Synthetic Protein Circuits Trigger Programmable Cystic Cell Death in ADPKD

Background: Pkd1 mutant renal epithelial cells are usually accompanied by a high level of Ras, which has been used to design a system to specifically induce apoptosis of Ras highly expressed cystic cells. We hypothesize that induction of cyst-lining cell death with this new system delays cyst growth in Pkd1 mutant mice. Methods: To precisely induce Pkd1 mutant cell death in vitro and in vivo, we generated caspase-cleavage dependent cell death systems, 1) a two-component circuit, composing a tobacco etch virus protease (TEVP) and a TEVP site linked large and small subunits of caspase-3 (Caspase-3L-tev-S), and 2) a three-component circuit, composing a Caspase-3L-tev-S and an inactive N-terminal and a C-terminal halves of TEVP, with each half fused to a Ras-binding domain (RBD). To test this system in vivo, we adapted each component of the three-component circuit in an adeno-associated virus (AAV) vector driven by a kidney-specific cadherin promoter. Cell death and the cleaved caspase 3 were determined by TUNEL assay and Western blotting. Results: We found that the transfection of the two-component circuit increased the cleavage of caspase 3 in HEK293T, Pkd1 wild type and mutant cells compared to those cells transfected with a single component. The transfection of the three-component circuit induced cell death and the cleavage of caspase 3 in Pkd1 null MEK and Pkd1 homozygous PN24 cells only but not in Pkd1 WT MEK and Pkd1 heterozygous PH2 cells. The two-component circuit contains a complete TEVP which can cleave the TEVP site in Caspase-3L-tev-S to initiate cell death in both wild type and Pkd1 mutant cells. The three-component circuit can only induce the death of Ras expressed Pkd1 mutant cells because the two split TEVPs need to bind to Ras to form a full TEVP in mutant cells to initiate cell death. Treatment with AAV-mediated three-component circuit delays cyst growth in Pkd1 mutant mice as seen by a decrease of cyst index, kidney weight/body weight rations and blood urine nitrogen (BUN) levels, and an increase of cell apoptosis and the cleavage of endogenous caspase 3 in Pkd1 mutant kidneys. Conclusion: This study supports that precisely inducing cell death in Ras-expressed Pkd1 mutant renal epithelial cells via a caspase cleavage dependent-Ras selective protein circuit delays cyst growth in Pkd1 mutant mice. Funding: NIDDK Support