The safety of several strategies using novel adeno-associated viral (AAV) vectors (serotypes-1,-5,-7,and -8) to skeletal muscle of large animals have been hampered by the immune response to the transgene

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. Dendritic cells (DC) are potent antigen-presenting cells that govern the immune responses. The involvement of proteases is critical to DC antigen processing and presentation. Proteases’ effects on cells are mediated by protease-activated receptor (PAR) a group of four G-protein-coupled receptors. Bone marrow-derived DC from PAR-2 knockout mice do not spontaneously develop to mature state and only do so upon stimulations of inflammatory signals, therefore, PAR-2 proved critical for DC activation. Work with selective PAR-2 agonists and knockout animals suggests a in vivo contribution of this receptor to several chronic inflammatory diseases and tumor development. In fact, small peptides functioning as PAR-2 antagonists are now being tested as anti-angiogenic agents. We sought to determine whether inhibiting specific targets on the T-cell activation cascade would provide a strategy to prevent the development of antibody following IM injection of AAV. To do this we evaluated the risk of inhibitior formation to clotting factor IX (FIX) in PAR-2 deficient mice compared with heterozygous (+/−) or normal (+/+) genotypes. Mice received IM injection of AAV-1 encoding human FIX (hFIX) at two different doses (low and high cohorts). FIX and antibody levels were monitored weekly. PAR-2(−/ −) mice(n=4) given the low dose exhibited circulating FIX levels of 500± 99ng/ml which remained stable for the duration of the experiment(10 weeks), and no antibody for FIX was detected. In three PAR-2(+/+) mice, FIX expression was transiently detected at week 2 followed by a period of 4 weeks with undetectable levels. This was due to the formation of FIX specific IgG-1 antibody, which results from activation of CD4+ T-cell mainly of Th2 subset, that peaked at week 6. These antibodies inhibit FIX clotting activity as determined by a functional assay at titre of 3–5.5BU (Bethesda Unit) in all 3 mice. High dose AAV-1 treated PAR-2(−/ −) mice(n=6) resulted in continuous expression of FIX(1,500±353ng/ml) in the absence of FIX antibody. In contrast, inhibitors of FIX were detected in all mice(5 per group) PAR-2(+/−) or PAR-2(+/+). All animals develop IgG-1 antibodies to FIX through 10 weeks of observation. At week 6 post-injection, FIX inhibitors were detected in 3/10 mice at levels of ~1 BU. Continuous follow-up showed that a decrease in antibody titers to FIX was associated with slowly increasing FIX antigen. In addition, splenocytes from PAR-2 (+/+) mice exhibit significant increase (p<0.05) in antigen-specific IFN-γ secretion detected by ELISpot whereas in PAR-2 (−/ −) mice no response was detected. Furthermore, in PAR-1(−/ −) and PAR-1(+/+) mice IM injected with AAV-1, FIX inhibitor was detected in all animals (n=4/group). This demonstrates a PAR-2-specific immunomodulatory mechanism. In summary, these data suggest that pharmacological inhibition of PAR-2 may provide a novel strategy to evade the immune response to the transgene in a variety of gene-based strategies.

Topics:
antigens, gene transfer techniques, immune response, receptor, par-2, antibodies, endopeptidases, peptide hydrolases, immunoglobulin g, intramuscular injections, receptor, par-1

Author notes

Corresponding author

2005, The American Society of Hematology
2005
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