(153) Investigating the Role of NELL1 as a Gene of Interest in Peyronie’s Disease

Abstract

Abstract Introduction Peyronie’s Disease (PD) is a superficial fibrosing disorder characterized by inelastic fibrous plaques within the tunica albuginea. We previously performed high-resolution array comparative genomic hybridization analysis of genomic DNA from men with both PD and Dupuytren’s Disease (DD), yielding candidate genes, including NELL1, with potential involvement in these conditions. NELL1 is a secreted growth factor that enhances osteoblastic differentiation and chondrogenesis and may contribute to calcification and inflammation in fibrotic plaques via its interaction with bone morphogenetic protein (BMP) within the TGF-β signaling pathway. Here, we describe the creation of lentiviral gRNA vectors for the up- and down-regulation of NELL1 in an in vitro setting, as well as PCR results for NELL1 regulation and preliminary results from RNA sequencing following CRISPR modification. Objective To determine the influence of NELL1 on extracellular matrix (ECM) deposition and downstream gene regulation. Methods For down-regulation of NELL1, gRNAs were delivered to HEK293 cells with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. For upregulation, gRNAs were delivered to HEK293 cells expressing the dCas9-VPR system adapted from Streptococcus pyogenes. Gene regulation via these systems was assessed using qRT-PCR. ECM deposition was evaluated using primary antibodies again Collagen I, Collagen III, and Elastin, and the appropriate secondary antibodies. RNA sequencing and analysis was performed by the High-Throughput Genomics and Bioinformatic Analysis core at the University of Utah. Results For up-regulation, four vectors were tested, with the most effective vector demonstrating a 40-fold increase in NELL1 expression via PCR testing. For down-regulation, three vectors were tested, with the most effective demonstrating a 95% reduction in expression via PCR testing (Figure 1). Non-target vectors did not change NELL1 expression levels. RNA sequencing results found over 10,000 differentially expressed genes between up-regulated and non-target cell lines, with significant changes in multiple pathways, including TNF-α signaling. Similarly, over 5,000 differentially expressed genes were found between down-regulated and non-target cell lines, with significant changes in inflammatory and G2-M checkpoint pathways, among others. Ongoing experiments are also testing the amount of extra-cellular matrix (ECM) deposited by up- and down-regulated cells and their non-target controls, in order to determine if changes in NELL1 expression influence ECM deposition. Conclusions We have demonstrated the creation of effective guides for the up- and down-regulation of NELL1, a gene of interest in Peyronie’s disease. We have also demonstrated, through RNA sequencing, that changes in NELL1 lead to large changes in gene and pathway expression. Ongoing experiments will determine if changes in NELL1 expression also affect ECM deposition. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Vault Health.