Preparation of wt AAV.
wt AAV was prepared with 293 cells grown as monolayers in Dulbecco modified Eagle medium, supplemented with 10% fetal bovine serum and 100 U of penicillin-streptomycin per ml, at 37°C under humidified air containing 5% CO2. The cells were grown to confluency on Cell Factories (Nunc) and infected with wt AAV seed stock at a multiplicity of infection (MOI) of 1. The cells were coinfected with Ad5 at an MOI of 3. After 48 h, the cultures were harvested, resuspended in phosphate-buffered saline (PBS), and frozen and thawed three times. The crude lysate was heated at 56°C for 15 min to inactivate Ad and then centrifuged for 5 min at 9,000 × g to remove cellular debris. The supernatant’s volume was raised to 10 ml with PBS and loaded onto a HiTrap heparin affinity column (Pharmacia Biotech) at a rate of 1 ml per min with a peristaltic pump (Bio-Rad). The column was then washed with 20 ml of 0.01 M sodium phosphate buffer (pH 8) at a rate of 1 ml per minute. The flowthrough was retained and later analyzed. Virus was eluted at a rate of 1 ml per min (15 ml total volume) with a salt gradient (0 to 1 M NaCl) in 0.01 M sodium phosphate buffer (pH 8). Fifteen fractions were collected and analyzed by PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). PCR-positive fractions were pooled, and CsCl was added to the pooled sample to a density of 1.41 g per ml (refractive index, 1.3710). The sample was then loaded onto an ultracentrifuge tube (Beckman) and centrifuged in an SW50 swinging-bucket rotor (Beckman) at 35,0000 rpm at 4°C for 24 h. The contents of the tube were then fractionated (500-μl fractions) and dialyzed against PBS by using an ultraconcentrating tube with a molecular mass cutoff of 50 kDa (Amicon). The dialyzed fractions were then analyzed for the wt AAV genome by PCR. The positive fractions were pooled and analyzed by SDS-PAGE.
Viral capsid proteins were analyzed by PAGE. A 5-μl volume from each fraction was denatured, mixed with 1× loading dye (final concentrations, 12.5 mM NaH2PO4, 35 mM Na2HPO4, 0.5% SDS, 0.5% β-mercaptoethanol, 75 μg of bromophenol blue per ml, and 3 M urea), and loaded onto a 10% polyacrylamide gel (Bio-Rad). Samples were electrophoresed at 100 V for 2 h at room temperature with a MiniProtein electrophoresis apparatus (Bio-Rad). The gel slab was then fixed, stained with Coomassie blue (2.5 mg/ml in 45% methanol–10% acetic acid) at room temperature overnight, and destained (in 30% methanol–6% acetic acid) for several minutes until the background cleared, to visualize protein bands.
QC-PCR to determine the physical titer of AAV.
The physical titer was assessed by a PCR-based protocol. A 1-μl sample was taken from the purified stock and treated with 10 U of DNase (Boehringer Mannheim) in 10 mM MgCl2–50 mM Tris-HCl (pH 7.5) (total volume, 100 μl) for 1 h at 37°C. The sample was then treated with proteinase K (Boehringer Mannheim) at a final concentration of 0.2 μg/ml for 1 h at 37°C, using the manufacturer’s recommended buffer conditions. Viral DNA was then purified by two phenol-chloroform extractions and one phenol extraction. The sample was precipitated with ethanol and then centrifuged at 10,000 × g for 15 min. The supernatant was carefully discarded. The DNA pellet was resuspended in 10 μl of distilled H2O. The sample was then serially diluted. A PCR cocktail containing 1 μl of serially diluted viral sample and different amounts of the internally deleted competitor template was prepared. The PCR mixture consisted of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 200 mM each dATP, dGTP, dCTP, and dTTP, 0.5 U of Taq polymerase (Promega), and 5 pmol of each amplification primer. The primers used for quantitative competitive (QC)-PCR were as follows: the 5′ primer sequence was 5′-TGGCCCACCACCACCAAAGCCCGCA-3′ hybridizing to wt AAV nucleotides (nt) 2283 to 2308, and the 3′ primer sequence was 5′-TGGCCCGCCTTTCCGGTTCCCGAGG-3′ hybridizing to wt AAV nt 2668 to 2693. Thirty-five cycles of PCR were performed with the following program: 96°C for 1 min, 72°C for 1 min, and 60°C for 1 min. The products were analyzed on a 1.5% agarose gel stained with ethidium bromide. These primers generated a 410-bp product from the wt AAV sequence and a 360-bp product from the standard template. Quantification was performed by comparing the PCR bands of the known standard template to the unknown concentration of the competitor.
Infectious-center assay to determine the biological titer of AAV.
