Treatment of neurological disorders

The invention provides for the use of recombinant AAV virions to deliver an AAV vector containing an EAAT gene such as EAAT2, to provide a therapeutic effect in neurological disorders such as ALS and epilepsy. The invention also provides for non-viral delivery systems to delivery EAAT2 for a therapeutic effect in ALS and epilepsy.

EXAMPLES 
 Example 1 
 Preparation of a Gene Transfer Vector The vector is an adeno-associated vector expressing the human 1.8 kb EAAT2 cDNA under the control of the 2.2 kb glial fibrillary acidic protein (GFAP) promoter. The EAAT2 gene and the promoter are flanked by the AAV 145 bp inverted terminal repeats (ITR). The vector can be constructed using standard recombinant DNA techniques (See Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)). 
 Example 2 
 Packaging Protocol for the Production of Recombinant Virions Recombinant AAV-GFAP-EAAT2 virions (rAAV-GFAP-EAAT2) can be generated using the helper-free packaging system as previously described by During, M. J. et al. (1998) Nature Med. 4:1131-113, with a slight modification. Human embryonic 293 kidney cells (from a Master Cell Bank), passages &num;4-12 only, were used for packaging. 1.5×10 7 cells were seeded into 40×15 cm dishes overnight in complete DMEM (Gibco) containing 10% fetal bovine serum (Hyclone), 0.1 mM MEM non-essential amino acids solution (Gibco), 1 mM MEM sodium pyruvate (Gibco), 0.05% Penicillin-Streptomycin (5,000 units/ml, Gibco). When close to 70% confluent (2-3 hours prior to transfection), the cells were fed with fresh Iscove modified DMEM (Gibco) containing 10% fetal bovine serum without antibiotics. All plasmids were prepared by alkaline lysis, further purified by HPLC (BioCAD, Sprint, PerSeptive Biosystems), and concentrated by 2 volumes of 100% ethanol (AR grade, BDH). A11 HPLC elute buffers (Buffer A: 250 mM TrisHCl, 10 mM EDTA, pH 8.0; Buffer B: 25 mM TrisHC1, 1 mM EDTA, 2M NaCl, pH, 8.0; Buffer C: Milli Q water) used for purification was autoclaved and filtered prior to usage. For each 15 cm tissue culture plate, a total of 60 &mgr;g of plasmid DNA is dissolved in 3.0 ml of 0.25M CaCl 2 and then quickly mixed with 3.0 ml of HEPES-buffered saline (50 mM HEPES, 280 mM NaCl, 1.5 mM Na 2 HPO 4 &lsqb;pH 7.10&rsqb;), incubated for 2 min and added to the cells. At 6-8 hours after transfection, the medium was aspirated and cells were washed with IMDM supplemented with 10% fetal bovine serum without antibiotics; the washing medium was aspirated and replaced with fresh IMDM (Gibco) containing 10% fetal bovine serum with trace antibiotics. The cells are harvested 48 hours after transfection. After low-speed centrifugation on a tabletop centrifuge, the cell pellets were resuspended in 150 mM NaCl, 20 mM Tris pH 8.0. Cell debris was removed with low speed centrifugation. The clarified supernatant was collected into a 25 ml polypropylene tube containing 0.5% sodium deoxycholate and Benzonase 5 U/ml. The cells were then incubated in a 37° C. waterbath for 30-40 min, before removing cell debris by centrifugation at 3000 g×15 min, 4° C. Supernatants were heated at 56° C. for 15 min, then frozen in a dry ice/ethanol bath, and thawed before removal of cell debris by centrifugation at 3000 g×15 min, 4° C. Supernatants were filtered through a 32 mm 0.45 um Acrodisc syringe filter to remove any particulate matter, prior to heparin column purification. Supernatants were run at a flow rate of 2 ml/min through a heparin column (1 ml HiTrap heparin columns, Sigma &num;5-4836). Columns were washed with 20 ml 100 mM NaCl, 20 mM Tris (pH 8.0). The elution fractions are spun down to as small a volume as possible. The fractions were dialyzed overnight against 1XPBS with 1 mM MgCl 2 , using a dialysis membrane (Pierce Slide-A-Lyzer dialysis cassettes, 3500 MWCO). Purified virus on a protein gel was stained with silver stain to determine the purity. 
