(212) INVESTIGATING THE ROLE OF ALCAM AS A GENE OF INTEREST IN ERECTILE DYSFUNCTION

Abstract Introduction Erectile Dysfunction (ED) is a common and psychologically damaging condition, impacting 20% of all men and 33% of men over age 50, increasing at 10% per additional decade of age, and costing billions of dollars per year to treat. Since the introduction of PDE5 inhibitors (PDE5i) in 1998, no novel therapies for ED have been introduced, despite substantial research to understand ED molecular mechanisms. Identification of genes associated with ED would provide a deeper understanding of underlying mechanisms and could provide targets for additional therapies. We previously identified Activated Leukocyte Cell Adhesion Molecule (ALCAM) as a candidate ED gene in men with diabetes. In order to understand the potential role of ALCAM in ED development, we have created lentiviral gRNA vectors for the up- and down-regulation of ALCAM in an in vitro setting. Subsequent to up- and down-regulation of ALCAM, we will perform PCR in order to identify changes in down-stream regulation of genes and pathways that may be implicated in ED progression. Objective To determine the down-stream influence of ALCAM regulation on genes and pathways that may be implicated in ED progression. Methods For down-regulation of ALCAM, gRNAs were delivered to fibroblasts with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. For upregulation, gRNAs were delivered to fibroblasts expressing the dCas9-VPR system adapted from Streptococcus pyogenes. Gene regulation via these systems was assessed using qRT-PCR. RNA sequencing and analysis will be performed by the High-Throughput Genomics and Bioinformatic Analysis core at the University of Utah. Results Final values for vector efficiency for up- and down-regulation of ALCAM are pending. Final vectors used will be chosen from four potential vectors each for up- and down-regulation. RNA sequencing will be performed pending final modification of cells with chosen vectors, though results from similar studies suggest that differential gene expression will range from 5,000-10,000 differentially regulated genes which will then be evaluated with pathway analysis. Conclusions Previous studies by our group have identified ALCAM as a potential gene of interest in the regulation of ED. Analysis of down-stream regulation of genes and pathways implicated in ED progression following regulation of ALCAM will shed additional light on the potential role of ALCAM in ED pathogenesis. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Paterna, Contraline.