108. Versican G1 Domain Stimulates Adenoviral Transgene Expression in a JAK/STAT Dependent Manner

Abstract

Gene therapy using either adeno-associated (AAV) or adenoviral (AdV) vectors has been shown to be effective in the ocular environment. Understanding the mechanisms of this enhancement could benefit gene therapy protocols in general. Vitreous, the gelatinous material filling the posterior eye, enhances the expression of transgenes delivered by AdV and AAV. Vitreous has no effect on vector internalization but is associated with increased transgene mRNA levels and mediates enhancement in hyaluronan-dependent and independent-manners (Chaudhuri et al. Mol Therapy 2007). We hypothesize that versican, a hyaluronan-binding proteoglycan expressed in vitreous, has a role in both mechanisms. Furthermore, we define an intracellular pathway through which the increased gene expression occurs.Versican-containing supernatant (VCS) was produced by culturing versican-secreting ACHN cells or HepG2 cells transfected to transiently express the recombinant versican G1 domain either lacking (G1-) or containing (G1+) the hyaluronan-binding region. Y79 or Weri retinoblastoma cells, or SKNDZ neuroblastoma cells that lack a cellular hyaluronan binding receptor were transduced in the presence of bovine vitreous, VCS, or either G1 domain with AdV vectors delivering a luciferase reporter transgene.Incubation of target cells with vitreous enhanced AdV or AAV gene expression greater than 4-fold (p<0.001) when Y79 cells, Weri cells, or SKNDZ cells were used as targets. Boiling vitreous prevented this enhancement. Thus, heat-stable hyaluronan alone was not responsible. Incubation with dasatanib (a Src kinase inhibitor), VCS, G1+, or G1- mimicked the effects of vitreous. Small-molecule inhibition of JAK1/2 or STAT3/5 using ruxolitinib or C188-9 mitigated the enhancement of transgene expression mediated by vitreous, dasatanib, VCS or either G1 domain, while inhibition of the mTOR pathway using rapamycin or everolimus showed no effect on enhancement. Vitreous or VCS treatment enhanced STAT3 phosphorylation in comparison to untreated cells.These results support a model in which Src kinase activity inhibits AdV or AAV viral vector mediated transgene expression. This inhibition can be overcome by hyaluronan-independent and dependent mechanisms mediated by the G1 domain of versican through activation of the JAK/STAT signaling pathway. Therefore, the versican G1 domain could be used as an adjuvant to enhance gene therapy protocols.