodchuck hepatitis virus post-transcriptional regulation element (WPRE).

10. A recombinant adeno-associated viral vector (rAAV) comprising AAV 5' and 3' ITRs flanking a human excitatory amino acid transporter 3 (EAAT3) gene which is operatively linked to a promoter suitable for expression of the EAAT3 in GABAergic neurons of the hippocampus.

1 1. The vector of claim 10, wherein the promoter is glutamic acid decarboxylase 65 (GAD65).

12. The vector of claim 1 1 , further comprising the 3' Woodchuck hepatitis virus post-transcriptional regulation element (WPRE).

13. A pharmaceutical composition for treating brain seizures, the composition comprising: an recombinant adeno-associated viral vector (rAAV) and a pharmaceutically acceptable carrier, wherein the rAAV comprises rAAV 5' and 3' ITRs flanking a human excitatory amino acid transporter 3 (EAAT3) gene which is operatively linked to a promoter suitable for expression of the EAAT3 in GABAergic neurons of the hippocampus.

14. The pharmaceutical composition of claim 13, wherein the promoter is glutamic acid decarboxylase 65 (GAD65).

15. The pharmaceutical composition of claim 13, wherein the rAAV further comprises the 3' Woodchuck hepatitis virus post-transcriptional regulation element (WPRE).

16. Use of a recombinant adeno-associated viral vector (rAAV) in the manufacture of a medicament for the treatment of brain seizures, wherein the rAAV comprises rAAV 5' and 3' ITRs flanking an excitatory amino acid transporter 3 (EAAT3) which is operatively linked to a promoter suitable for expression of the EAAT3 in GABAergic neurons of the hippocampus.

17. The use of claim 16, wherein the brain seizures are epileptic seizures.

18. The use of claim 16, wherein the promoter is glutamic acid decarboxylase 65 (GAD65).

19. The use of claim 16, wherein the rAAV further comprises the 3' Woodchuck hepatitis virus post-transcriptional regulation element (WPRE).