Supplementary Material for: Therapeutic Challenge of Minicircle Vector Encoding Klotho in Animal Model

Background: Klotho treatment is a promising approach against kidney injury, but its clinical application is still undetermined. We developed a novel strategy to allow self-production of Klotho protein, using minicircle (MC) technology, and evaluated its feasibility in therapeutic Klotho delivery. Methods: We engineered MC vectors to carry cassette sequences of Klotho and verified the self-production of Klotho protein from in HEK293T cells. We evaluated the location and persistence of delivered MC in vivo, and the duration of Klotho protein production from MCs by serial measurement of Klotho protein in blood. We subsequently evaluated the therapeutic potential of Klotho-encoding MCs in experimental model of renal injury. Results: We confirmed the production of Klotho from MC by its significant availability in cells transfected with the MC, as well as in its conditioned medium, compared to that in cells transfected with parent vector. MCs were delivered in vivo by hydrodynamic injection via tail vein. After a single injection of MCs, red fluorescence protein was detected until 30 days in liver, and Klotho protein was maintained until 10 days in the blood, suggesting the production of Klotho protein from MCs via protein synthesis machinery in liver. Therapeutic effect of MC was confirmed by functional and histological improvement seen in mouse model of acute ischemia-reperfusion injury and unilateral ureteral obstruction. Conclusion: Together, these findings implied that self-generated Klotho protein, using MC technology, is functionally active and relevant as a therapeutic approach in renal injury.