The primary cilium is an ancient, conserved organelle that has garnered significant attention in recent years. This is in part because of our expanding appreciation for its role(s) in a host of key cellular processes and in part because of the identification of genetic lesions that affect ciliary structure and function in >100 human genetic disorders. Our aspirational goals have been to understand ciliary functions; to improve the diagnostic and prognostic value of genetic variation in ciliopathies; and to inform rational therapeutic design for a group of disorders bereft of treatment options. During the previous funding period, we made significant progress on several fronts. These included a) the implementation of in vivo signaling assays to decipher the pathogenic potential of newly-discovered alleles, which in turn assisted the cloning of ciliopathy loci; b) the demonstration that ciliopathy loci contribute to the genetic burden of complex traits; and c) the description of the phenomenon of cis-complementation, wherein species-specific alleles can shield the pathogenic effect of missense variation in the same protein. Most poignantly, several basal body proteins regulate proteasomal function, in part through the regulation of the composition of proteasomal subunits. These data led to the realization that numerous paracrine pathways are affected by ciliary dysfunction through the mis-regulated degradation of key effector molecules. In parallel, and in departure from our long-term work in cloning genes that contribute and/or exacerbate ciliopathy phenotypes, we used the amalgam of our in vitro and in vivo tools to design screens that would identify suppressors of ciliary dysfunction that thus offer an alternative approach to rational therapeutics. Through a genome-wide RNAi screen for bbs4-induced deficient signaling, we identified and validated in vivo 11 suppressors. Among these was the ubiquitin specific peptidase USP35, suppression of which in cells and CRISPR-mediated deletion in zebrafish embryos could rescue bbs4-induced pathologies such as rhodopsin aggregation in photoreceptors and renal convolution defects. Based on these observations, we propose to extend our studies on the suppressor potential of USP35 by testing whether (and when) ablation of this locus in ciliopathy mouse models can rescue or ameliorate key ciliopathy pathologies such as retinal degeneration and renal function. Moreover, given that the 11 discovered suppressors are natural candidates for harboring protective alleles in humans, we will merge next-gen analysis in our extensive ciliopathy cohort with systematic allele functional testing to ask whether we observe an enrichment of deleterious alleles for these candidates in individuals with mild ciliopathies or no disease at all. Finally, we will develop tools to perform efficient CRISPR/Cas9-mediated ciliopathy suppressor screens in patient-derived cells as a means to both identifying new such molecules and developing scalable tools of broader utility. Together, our studies will inform the largely unknown genetic properties of genetic suppressors; will contribute to the more systematic identification of such genes; and will potentially lead to the development of rational therapeutic antagonists.
Establishing the link between the basal body and the cilium with Wnt signaling represents a potential paradigm shift in renal cystic disease and will likely lead to a profoundly novel means of approaching treatment and prevention.
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