(175) Exploring the Downstream Effects of Regulation of Genes of Interest in Peyronie’s Disease

Introduction We previously performed high-resolution array comparative genomic hybridization analysis of genomic DNA from men with both Peyronie’s Disease (PD) and Dupuytren’s Disease (DD), yielding candidate genes, including NELL1 and CTDSPL, with potential involvement in these conditions. NELL1 and CTDSPL both have known involvement in the TGF-β pathway, with roles in epithelial-mesenchymal transition and myofibroblast differentiation, processes integral to inflammation and fibrosis. Here, we provide results of up- and down-regulation of NELL1 and CTDSPL in an in vitro setting. Objective The objective of this work is to examine the downstream effects of NELL1 and CTDSPL regulation. Methods NELL1 and CTDSPL regulation was performed using CRISPR techniques. For down-regulation, gRNAs were delivered to cells with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. For upregulation, gRNAs were delivered to cells expressing the dCas9-VPR system adapted from S. pyogenes. Gene regulation via these systems was assessed using qRT-PCR. RNA sequencing and analysis was performed by the High-Throughput Genomics and Bioinformatic Analysis core at the University of Utah. Results Multiple guides were tested for up- and down-regulation efficiency. Down-regulation guides led to up to a 95% and 86% reduction in NELL1 and CTDSPL expression, respectively. Up-regulation led to a 40-fold and 56% increase in NELL1 and CTDSPL, respectively. Down-regulation of both genes is suspected to increase fibrosis based on interactions in the TGF-β pathway. Down-regulation of NELL1 was associated with an increase in inflammatory response pathways and a decrease in G2-M pathways, while down-regulation of CTDSPL was associated with changes in pathways related to DNA repair and damage response, and ECM organization. Conclusions Previous work has identified NELL1 and CTDSPL as genes of interest in PD. By evaluating the downstream effects of NELL1 and CTDSPL regulation, we will gain a more complete understanding of the biological processes that these genes may help regulate, shedding light on the possible mechanism of development of PD. Results to date support the hypothesis that these genes can influence ECM organization and/or aberrant fibrosis. Additional work is being conducted using animal knock-out models to measure the amount of fibrosis in multiple organs compared to wild-type mice. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Contraline, Paterna