AAV vectors had been administered intramuscularly into two web-sites, quadriceps and tibialis anterior of one particular hind limb, as previously described. Plasma samples had been col lected by tail bleed into citrate buffer as described. AAV vectors ssAAV vector expressing human F. IX cDNA in the CMV IE en hancer promoter was as published. For construction of scAAV, the human F. IX coding sequence was cloned into an scAAV CMV GFP construct, replacing the GFP se quence. This construct includes a little B globin IgG chimeric intron. Vector genomes had been packaged into AAV serotype 1 capsid by triple transfection of HEK 293 cells. Vector particles were purified by iodixanol gradient centrifugation, and vector titers determined by dot blot hybridization and confirmed by Western blot utilizing a reference standard of recognized titer for comparison.
Analysis of plasma samples Plasma was analyzed for hF. IX expression, anti hF. IX IgG1, and anti AAV1 IgG2a by enzyme linked immuno sorbent assay as previously described. For the anti capsid antibody ELISAs, sample wells have been coated with two. five 109 vg properly intact AAV1 particles. The assay for anti hF. IX IgG1 was sensitive to 200 ng mL. Anti hF. IX selleck inhibitory activity was assessed working with the Bethesda assay, as previously described. One particular Bethesda unit represents the inhibition of 50% of clotting activity. Clotting assays were performed on a Begin Hemostasis Analyzer. ELISPOT assays Enzyme linked immunosorbent spot assays have been performed to enumerate hF. IX distinct CD8 T cells in mouse spleens, as previously described.
Briefly, splenocytes have been plated at 1 106 cells nicely, and stimulated with media Zosuquidar P-glycoprotein inhibitor alone, staphylococcal enterotoxin B, or the immunodominant CD8 epitope of hF. IX for the C3H HeJ background. Analyses were performed in triplicate on indivi dual mice. Soon after stimulation for 20 hours, plates have been harvested and IFN spot forming units had been detected and counted employing the ImmunoSpot Analyzer. Results were calculated as spot forming units per 106 total cells. Immunohistochemistry Immunohistochemistry was performed applying fluorescent antibodies on frozen and cryosectioned tissue, as pre viously described. Briefly, muscle tissue was har vested and frozen in liquid N2 cooled two methylbutane. Cryosections of tissue had been fixed in acetone at space temperature, blocked with 5% donkey serum, and stained with rat anti CD8 and goat anti hF. IX. Secondary anti body donkey anti rat Alexa Fluor 488 and donkey anti goat Alexa Fluor 568 had been utilized for detection. Fluorescence microscopy was performed with a Nikon E800 microscope. Statistics Outcomes are reported as suggests SEM. Substantial dif ferences between groups had been determined with unpaired Students t test. P values of 0. 05 had been regarded sig nificant.