 Example 3 
 Preparation of Primary Neuronal Cultures In order to obtain neuronal cultures, primary cortical, striatal, hippocampal and nigral (mesencephalic) cell cultures can be prepared from E15 pregnant Wistar rats. The embryos from two litters (approximately 24 embryos in total) can be removed into a dish of warm dissecting medium (Ca 2&plus;− and Mg 2&plus;− free Hank's balanced salt solution containing 0.6% glucose, 100 U/ml penicillin, 100 &mgr;g/ml streptomycin, 15 mM HEPES). The appropriate brain regions can be dissected out and collected into tubes containing fresh dissecting medium. Tissue can then be digested in a trypsin solution (0.25% trypsin, 200 &mgr;g/ml DNase in dissecting medium) for 15 minutes at 37° C. in a shaking waterbath. The trypsin reaction can be terminated by the addition of trypsin inhibition medium (100 &mgr;g/ml soybean trypsin inhibitor, 20% fetal bovine serum, 200 &mgr;g/ml DNase in dissecting medium), and the tissue can be washed twice in wash medium (dissecting medium containing 10% fetal bovine serum and 200 &mgr;g/ml DNase). A cell suspension can then be obtained by triturating the tissue using a small-bore, fire-polished Pasteur pipette until tissue clumps are no longer visible, and then the suspension can be filtered through a 100 &mgr;m nylon filter to eliminate any remaining cell clumps. The cells can then be pelleted at 400 g, resuspended, counted and plated onto poly-1-lysine-coated dishes at a density of 250,000 cells/well in 24 well-plates and at a density of 100,000 cells/well in 96 well-plates in Neurobasal™ medium (Gibco BRL) containing B27 supplement (Gibco BRL) and 0.5 mM 1-glutamine. Medium can be replenished every 48 hours. 
 Example 4 
 Vector Characterization (a) Determination of rAAV Titer with Optical Density (O.D.) Reading Estimation of viron concentration was accomplished by UV spectrophotometric analysis of extracted viral genomic DNA, using the formula that one absorbance unit at 260 nm is equal to 10 12 viral particles per ml (Curiel et al., 1991), where 1 mg viral protein is equal to 3.4-3.5×10 12 viral particles. (b) Determination of rAAV Titer with ELISA A sandwich ELISA technique was used to determine rAAV titres. A monoclonal antibody specific for AAV assembled capsids was coated onto microtiter strips and was used to capture AAV particles. A biotin-conjugated monoclonal antibody to AAV was bound to the immune complex, and streptavidin peroxidase conjugate reacted with the biotin molecules. Addition of substrate solution resulted in a color reaction which was proportional to specifically bound virus particles, and allowed the quantitative determination of an unknown particle titer (Wistuba et al., 1997). The AAV titration ELISA kit was provided by Progen (Germany). One 100 &mgr;l of ready-to-use wash buffer, positive and negative controls and dilutions of standard and samples were pipetted into appropriate wells of the microtiter strips, and strips were sealed with adhesion foil. After incubation for 1 hour at 37° C., the solution was removed and each well was rinsed 3 times with 200 &mgr;l of washing buffer for 30 seconds. 100 &mgr;l of ready-to-use biotin conjugate was added, and strips were sealed with adhesion foil and incubated for one hour at 37° C., washing steps were repeated as above. 100 &mgr;l of ready-to-use streptavidin conjugate was added, and strips were sealed with adhesion foil and incubated for one hour at 37° C. Washing steps were repeated as described above. 100 &mgr;l of substrate was pipetted into each well and incubated at room temperature for 10 min. The reaction was stopped by adding 100 &mgr;l of stop solution into each well. Absorbance is measured photometrically at 450 nm. (c) Determination of AAV Particle to Transducing Unit Ratio To test for the AAV transducing unit ratio, 5×10 4 293 cell per well were seeded onto collagen-coated 24 well plates. The cells were grown in DMEM (Gibco) containing 10% fetal bovine serum (Hyclone), 0.1 mM MEM non-essential amino acids solution, 1 mM MEM sodium pyruvate, 0.05% Penicillin-Streptomycin (5,000 units/ml), at 5% CO 2 and 37° C. overnight. 