Control of plasma
cholesterol levels is a major therapeutic strategy for management of
coronary artery disease (CAD). Although reducing
LDL cholesterol (
LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25-40% reduction in cardiovascular events. Epidemiological studies have shown that a high
LDL-c level is not the only risk factor for CAD; low
HDL cholesterol (HDL-c) is an independent risk factor for CAD.
Apolipoprotein A-I (
ApoA-I) is the major
protein component of HDL-c that mediates reverse
cholesterol transport from tissues to the liver for excretion. Therefore, increasing
ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide
siRNA screening, targets that regulate hepatocyte
ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for
ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the
siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted
enzymes,
proteases,
G-protein-coupled receptors, metabolic
enzymes, ion transporters, and
proteins of unknown function. Repression of
farnesyltransferase (FNTA) by
siRNA and the
enzyme inhibitor manumycin A caused elevation of
ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and
cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.