Animals and animal procedures. Rhesus macaques (Macaca mulatta) were housed and handled in accordance with the guidelines set by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (DHHS publication no. NIH 85-23). Animals were screened for anti-AAV8 NAb before inclusion in the study and only naive (seronegative) animals were selected.
AAV vectors, rtx, and diphenhydramine were given intravenously as a bolus. CsA was given mixed with food. All experiments performed were approved by the Animal Care and Use Committees of the Children's Hospital of Philadelphia and the National Heart, Lung, and Blood Institute.
Empty capsid-free AAV vectors preparations were obtained by triple transfection followed by cesium chloride gradient-based gradient purification as previously described.49
Vectors were resuspended in phosphate-buffered saline supplemented with F68. Vectors were titered by gel electrophoresis followed by silver staining and by quantitative real-time PCR. The expression cassette driving the expression of coagulation human F.IX under the control of the hepatocyte-specific promoter human α-1 antitrypsin (hAAT) has been previously described.10
For the purpose of the study, the same expression cassette was packaged into AAV serotype 6 and 8 vectors and delivered intravenously in saline solution.
CsA (Neoral; Novartis Pharmaceuticals, East Hanover, NJ) was given orally at doses up to 25 mg/kg twice daily for about 9 weeks as previously described.25
Rituximab (Genentech-Roche, South San Francisco, CA) was given intravenously at a dose of 20 mg/kg (equivalent to ~375 mg/m2
Regimens based on a similar dose of rtx in addition to other immunosuppressants have been previously tested in patients with acquired hemophilia, showing good efficacy and tolerability profile.26,27
Animals received three doses of rtx at intervals of 3 and 4 weeks between the first and the second dose, and the second and the third dose, respectively. Rtx was coadministered with the antihistamine drug diphenhydramine given intravenously at a dose of 4 mg/kg to prevent allergic reactions against the infused monoclonal antibody rtx.
Flow cytometry. Frequency of CD20+ B cells in peripheral blood was determined by staining PBMC with an anti-CD20 antibody (Biolegend, San Diego, CA) followed by flow cytometry. Frequency of CD4+CD25+FoxP3+ Treg cells was determined with an antibody set from eBioscience (San Diego, CA) following the manufacturer's protocol. Samples were acquired on a BD Canto II flow cytometer (BD Bioscience, San Jose, CA) and analyzed with FlowJo version 8.8.3 software (Tree Star, Ashland, OR).
Factor IX antigen and anti-F.IX antibody determination.
Human F.IX antigen levels in plasma were determined as previously described24
using an enzyme-linked immunosorbent assay specific for the human protein, which did not cross-react with the endogenous rhesus F.IX. Anti-hF.IX antibodies were measured as described24
with a capture assay in which human F.IX was coated onto enzyme-linked immunosorbent assay plates, test sera were added, and IgG specific for human F.IX were detected with a goat anti-rhesus IgG (H+L) antibody (Southern Biotech, Birmingham, AL).
Inhibitory antibodies to the human F.IX transgene product were determined with a modified Bethesda assay24
in which test plasma was heat-inactivated at 56 °C for 1 hour before testing to eliminate the activity of the endogenous rhesus F.IX.
Anti-AAV antibody determination.
Anti-AAV IgG levels were determined with a capture assay as described by Mingozzi and colleagues.24
Anti-AAV NAb titers were determined using a modified version of a previously described in vitro
Briefly, an AAV vector carrying a self-complementary genome for the expression of the Renilla
luciferase transgene under the control of the cytomegalovirus promoter/enhancer was incubated with semi-log dilutions of test sera. Luciferase activity on cell lysate was measured with a commercially available kit from Promega (Madison, WI). NAb titers were expressed as the reciprocal dilution at which 50% inhibition of luciferase activity was measured compared to the maximum activity (no inhibition).
ELISpot assay. Blood was collected from animals by venipuncture and PBMC were isolated from animals using heparinized DB Vacutainer CTP tubes (BD, Franklin Lakes, NJ). PBMC were cryopreserved until tested.
T-cell reactivity to either the human F.IX transgene or to alternate AAV serotypes was measured with an IFN-γ ELISpot assay as previously described,50
using an antibody set for human IFN-γ which showed high levels of cross-reactivity with the monkey protein (MabTech, Mariemont, OH).
As test antigens, recombinant human F.IX (Benefix; Pfizer, New York, NY) and purified AAV2, AAV6, and AAV8 empty capsids were used at a concentration of 25 µg/ml. Results were expressed as spot forming units per million PBMC plated in the assay; a positive response to an antigen was defined as a spot count of at least 50 spot forming units/million PBMC and threefold the count of the medium-only negative control.
Statistical analysis. Statistical analysis was performed with the Prism Version 5.0b (GraphPad Software, La Jolla, CA). Two-tailed unpaired t-test was used to compare means, P values <0.05 were considered significant.