title: Artificial Induction of Native Aquaporin-1 Expression in Human Salivary Cells.
abstract: Gene therapy for dry mouth disorders has transitioned in recent years from theoretical to clinical proof of principle with the publication of a first-in-man phase I/II dose escalation clinical trial in patients with radiation-induced xerostomia. This trial used a prototype adenoviral vector to express aquaporin-1 (AQP1), presumably in the ductal cell layer and/or in surviving acinar cells, to drive transcellular flux of interstitial fluid into the labyrinth of the salivary duct. As the development of this promising gene therapy continues, safety considerations are a high priority, particularly those that remove nonhuman agents (i.e., viral vectors and genetic sequences of bacterial origin). In this study, we applied 2 emerging technologies, artificial transcriptional complexes and epigenetic editing, to explore whether AQP1 expression could be achieved by activating the native gene locus in a human salivary ductal cell line and primary salivary human stem/progenitor cells (hS/PCs), as opposed to the conventional approach of cytomegalovirus promoter-driven expression from an episomal vector. In our first study, we used a cotransfection strategy to express the components of the dCas9-SAM system to create an artificial transcriptional complex at the AQP1 locus in A253 and hS/PCs. We found that AQP1 expression was induced at a magnitude comparable to adenoviral infection, suggesting that AQP1 is primarily silenced through pretranscriptional mechanisms. Because earlier literature suggested that pretranscriptional silencing of AQP1 in salivary glands is mediated by methylation of the promoter, in our second study, we performed global, chemical demethylation of A253 cells and found that demethylation alone induced robust AQP1 expression. These results suggest the potential for success by inducing AQP1 expression in human salivary ductal cells through epigenetic editing of the native promoter.
authors.affiliation: 1 Gene Therapy Program, Allegheny Health Network, Pittsburgh, PA, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.affiliation: 2 Department of Biological Sciences, University of Delaware, Newark, DE, USA.|3 Center for Translational Cancer Research, Helen F. Graham Cancer Center & Research Institute, Newark, DE, USA.
authors.forename: S
authors.initials: S
authors.lastname: Pradhan-Bhatt
authors.affiliation: 4 The University of Texas Health Science Center at Houston School of Dentistry, Houston, TX, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Farach-Carson
authors.affiliation: 1 Gene Therapy Program, Allegheny Health Network, Pittsburgh, PA, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Passineau
mesh_terms: D051398:Aquaporin 1 / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D057890:Epigenomics; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012469:Salivary Glands / Q000166:cytology / Q000378:metabolism*; D012984:Software; D014987:Xerostomia / Q000378:metabolism* / Q000628:therapy
chemical_list: D051398:Aquaporin 1
keywords: epigenetic repression; genetic therapy; genetic vectors; progenitor cells; salivary glands; xerostomia