0.5 &mgr;l of AAV/GFAP EAAT2 virus was added and incubated for 48 hours. For rat primary neurons and glia, E15 animals were used for nigra and cortex preparation, while E18 animals were used for hippocampal and striatal primary cell preparation. 250,000 harvested cells per well were pipetted into poly-1-lysine-treated 24 well plates, and incubated at 5% CO 2 and 37° C. for 24-48 hours. Medium B (15% FCS, 0.6% glucose, 100U per ml pen/strep in DMEM/F12) was added on cultures. After 3 days incubation, 0.5 &mgr;l of AAV-NSE-EAAT2 virus was added to cortical culture, and to nigral, hippocampual and striatal cultures after 4-5 days incubation. All media was replaced with fresh culture medium one day before addition of virus; cultures are incubated at 5% CO 2 and 37° C. for 3 days, then cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 15 minutes and washed with phosphate buffered saline (PBS) containing TritonX100. (d) Determination that rAAV is Stable to Dialysis To determine rAAV virus stability during the dialysis processing, rAAV-CMV-lacZ virus was used for comparison. After ultracentrifugation, rAAV-CMV-lacZ from the same collection tube was divided and processed into two fractions, with and without dialysis, 1×10 6 particles of rAAV virus per well, & added to 6-well plates seeded with 5×10 5 293 cell for 24 hours. After incubation at 37° C. for 2 days, enzyme activity is determined by &bgr;-Gal ELISA (Boehringer Mannheim). For rAAV-GFAP-EAAT2, 5×10 4 human 293 cells were seeded onto a collagen-coated 24-well plate for 24 hours and 1×10 5 virus particles with or without dialysis were added into each well & incubated for 3 days prior to fixation for immunocytochemistry. The recovery rate of rAAV-GFAP-EAAT2 virus during the dialysis is run in a separate experiment, and is estimated by adding trace of hemoglobin, 14 C-methylated (Sigma) into samples for dialysis. A part of sample is taken to measure radioactivity by liquid scintillation counter (Wallac) before and after dialysis. (e) Determination of 293 Cell Protein Level Approximately 5-10×10 8 particles were separated on a 10% SDS-PAGE gel and stained with Coomasie R250. This analysis shows the correct virion protein composition and presence of less than 1% contaminating cellular proteins. Protein bands were captured with a video camera and saved to a Macintosh computer file. Integrated densities from each band were measured and analyzed with the NIH Image software. Silver staining was also performed to analyze the protein content of vector stocks, which show ˜99% purity. (f) Determination of Infectious Adenovirus Lack of contamination with infectious adenovirus—2×10 6 293 cells (containing the E1A gene) were infected with 20 microliters (2×10 10 particles) of the rAAV and incubated for three days at 37° C. in 5% CO 2 . No cytopathic effect was obtained. An immunofluorescent assay was also employed, in which the infected cells were stained for adenovirus hexon protein in an immunofluorescence assay (Chemicon). The sensitivity of this test is 6 adenoviral particles in the presence of 6×10 10 control rAAV, with no signal apparent in the vector stock. (g) Analysis of Wild-Type AAV The presence of wild-type AAV was determined by a PCR assay (see Snyder et al., 1997). 5×10 10 rAAV particles (200 &mgr;l) were treated with DNase I (100 units/ml for 30 min at 37° C.) to degrade any unencapsulated DNA, with proteinase K (0.5 mg/ml for 60 minutes at 37° C.) to liberate the rAAV genomes, which were phenol extracted ×2, ethanol precipitated, and dissolved in 30 &mgr;l of water. 