(635c) Identifying Proteins That Regulate Endocytosis of Nucleic Acid Therapeutics

Nucleic acids can be used to treat diseases of improper protein expression. Messenger RNA (mRNA) and plasmid DNA (pDNA) can be used to increase the expression of a protein, and small interfering RNA (siRNA) can be used to decrease the expression of a target protein. Nucleic acids and other macromolecular therapeutics cannot cross cell membranes due to electrostatic repulsion; therefore, target cells must actively transport nucleic acids into the cell, a process called endocytosis, to have a therapeutic effect. Endocytosis has been classified into six different mechanisms that have been shown to be used by different cell types for different therapeutics. Moreover, the mechanism of endocytosis directly controls the efficacy of the endocytosed material. Clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis have been well studied; however, the regulation of more recently discovered endocytic pathways (Graf1-mediated, Flotillin-mediated, and Arf6-depenedent endocytosis) remains largely unknown. In this work, we used gene expression data and computational methods to determine the regulating proteins among the endocytic pathways. ReliefF was used to find condition-specific differentially expressed genes in NCI-H1299 (human non-small cell lung carcinoma), HEK293 (human embryonic kidney), HeLa (human cervical cancer), and HepG2 (human hepatocytoma) that relate to endocytosis. Fisher’s Exact Test for overlap of differentially expressed genes with cellular pathways and combinatorial z-scores were used to identify potential pathways and regulatory molecules of endocytosis, respectively. Computationally predicted regulators of endocytosis were confirmed in cell culture experiments.