Evaluation of Targeted Rescue Therapy of Polycystin-1 Missense Mutations in ADPKD by Upregulation or Activation of the Transcription Factor XBP1Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is the most common renal monogenic disorder. It is caused by mutations in one of two genes, PKD1 and PKD2, coding respectively the polycystin-1 (PC1) and polycystin-2 (PC2). The focal nature of the cysts in ADPKD results from a loss of function of both alleles of either PKD1 or PKD2. In addition, these mutations disrupt cilia function, in which PC1 dosage plays a central role as a modulator of disease progression.Justification: Evidence of a common mechanism between autosomal dominant polycystic liver disease (ADPLD) and ADPKD has provided new insights into the genesis of cyst formation. ADPLD is characterized by multiple liver cysts that resemble ADPKD without kidney cysts. ADPLD results from mutations in PRKCSH or SEC63, genes that code the glucosidase II² (GII²) and SEC63p, proteins related to the endoplasmic reticulum. Recent evidence has shown that SEC63p is required for adequate expression of PC1 and PC2 and that PC1 is the rate-limiting component of this complex. Since GII² and SEC63 have a role in unfolded protein response (UPR), it has been hypothesized that UPR could interfere with cyst progression. It was shown that SEC63p deficiency activates the IRE1±-XBP1 branch of UPR, resulting in up-regulated transcription of chaperone proteins, which work to overcome the burden of unfolded proteins. In addition, the inactivation of XBP1 accentuated the cystic kidney phenotype of Sec63 mutant mice in a PC1-dosage dependent manner, indicating that XBP1 activation has a protective role. Such findings suggest that chaperone therapy could be useful when extrinsic gene mutations result in reduced function of PC1. Aims: To address whether reduced functional PC1 resulting from missense mutations are amenable to rescue by chaperone therapy.Specific aims:1) To generate modified cell lines and mouse models using genome-editing technology. We plan to develop modified cell lines and mouse models using CRISPR-Cas9 technology. To investigate whether PC1 resulting from missense mutations are amenable to rescue by chaperone therapy, two patient-based mutations were chosen: p.R2220W and p.E2771K.2) To perform in vitro studies evaluating expression and trafficking of mutant Polycystin-1: The inactivation of endogenous Pkd1 and Pkd2 in LLC-PK1 cells was developed as a functional bioassay to address expression and trafficking of mutant PC1 by using fluorescent staining methods. 3) To investigate the effects of mutant Polycystin-1 on cilia-expressing cells: We plan to inactivate Pkd1 and Pkd2 in IMCD3 cells for primary cilia studies. This will allow the insertion of the above-mentioned mutations and epitope tags into Pkd1 enabling studies on cellular phenotypes.4) To generate genetically modified mice with patient-based, missense Pkd1 mutations: We plan to generate mouse models with the same patient-based missense mutations described above using CRISPR-Cas9 system.5) To evaluate whether overexpression of XBP1 rescues the biogenesis and trafficking of mutant Polycystin-1: We will examine the in vitro effects of active XBP1 (XBP1s) expression on PC1 mutants with N- and C-terminal FLAG and HA epitope tags. To generate in vivo XBP1s overexpression we use a model in which XBP1s is knocked into the ROSA26 locus. In this case, activation of Cre recombinase removes a stop cassette that turns on XBP1s expression. In this scenario, we will generate ROSA-XBP1s; Pkd1 mutant-BAC mice and cross them with the Pax8-rtTA; TetO-Cre; Pkd1flox/flox mice. Doxycycline-inducible activation will inactivate the endogenous Pkd1 while activating XBP1s. To the extent that the XBP1s improves function of the mutant PC1 that is still being expressed from the kidneys of Pkd1-BAC models, we will be able to evaluate the benefits of chaperone therapy.
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