Purchase this article with an account.
Patricia Akinfenwa, Wesley S Bond, Cristhian J Ildefonso, Mary Hurwitz, Richard L Hurwitz; Versican G1 Domain: Improving Viral-mediated Gene Therapy by Activating JAK/STAT Signaling. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1091.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Vitreous enhances adenoviral (AdV) and adeno-associated (AAV)-mediated gene expression in hyaluronan-dependent and independent-manners. We hypothesize that versican, a proteoglycan that binds hyaluronan highly expressed in vitreous, is responsible for both mechanisms. Furthermore, we will 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, a domain that does not contain glycosylation sites. Y79 and Weri retinoblastoma cells and SKNDZ cells that lack hyaluronan binding domains were transduced in the presence of vitreous, VCS, or G1with AdV vectors delivering the luciferase reporter gene.
Incubation of target cells with vitreous, dastatanib (a Src kinase inhibitor), VCS or G1 enhanced AdV gene expression 4-6-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 mimicked the effects of vitreous. Small-molecule inhibition of JAK1/2 and STAT3/5 using ruxolitinib and C188-9, respectively, mitigated the enhancement of transgene expression mediated by vitreous, while inhibition of the mTOR pathway using Rapamycin or Everolimus showed no effect on enhancement. Both vitreous and versican treatment enhanced STAT3 phosphorylation (p<0.05).
Versican appears to be a bi-functional molecule that can regulate hyaluronan-dependent and -independent mechanisms of viral gene expression through the G1 and hyaluronan binding domains. Versican enhances the expression of transgenes delivered by adenoviral vectors through activation of the JAK/STAT signaling pathway. This enhancement appears to be inhibited by src kinase. Therefore, utilization of the versican G1 domain, together with the hyaluronan binding domain, could be used to enhance gene therapy protocols.
This PDF is available to Subscribers Only