You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2018MP09-15 VALIDATION OF EX-VIVO MURINE BLADDER MODEL TO TEST RHOGDI-BASED BLADDER GENE THERAPIES FOR BENIGN AND MALIGNANT BLADDER PATHOLOGIES Gregory Joice, James Bell, Justin La Favor, Takahiro Yoshida, Gonzalo Torga, Kelly Harris, Xiaopu Liu, Matthew Kiedrowski, Marc Penn, Trinity Bivalacqua, and Nikolai Sopko Gregory JoiceGregory Joice More articles by this author , James BellJames Bell More articles by this author , Justin La FavorJustin La Favor More articles by this author , Takahiro YoshidaTakahiro Yoshida More articles by this author , Gonzalo TorgaGonzalo Torga More articles by this author , Kelly HarrisKelly Harris More articles by this author , Xiaopu LiuXiaopu Liu More articles by this author , Matthew KiedrowskiMatthew Kiedrowski More articles by this author , Marc PennMarc Penn More articles by this author , Trinity BivalacquaTrinity Bivalacqua More articles by this author , and Nikolai SopkoNikolai Sopko More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.341AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Gene therapy is an attractive option for treating malignant and bladder pathologies. Novel non-viral gene transfer technologies are being developed to improve the restrictively low rate of tissue gene transfer. In-vivo gene transfection is resource intensive and is not ideal for screening novel approaches. The RhoA pathway is involved in smooth muscle regulation, cancer invasion and metastasis. RhoGDI is an inhibitor of the RhoA pathway. We validated ex-vivo bladder gene transfer to facilitate high throughput assessment of novel methods and gene targets for treating bladder pathology. METHODS Basic Local Alignment Search Tool was used to identify human RhoGDI coding sequences and to compare between rats and humans, which were cloned into a eCMV-based expression vector. NBTII rat bladder cancer cell lines were transfected using FuGENE (Promega, USA) and human protein expression and interaction with endogenous RhoA were tested using flow cytometry, immunofluorescence, and RNA expression analysis. Bladders were harvested from female Lewis Rats (~250g) and sectioned sagittally and cultured for 72-hours following transfection with RhoGDI. Transfected bladder tissues were analyzed as described above. Non-transfected cultured bladder segments were analyzed using myography for viability and intact smooth muscle physiology in response to 120 mM KCl and 30uM carbachol. RESULTS Human and rodent RhoGDI protein homology is 96%. Human RhoGDI was successfully detected exclusively in transfected NBTII cells with a top efficiency of 26%. qPCR analysis demonstrated rodent RhoA and RhoGDI levels were not impacted but ROCK1 and ROCK2 mRNA were significantly decreased by 23.6% (p=0.034) and 40.0% (p=0.015), respectively following human RhoGDI transfection as compared to transfection control NBTII cells. Ex-vivo cultured bladder strips successfully contracted to KCl (mean 0.88+/-0.48 mN/mg tissue) and carbachol (1.82+/-0.97 mN/mg tissue) stimulation. Human RhoGDI was detected in transfected ex-vivo cultured bladder segments. Similar to NBTII cells, qPCR analysis demonstrated rodent RhoA and RhoGDI levels were not impacted but ROCK1 and ROCK2 mRNA were significantly decreased by 15.0% (p=0.035) and 22.4% (p=0.010), respectively following human RhoGDI transfection as compared to transfection control bladder strips. CONCLUSIONS Ex-vivo bladder culture, transfection, and physiological assessment are feasible and may provide a high-throughput method to test novel gene transfer technologies before in-vivo testing. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e112 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Gregory Joice More articles by this author James Bell More articles by this author Justin La Favor More articles by this author Takahiro Yoshida More articles by this author Gonzalo Torga More articles by this author Kelly Harris More articles by this author Xiaopu Liu More articles by this author Matthew Kiedrowski More articles by this author Marc Penn More articles by this author Trinity Bivalacqua More articles by this author Nikolai Sopko More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...