Protease−Activated Receptor 2 (PAR−2) as a Novel Target To Prevent Inhibitor Formation to FIX.
Authors: Baila Stefano , Freguia Christian Furlan , Iacobelli Nicholas , Dunn Danielle , Schuettrumpf Joerg , Mingozzi Federico , Andrade-Gordon Patricia , Arruda Valder R. 
Published: April 2020

Direct intramuscular injection (IM) of adeno−associated viral (AAV) serotype 2 in humans with hemophilia B (HB) is a promising therapeutic strategy since muscle biopsies obtained &gt;3 years after vector injection demonstrated stable local gene expression. However to achieve therapeutic FIX levels using AAV−2 would required hundreds IM injections. The use of alternate AAV serotypes is an attractive strategy since AAV−1 or AAV−6, resulted in a &gt;10−fold increase in transgene levels compared to AAV−2 in large animals, but the immune response to the transgene product has been consistently demonstrated as a major limitation of this strategy. There is growing evidence that blood proteases play an important role in modulating inflammatory and immune responses through activation of PARs. Mice lacking PAR−1(−/−) or PAR−2(−/−) alleles presented amelioration of immune− or infection−mediated diseases. Here we sought to determine whether inhibition of PARs could be used as a strategy to prevent immune responses to the FIX following AAV−mediated gene transfer to skeletal muscle. We used PAR−1 and PAR−2 knockout mice on C57Bl/6 background and littermate mice received IM injection of AAV1−CMV−hFIX. At dose 5x1011vg/kg, PAR−2 (−/−) mice (n=5) exhibited circulating FIX levels of 500± 99ng/ml (8–10%) which remained stable for the duration of the experiment (10 weeks), and no antibodies for FIX were detected (n=5). In contrast, all PAR−2 (+/+) mice (n=4) developed antibodies to FIX which inhibits FIX clotting activity, as determined by Bethesda assay (2.1± 0.6 BU). However, when similar vector doses were delivered to PAR−1(−/−) or PAR−1(+/+) (n=4/genotype) mice, antibodies to FIX developed in all animals. We next tested a higher vector dose in PAR−2 models. At dose 1x1012vg/kg, PAR−2(−/−) mice(n=7) resulted in FIX levels of 1,500±353ng/ml, and again no antibodies for FIX were detected. At the same dose, 6 out of 10 mice of PAR−2 (+/+)/(+/−) developed inhibitory antibodies (1.8± 0.7 BU). Further increase in the vector dose to 5 x 1012 vg/kg resulted in the development of inhibitor to FIX in both PAR−2 (−/−) (4/11 mice, 36%) and PAR−2(+/+)/(+/−) (10/17 mice, 60%). This suggests a threshold value in the protective effect in the PAR−2 (−/−) model. We sought to assay for FIX−specific T−cell by ELISPOT assay to quantify IFN−γ secretion from splenocytes of PAR−2 (−/−) and PAR−2 (+/+) mice injected at 5x1011 or 1x1012vg/kg. No difference in IFN−γ secretion was observed between PAR−2 (−/−) and their controls. Moreover, upon repeated challenges with FIX protein following vector injection antibody to FIX was detected in only 1/4 PAR−2 (+/+) mouse and none of 5 PAR−2 (−/−). Thus, PAR−2 inhibition does not compromise the tolerance to FIX.

In a different model, intravenous injection of FIX protein into normal mice upon simultaneous activation of PAR−2 by using specific agonist peptide the rates of FIX antibody formation were comparable with those of a control peptide group. Thus, PAR−2−mediating antibody formation to FIX may differ among distinct immunologic challenges. Together, these data suggest that PARs play a role in the immune response to FIX and that inhibition of PAR−2 (but not PAR−1) could be a novel target in preventing inhibitor formation in hemophilia gene therapy and potentially for protein−based therapy.