Both hemostatically normal and HB dogs from the colony at the University of North Carolina at Chapel Hill were used in this study. This HB canine model is characterized by the presence of a missense mutation within the region of the gene encoding the catalytic domain of FIX, resulting in complete absence of FIX antigen or activity in plasma, mimicking severe human HB.31
At the time of gene transfer, all animals were naive for antibodies to AAV capsid, with the exception of dogs used in re-administration experiments. Sedated dogs were administered AAV vectors by either i.m. injection or ATVRX, as previously described.30
At the time of vector administration, all HB dogs were supplemented with pooled normal canine plasma at doses calculated to achieve at least 10–20% of normal cFIX plasma activity. Dogs received 6 weekly intravenous infusions of cyclophosphamide (200 mg/m2
of body surface area) starting 1 week before vector delivery as previously described.28
Animals were bled periodically to obtain plasma and PBMCs. All procedures were approved by the Institutional Animal Care and Use Committee at the Children's Hospital of Philadelphia and the University of North Carolina at Chapel Hill.
AAV vectors were produced by triple transfection of HEK293 cells followed by cesium chloride gradient–based purification as previously described.13
All vector preps used were devoid of empty capsids. Vectors were titered by dot-blot hybridization or real-time PCR; titers were expressed as vg/ml. An AAV expression cassette with the cFIX complementary DNA under transcriptional control of the cytomegalovirus immediate-early enhancer–promoter, a chimeric β-globin/cytomegalovirus intron, and the human growth hormone polyadenylation signal13
was used in most of the experiments in HB and normal dogs. An AAV “null” vector carrying an antisense copy of the β-galactosidase complementary DNA under the control of a cytomegalovirus promoter was used in a normal dog. Vectors were pseudotyped with either AAV-1, AAV-2, or AAV-6 capsids.
cFIX antigen detection. cFIX antigen levels were measured by immunoassay using a polyclonal matched-pair antibody set for cFIX enzyme-linked immunosorbent assay (Affinity Biologicals, Ancaster, Ontario, Canada) following manufacturer's instructions. Serial dilutions of normal pooled canine plasma were used as standards. With this methodology, cFIX was undetectable in untreated HB animals.
The presence of cFIX inhibitory antibodies was determined as previously reported by Bethesda assay.22
One Bethesda unit represents an amount of antibody sufficient to neutralize 50% of FIX activity. Antibody subclasses against cFIX were evaluated by enzyme-linked immunosorbent assay capture immunoassay. Briefly, microtiter plates were coated with 1 µg/ml of purified plasma-derived cFIX (Enzyme Research, South Bend, IN). Plates were washed and blocked for 2–4 hours with 3% bovine serum albumin 0.05% Tween 20 in phosphate-buffered saline. Samples were diluted 1/20 or 1/40 in LowCross-Buffer (Candor Bioscience, Weissensberg, Germany) and incubated for 2 hours at 37 °C. Antigen-antibody complexes were detected by 2-hour incubation at 37 °C with horseradish peroxidase–conjugated goat anti-dog IgG1 or sheep anti-dog IgG2 (Bethyl Laboratories, Montgomery, TX). Similarly, to detect antibody responses against viral proteins, plates were coated with serotype-specific AAV empty capsids at 1 µg/ml. Samples were diluted 1/200 to 1/800 in 3% bovine serum albumin 0.05% Tween 20 in phosphate-buffered saline and incubated overnight at 4 °C. In both cases, a standard curve generated by coating serially diluted dog reference serum with known quantities of IgGs (Bethyl Laboratories) was used for quantification of results.
