Analyses were performed working with GraphPad Prism. Results The vector genome impacts the CD8 T cell response to F. IX in null mutation mice To assess the effect of a scAAV genome around the immune response to F. IX, we injected hemophilia B C3H HeJ mice intramuscularly with 1011 vector genomes of ss or scAAV serotype 1 vectors expressing human F. IX beneath the manage of a cytomegalovirus promoter. These HB mice possess a targeted dele tion in the murine F9 gene and thus lack tolerance to F. IX antigen. In earlier studies, we located that ssAAV2 CMV hF. IX induced neutralizing anti body and CD8 T cell responses against hF. IX upon i. m. injection in this strain. Here, we utilized serotype 1 vec tor, since it is superior for muscle gene transfer and is therefore in clinical trial use for muscle gene transfer for 1 antitrypsin deficiency and for lipoprotein lipase defi ciency.
Plasma was then collected 1, 2, and 4 weeks post injection to assess circulating expression of hF. IX also as antibody responses for the transgene item. One particular week following vector injection, expression of hF. IX was de tected in mice that received ss or scAAV1. At two weeks and thereafter, even though, circulating DNA adenine methyltransferase hF. IX was not detected in either group of animals. Corresponding with the loss of hF. IX expression in plasma, antibodies against hF. IX had been very first detected 2 weeks post injection by ELISA. Constant with prior findings, these have been of your IgG1 subclass, whereas levels of IgG2a and IgG2b have been comparatively pretty low or nonexistent. Typical anti hF. IX titers had been almost identical for both ss and scAAV vectors.
To assess the functionality of this humoral immune response, we performed the Bethesda assay, which measures the capacity of hF. IX certain anti bodies to stop plasma clotting activity. Inhibitor titers lagged behind the detection of anti hF. IX IgG1, with no tiny or no inhibition of clotting detected just after two weeks. Immediately after 4 weeks, typical titers of 20 BU had been measured regardless purchase NLG919 whether mice received ss or scAAV1. Two and 4 weeks post injection, splenocytes have been harvested to measure the CD8 T cell response to hF. IX by ELISPOT. Both vectors induced a measurable antigen precise response. On the other hand, mice that received scAAV1 had a significantly higher variety of IFN spot forming units when stimulated together with the immunodominant CD8 epitope of hF. IX at two weeks.
Four weeks post injection, all animals nevertheless showed a response, which was comparable for ss and scAAV1 treated mice at this later time point. Background SFU had been larger at two weeks, possibly as a consequence of ele vated immune activity at this time point. In an effort to assess no matter whether activated hF. IX specific CTLs infiltrated the transduced tissue, immunohistochemical analyses of injected muscle tissues have been performed. Two weeks post injection, mice that received either ss or scAAV1 had important CD8 T cell infiltration, though there was more evidence of regional hF.