AAV-Klotho in vivo dose response study

Female C57Bl/6 mice (9–12 weeks old) with a body weight of 19–21 g were purchased from Charles River Laboratories. AAVs were diluted to the desired concentrations in Dulbecco’s phosphate-buffered saline (DPBS) and administered into the tail vein under light isoflurane anesthesia. The doses tested were 1 × 10⁸, 3 × 10⁹, and 1 × 10¹⁰ vg/mouse AAV-Kl compared with 1 × 10¹⁰ AAV-GFP control. The final volume for injection was 100 μL per mouse. Three weeks after injection with AAV-GFP, AAV-Kl or DPBS, animals were sacrificed and blood samples were collected for the detection of circulating Klotho levels by MSD-ELISA. Livers were also collected to quantify AAV vector genomes. For vector DNA isolation flash-frozen liver samples, were homogenized in 900 μL of RLT buffer (79216, Qiagen), using a Precellys-24 homogenizer and ceramic bead tubes (KT03961-1-009.2, VWR) at 6000 rpm for 30 s. Homogenates were further processed by applying standard Phenol-chloroform extraction. Finally, vector DNA was purified by using the AllPrep DNA/RNA 96 kit (80311, Qiagen) according to the manufacturer’s instructions. Detection of AAV vector genomes was performed by qPCR using primers specific for the LP-1 promoter (forward: GACCCCCTAAAATGGGCAAA. reverse: TGCCCCAGCTCCAAGGT). For quantification, a standard curve generated by serial dilutions of the respective Klotho expression plasmid was used. qPCR runs were performed on an Applied Biosystems ViiA 7 Real-Time PCR System. Animal experiments in this study were approved by the local German authorities (Regierungs-präsidium Tübingen) and conducted in compliance with the German and European Animal Welfare Acts.