The infectious-center assay was used as a means of quantifying the infectivity of purified wt AAV. The assay measures the ability of the virus to infect, uncoat, and replicate. 293 cells were plated in a 96-well plate at 50%. At 24 h later, the wells were infected at serially diluted amounts of the purified wt AAV and with Ad at an MOI of 10. The cells were then incubated for 24 h and harvested with trypsin-EDTA solution. The cells from individual wells were suspended in 5 ml of PBS and vacuum filtered onto wet nylon membrane filters (Whatman). They were lysed by placing the membrane for 5 min (cell side up) on filter paper saturated with 10% SDS, processed, and hybridized at 60°C with an AAV [32P]DNA probe specific for wt AAV (AAV2) radiolabeled by random priming (Boehringer Mannheim). Individual spots corresponding to infectious centers were visualized by autoradiography and counted manually.
Vector construction and production.
The recombinant AAV, rAAV-UF5, expressing the humanized green fluorescent protein (hGFP) transgene driven by a cytomegalovirus promoter was packaged as previously described by Zolotukhin et al. (43
) with Ad5-infected 293 cells cotransfected with a helper plasmid (to provide rep
from an ori construct) and a vector plasmid containing the cDNA flanked by AAV inverted terminal repeats. The vector was purified by two successive CsCl ultracentrifugation steps.
Eleven female rhesus macaques ranging from 2 to 3 years of age and weighing between 2.8 and 4.2 kg were obtained (Covance Research). Sera from the animals were assayed for AAV antibodies prior to purchasing. The animals were housed at the University of Florida Animal Facility. The animals were sedated during all procedures by administration of 10 mg of Ketamine per kg intramuscularly. wt AAV (1010
IU) was administered intravenously (into the right femoral vein) to two animals in a 3-ml suspension of bacteriostatic 0.9% sodium chloride. Three animals received 1010
IU of wt AAV into the left quadriceps muscle (6.5 cm from the patella) in a 500-μl suspension of bacteriostatic 0.9% sodium chloride. Three other animals received 5 × 109
IU of wt AAV in a 250-μl suspension of bacteriostatic 0.9% sodium chloride into each nostril (for a total dosage of 1010
IU). Two animals received coadministrations of wt AAV and a mutant form of Ad. These two animals received 5 × 109
IU of wt AAV in a 250-μl suspension of bacteriostatic 0.9% sodium chloride into each nostril (for a total wt AAV dosage of 1010
IU) plus 5 × 107
PFU of AdHR405 per nostril (for a total AdHR405 dosage of 108
PFU). AdHR405 is a host range mutant form of Ad selected for growth on monkey cells (1
). The control animal was given 250 μl of bacteriostatic 0.9% sodium chloride into each nostril and was sedated regularly along with the other animals. The animals were bled every 7 days throughout the study.
Genomic DNA analysis.
High-molecular-weight DNA was extracted from animal tissue by using QIAamp tissue kits (Qiagen). The DNA concentration was determined by spectrophotometric analysis of the optical density at 260 nm. The DNA (30 μg) was digested for 24 h with KpnI (New England BioLabs) under conditions recommended by the manufacturer. The DNA was then separated by agarose gel electrophoreses (1% agarose) in TBE buffer (10 mM Tris borate, 2 mM EDTA [pH 8]). The agarose gel was acid treated for 20 min with 0.2 N HCl and denatured for 15 min with 1.5 M NaCl–0.5 M NaOH. The agarose gel was then neutralized with 3 M NaCl–0.5 M Tris and then blotted via capillary forces by using 20× SSC (1× SCC is 0.15 M NaCl plus 0.015 M sodium citrate) (Sigma) onto nylon membranes. The nylon membrane was then baked for 2 h at 80°C under vacuum. The membranes were hybridized at 60°C with an AAV [32P]DNA probe specific for wt AAV (AAV2) radiolabeled by random priming (Boehringer Mannheim). The hybridization solution contained 6× SSC, 0.5% SDS, 1× Denhardt’s solution, 20 μg of herring sperm DNA per ml, and 0.01 M EDTA. The membrane was washed in large volumes of 2× SSC–0.1% SDS at 60°C, dried, placed in an X-ray cassette (Kodak), and exposed to X-ray film (Kodak) for several days.
DNA PCR for detection of viral genomes and site-specific integration.
Genomic DNA samples from peripheral blood mononuclear cells (PBMCs) and from the sites of virus administration were purified with QIAamp blood kits (Qiagen). High-molecular-weight DNA was extracted from animal tissue by using QIAamp tissue kits. PCR was carried out with 100 ng of genomic DNA (or 1 μl of fractioned material) added to 50 μl of total PCR cocktail (ingredients and primers are given above). Thirty-five cycles of PCR were performed with the following program: 96°C for 1 min, 72°C for 1 min, and 56°C for 1 min. The products were analyzed on a 1% agarose gel, stained with ethidium bromide, transferred to nitrocellulose, and hybridized with a wt AAV-specific probe radiolabeled by random priming (Boehringer Mannheim).