One-color ELISpot assays were used to measure IFN-γ or IL-10 responses to cFIX or AAV capsid proteins in PBMCs isolated from AAV-treated dogs. Peptide libraries of 15-mers overlapping in sequence by 10 amino acids and spanning the sequence of cFIX and of the AAV capsid serotype-specific VP-1 protein were synthesized (Mimotopes, Clayton, Australia). Peptides were arranged into a matrix as previously described,26
or alternatively assembled into pools of 10–24 peptides each. ELISpot assays were performed as previously described.55
Briefly, a 1/60 dilution of anti-canine IFN-γ or anti-canine IL-10 antibody (R&D Systems, Minneapolis, MN) was used to coat ELISpot plates (Millipore, Bedford, MA) overnight at 4 °C. After blocking of free binding sites for 2 hours, 100 µl of culture media (2-mixed leukocyte culture 4% heat-inactivated fetal bovine serum20
) containing the antigens were added to each well. Media containing a mixture of 0.05 µg/ml of phorbol 12-myristate 13-acetate (Sigma, St Louis, MO) and 1 µg/ml of ionomycin (Sigma) served as positive control, while plain media served as negative control. In all experiments, the positive control gave a robust response (>1,000 spot-forming units/106
PBMCs), confirming good cell viability. Cryopreserved PBMCs were thawed, washed, and adjusted to a concentration of 2–2.5 × 106
cells/ml in culture media, and 100 µl of the suspension was carefully added to each well. Each condition was assayed in triplicate. Cultures were incubated for 24 hours (IFN-γ) or 48 hours (IL-10) at 37 °C, 10% CO2
. Plates were subsequently washed, and a biotinylated anti-canine IFN-γ antibody or anti-canine IL-10 antibody diluted 1/60 in phosphate-buffered saline 1% bovine serum albumin was added. Finally, plates were washed and developed after addition of streptavidin–alkaline phosphatase (Mabtech, Cincinnati, OH) and a chromogenic substrate (BCIP/MBT; KPL, Gaithersburg, MA). The number of spots was determined using an ELISpot reader (CTL, Cleveland, OH) and analyzed with Immunospot version 3.2 software (CTL). A response was considered to be positive if the average number of spot-forming units per million PBMCs was at least 50 and also thre times greater than the media control.
T-cell expansion experiments and flow cytometry.
A volume of 1 × 106
PBMCs were resuspended in ~500 µl of 4% fetal bovine serum 2-mixed leukocyte culture media,20
along with an equal volume and concentration of irradiated feeder cells, and cultured at 37 °C and 5% CO2
. Irradiated feeder cells were from the same initial PBMC population. Peptides were added at a concentration of 10–50 µg/ml on day 0 to stimulate PBMCs for 1 week. To sustain culture growth, canine IL-2 (R&D Systems) was added every 2 days at a 10 ng/ml concentration. On the final day, all cells were re-stimulated with peptide 68, and monensin was added (Golgi Stop, BD Biosciences, San Jose, CA) according to manufacturer's directions. After a further 4–5 hours of incubation, cells were harvested into fluorescence-activated cell sorting tubes, washed twice with FACS buffer (2% fetal bovine serum in phosphate-buffered saline, 0.05% sodium azide), and stained with anti-canine CD4-FITC (AbD Serotec, Kidlington, UK) for 1 hour at 4 °C. Cells were washed twice with FACS buffer, then incubated in Fixation/Permeabilization Buffer (eBioscience, San Diego, CA) for 1 hour at 4 °C. Cells were washed twice with Permeabilization Wash Buffer (eBioscience), and incubated in biotin-binding NeutrAvidin (Invitrogen, Carlsbad, CA) for 30 minutes at room temperature. After two more washes in Permeabilization Wash Buffer, cells were intracellularly stained with previously conjugated anti-canine IL10-biotin (R&D Systems) to streptavidin–phycoerythrin (Invitrogen), along with anti-Foxp3-APC (eBioscience) for 1 hour at 4 °C. Finally, cells were washed twice with Permeabilization Wash Buffer, and resuspended in 4% paraformaldehyde. For the suppression assay, PBMCs were expanded in the presence of cFIX or Peptide 68 for 5 days (suppressor cells), and plated at a 5:1 T suppressor:T effector ratio with unexpanded autologous effector cells labeled with carboxyfluorescein succinimidyl ester (Molecular Probe, Invitrogen, Carlsbad, CA) to distinguish them from the suppressor cell population. This was done in anti-CD3 coated plates to stimulate target T-cell IFN-γ production (0.5 µg/well for 96 wells), with 4 × 105
of expanded suppressor cells in 100 µl and 1 × 105
effector cells in 100 µl. Cells were incubated for 1 hour, after which monensin was added. After an additional 4-hour incubation at 37 °C, 5% CO2
, cells were harvested and stained with anti-CD4 PacBlue, anti-CD8 PE, and anti-IFN-γ Alexa 647 antibodies (AbD Serotec, Kidlington, UK). IFN-γ secretion was measured on CFSE+
T-cells. All samples were read on a FACS CantoII flow cytometer (BD Biosciences); data were analyzed using the FACS Diva Software (BD Biosciences) and Flowjo (Treestar, Ashland, OR).
