AAV vectors were administered intramuscularly into two web pages, quadriceps and tibialis anterior of one particular hind limb, as previously described. Plasma samples have 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 small B globin IgG chimeric intron. Vector genomes were 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 using a reference normal of known titer for comparison.
Evaluation 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 had been coated with 2. 5 109 vg nicely intact AAV1 particles. The assay for anti hF. IX IgG1 was sensitive to 200 ng mL. Anti hF. IX selelck kinase inhibitor inhibitory activity was assessed utilizing the Bethesda assay, as previously described. One Bethesda unit represents the inhibition of 50% of clotting activity. Clotting assays had been performed on a Start off Hemostasis Analyzer. ELISPOT assays Enzyme linked immunosorbent spot assays were performed to enumerate hF. IX particular CD8 T cells in mouse spleens, as previously described.
Briefly, splenocytes have been plated at 1 106 cells effectively, and stimulated with media selleck chemicals Vemurafenib 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 were detected and counted working with the ImmunoSpot Analyzer. Results have been 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 2 methylbutane. Cryosections of tissue were fixed in acetone at room 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 employed for detection. Fluorescence microscopy was performed using a Nikon E800 microscope. Statistics Results are reported as signifies SEM. Important dif ferences amongst groups have been determined with unpaired Students t test. P values of 0. 05 were regarded as sig nificant.