To detect site-specific integration, DNA samples that were positive for AAV sequences by the internal PCR described above were also analyzed by the PCR dot blot methods described by Yang et al. (41
), in which the 5′ PCR primer was chosen from within the 3′ end of the wt AAV sequence (5′-ATAAGTAGCATGGCGGGTTA-3′) and was directed outward from the proviral insert while the 3′ primer was chosen from within the AAVS1 site (5′-GCATAAGCCAGTAGAGCTCA-3′). Homology between the primer sequence and the rhesus sequence was confirmed by sequence alignment. PCR products were immobilized on nylon membranes by using a Schleicher and Schuell Minifold II dot blot manifold, and a random-primed 32
P-labeled probe from the end of the AAV genome [the 180-bp Pvu
I fragment from pSub201(+)] was used for the hybridization. The specificity of this signal for AAV-chromosomal junctions was confirmed by comparison with a control in which only the internal AAV probe was used.
Fluorescence in situ hybridization (FISH) analysis.
Metaphase chromosomes and interphase nucleus preparations were prepared by a mitotic shakeoff method (25
). Hypotonic fixation and slide preparation were performed by standard cytogenetic methods. A wild-type AAV probe was labeled and hybridized to each preparation as previously described (25
). Photomicrographic images of nuclear signals were acquired by using a cooled charge-coupled device camera under the control of the Metamorph software package.
293 cells were plated in a 96-well plate at 50 to 75% confluency (5 × 103 cells per well). The cells were cultured overnight at 37°C in humidified air containing 5% CO2. The following day, serial dilutions of animal serum (day 0 and day 21) were incubated with 105 IU (equivalent to an MOI of 10) of recombinant AAV expressing the hGFP transgene (rAAV-UF5). The dilutions were performed in Hanks balanced salt solution a 100-μl total volume. The sample was then incubated at 37°C for 1 h. After 1 h, the medium from the previously plated cells was removed and 100 μl of Dulbecco modified Eagle medium supplemented with 20% heat-inactivated fetal bovine serum and 200 U of penicillin-streptomycin per ml containing 2 × 105 PFU of Ad was added. Additionally, 100 μl of the serially diluted serum plus rAAV-UF5 solution was added to the wells. The cells were cultured for 24 h at 37°C in humidified air containing 5% CO2. After 24 h, the transgene product was visualized under a fluorescence microscope. The end point was defined as the dilution of serum which inhibited the transgene efficiency by at least 10-fold.
Antigen-specific lymphocyte proliferation assay to assess cell-mediated immunity to AAV.
Heparinized whole blood (5 ml) was collected and diluted 1:1 in Hanks buffered salt solution in a conical centrifuge tube. Ficoll-Hypaque (5 ml; Pharmacia) was slowly layered at the bottom of the conical tube. The tube was then centrifuged for 30 min at 500 × g at room temperature. The layer above the clear layer was carefully removed with a sterile transfer pipette. The removed material was transferred to a centrifuge tube containing 10 ml of Hanks buffered salt solution and centrifuged for 10 min at 500 × g at room temperature. The supernatant was removed, and the cell pellet was washed again with 10 ml of Hanks buffered salt solution and recentrifuged for 10 min at 500 × g at room temperature. The supernatant was discarded, and the cell pellet was resuspended in 2 ml of RPMIC+ medium (CellGrow). The cells were counted by a Trypan blue exclusion method. β-Mercaptoethanol was added at 2 μl per ml of cell suspension (adjusted to account for 106 cells per ml). Two 96-well plates were set up for every animal, one for the antigen (VP3 capsid proteins) and a second for the mitogen phytohemagglutinin as a positive control. Cells were plated on the wells, and the respective agent was added and incubated for 3 days at 37°C in humidified air containing 5% CO2. On day 3, 20 μl of a 1:20 [3H]thymidine dilution was added to the mitogen-treated plate. On day 4, the mitogen-treated cells were removed and the level of radioactivity was determined. On day 5, 20 μl of a 1:20 [3H]thymidine dilution was added to the antigen-treated plate. After 24 h, the plate was harvested and the level of radioactivity was determined by liquid scintillation counting.
Tissue preparation and histology.
Tissues from the lungs, nasal passages, trachea, thymus, bronchial lymph nodes, heart, liver, spleen, pancreas, kidney, jejunum, mesenteric lymph nodes, gonads, brain, and muscles were isolated aseptically and placed in 4% paraformaldehyde for 24 h at 4°C. The tissues were then embedded in paraffin, and 10-μm sections were made. The sections were then stained with hematoxylin and eosin, coverslipped, and photographed with a Zeiss Axioskop upright microscope.