3 &mgr;l volume (˜6×10 9 rAAV particles) is subjected to PCR along with positive and negative controls. The PCR primers were “D1” (5′-ACTCCATCACTAGGGGTTCC-3′) in the AAV ITR; “D2” (5′-GGTAATGATTAACCCGCCATGCTACTTATC-3′) also in the ITR; “AAV2S2” (5′TCAGAATCTGGCGGCAACTCCC-3′) in the AAV rep gene; “Splice1” (5′-TCGTCAAAAAGGCGTATCAG-3′) in the AAV splice region; “CAP2” (5′-TCCCTTGTCGAGTCCGTTGA-3′) in the AAV cap gene; and “CAP1” (5′-CAGAAGGAAAACAGCAAACG-3′) in the AAV cap gene. PCR is carried out under standard conditions with Taq polymerase for 25 cycles using the following primer pairs: D1 and AAV2S2 to detect left end sequences in wild-type AAV; D2 and AAV2S2 to specifically detect left end sequences in wild-type AAV arising from recombination between the vector and helper plasmids; Splice1 and CAP2 to detect internal sequences; and CAP1 and D2 to detect right end sequences in both wild-type AAV virus and wild-type AAV arising from recombination between the vector and helper plasmids. The products are separated on a 2% agarose gel and stained with ethidium bromide, and no bands indicative of a wild-type AAV contamination are detected to a sensitivity of one wild-type in 10 9 rAAV. The presence of wild-type AAV was also determined by a modified replication center assay as described by Snyder et al. (1996). Human 293 cells were seeded in a 24-well plate. All wells (1×10 5 cells) were then co-infected with adenovirus at an MOI of 20 and also 10, 5, 2.5 or 1 ul of the rAAV stock. As a positive control, Ad-infected cells were infected with 1000, 100, 10 or 1 wtAAV particles, which were spiked into a rAAV stock already determined to be free of wild-type AAV. Cells were incubated for 24 hours; the cell culture supernatants (containing any detached cells) were removed and diluted into 10 ml PBS. The cells were detached from the plate with trypsin and combined with their corresponding supernatants. The single-cell suspension was vacuum filtered onto nylon membranes wetted with PBS, and the membranes were incubated cell side up on Whatman filter paper saturated with 0.5N NaOH/1.5M NaCl for 5 min at room temperature, followed by Whatman paper saturated with 1M Tris-HCl (pH 7.0)/2× SSC for 5 min at room temperature. The paper was probed with rep and cap sequences and exposed to film. No wild-type AAV was detected in the rAAV stock, to a sensitivity of 1 in 10 9 rAAV, indicating a complete absence of wtAAV contamination. (h) Delivery and Distribution The method of delivery is important in obtaining efficient transduction of target cells in the mammalian brain. A recent study Cunningham et al., (1999) shows that vector concentration is a significant parameter for enhancing transduction. Therefore the quality and concentration of vector stocks was a critical. With titers of >10 13 transducing particles/ml, delivery of 3 microliters at a single site can transduce a region extending several millimeters. Furthermore, microperfusion at slow flow rates using high titer will optimize expression for a given expression cassette and vector dose. 
 Example 5 
 In vivo Expression of EAAT2 Preliminary results from a transgenic mouse produced by a cross between a mutant ALS mouse and a mutant mouse that significantly overexpresses EAAT2, shows a substantial increase the life span of the animal. Overexpression of EAAT2 engineered with an amplified GFAP promoter in a 10X transgenic mouse was phenotypically normal. The 10xEAAT2 mouse was crossed with the familial ALS mouse model (SOD G93A). The resulting double-heterozygote offspring of that cross has now survived 12 months and is still showing no symptoms of the disease. This offspring is now 6.5 months beyond the normal life expectancy of a SOD g93A transgenic. The single-heterozygote SOD offspring of the cross that did not contain the over expression of EAAT2 died at the normal life span for a G93A mouse. For comparison purposes, data on a mouse on Rilutek (a glutamate release inhibitor) extended the life of the G93A mouse only by 2-3 weeks, and the longest a genetic cross has only extended the life of a G93A mouse by 3 months.