Cytokine profile. Serum samples were analyzed at baseline and at days 1, 3, 21, and 60 after vector delivery by multiplex assay on a Luminex 100 instrument at the Radioimmunoassay and Biomarker core of the University of Pennsylvania with the Milliplex Canine Cytokine/Chemokine Panel (Millipore, Billerica, MA) for the simultaneous detection and quantification of 14 canine cytokines. The sensitivity of this assay was 14.4 pg/ml for granulocyte–macrophage colony-stimulating factor, 1.6 pg/ml for keratinocyte-derived chemokine, 6.4 pg/ml for IL-2, 12.1 pg/ml for IL-6, 20.3 pg/ml for IL-8, 14.8 pg/ml for IL-15, 8.6 pg/ml for monocyte chemotactic protein -1, 4.4 pg/ml for IFN-γ, 2.4 pg/ml for interferon-γ inducible protein -10, 28.8 pg/ml for IL-4, 4.6 pg/ml for IL-7, 1.6 pg/ml for IL-10, 4.6 pg/ml for IL-18, and 0.4 pg/ml for TNF-α.
Biodistribution. Total DNA was isolated with either QIAamp DNA Mini Kit (Qiagen, Valencia, CA) or MasterPureDNA Purification Kit (Epicentre Biotechnologies, Madison, WI). Vector genome copy number in 100–200 ng of genomic DNA was determined by quantitative real-time PCR with a set of primers and probe placed across a splicing junction in the cFIX complementary DNA. Forward primer: 5′-AAC GTC ACC CAA CCG CTT AA-3′ reverse primer: 5′-ATG ATG GAA CCT CCG CAG AA-3′ probe: 5′-FAM-CCA GGT CAA TTC CCT TGG CAG GTC C-Quencher-3′ (Applied Biosystems, Foster City, CA). Serial dilutions of a linearized plasmid bearing the cFIX expression cassette used in the study supplemented with 200 ng of commercially available dog genomic DNA (Zyagen, San Diego, CA) were used to build a reference standard.
Histology. Biopsies of muscles were obtained after vector administration. Muscle tissue was either fixed in 10% formalin or frozen in cooled isopentane followed by liquid nitrogen. Cross-sections were stained with hematoxylin and eosin. For cellular infiltrate staining, muscle serial cryosections were incubated with an anti-CD4 monoclonal antibody (1:200 dilution; AbD Serotec, Oxford, UK), an anti-CD8 monoclonal antibody (1:50 dilution; AbD Serotec, Oxford, UK), or an anti-CD45 monoclonal antibody (1:200 dilution; AbD Serotec, Oxford, UK) followed by horseradish peroxidase staining. Images were obtained with an Eclipse E800 microscope (Nikon, Tokyo, Japan).
Table S1. Summary of IFN-γ responses monitored by antigen-specific ELISpot.
Figure S1. Characterization of muscle infiltrates in dogs H48 and M14.