In a “gene desert” region on human chromosome 9, a Scripps Translational Science Institute (STSI)-led team of researchers is taking advantage of the relatively new research tool, zinc finger nucleases, and other groundbreaking new approaches, such as induced pluripotent stem cells, to identify DNA variants that increase risk for coronary artery disease (CAD).
The 9p21 locus, even though devoid of genes, is the strongest genomic marker for CAD that has thus far been identified in multiple genome wide association studies (GWAS). These studies have revealed that the 9p21 locus contains single nucleotide polymorphisms (SNPs) associated with myocardial infarction (MI), abdominal aortic aneurysm, intracranial aneurysm as well as CAD.
While the causative functional variant for CAD has yet to be pinpointed, clues to its identity have been discovered by STSI and UC San Diego scientists. In the Feb. 10 2011 issue of Nature, the scientists reported that they had uncovered 33 distinct enhancers within the 9p21 locus and found that SNPs in one of the enhancers trigger a cascade of molecular actions that contribute to inflammatory signaling in human endothelial cells. (Because previous studies have associated inflammation, which can weaken artery walls, with the propensity for CAD, physicians often recommend a daily aspirin regimen for selected patients.)
In a press interview, STSI Director Eric J. Topol, M.D., one of the scientists who conducted this study, commented, “The enhancers trigger interactions with genes located at considerable distances from each other on the chromosome. This lesson of long-range interactions of a locus of the genome — not a gene — with something that is a gene that’s hundreds of thousands of bases away, is a vital lesson.”
Now, in the new study using zinc finger nucleases, Dr. Topol and other STSI researchers as well as their collaborators at The Scripps Research Institute (TSRI) and Sangamo Biosciences are trying to identify the intracellular network that connects 9p12 variation to CAD risk. (Illustrating this page is a schematic of the structure of Sangamo’s engineered zinc finger protein transcription factors and zinc finger nucleases.)
The findings may illuminate novel gene targets for drug discovery, just as previous studies by other labs on the network that includes the BRCA1/2 genes for repairing double-strand DNA breaks led to pharmacological inhibitors of the enzyme Poly ADP ribose polymerase (PARP) as “synthetic lethal” therapeutics against cancer.
The STSI/TSRI/Sangamo research team, whose program director is Dr. Topol, will generate at high-throughput, isogenic induced pluripotent stem cells (iPSCs) and use their differentiated progeny to understand the impact of human genetic variation on the risk of developing CAD.
The iPSCs will be produced from cells of CAD and MI patients enrolled in Scripps Health’s GeneHeart Study of 950 patients with non-ischemic cardiomyopathy, atrial fibrillation, aortic stenosis/mitral regurgitation as well as CAD and MI. STSI researchers have genotyped the patients’ tissue at the 9p21 locus and categorized the results for both health and carrier status or susceptible or protective haplotypes of this locus.
By using the designed zinc finger nucleases (ZFNs), Samuel Levy, Ph.D., Director of Genomic Sciences at STSI, said that the research team will be able to genetically engineer native loci in primary human cells at unprecedented efficiency and accuracy.
Dr. Levy and the other researchers on the team will use ZFN-driven genome editing of peripheral blood cells from the patients combined with novel small molecules. A turnkey approach to generate the iPSCs isogenically will be created to avoid the variability associated with conventional iPSC generation approaches.
The researchers plan to produce a large panel of two important types of iPSCs characterizing the 9p12 locus: one will contain susceptible alleles while the other will have the protective alleles. The iPSC panel, which will be diverse in genetic background but isogenic for an inducible pluripotency cassette and for endogenous reporters of cell fate, will be a unique, valuable resource for the scientific community.
Such a molecular characterization of isogenic, differentiated cells both with and without specific modifications will enable the first unbiased identification of the extent to which specific polymorphisms contribute to disease relevant cell phenotypes.
This research hopefully will lay the groundwork for a reliable screening test that will accurately detect both high and low genetic risks for CAD. The findings also may provide “drug-able” molecular targets.
In addition, this unique research program may provide a new approach for investigating other diseases that also are characterized by complex genetic architectures. “We deliberately structured the research program to ensure that this tool would be as applicable to any disease with complex genetics where cell base disease proxy phenotypes can be assayed,” said Dr. Levy.
In addition to Drs. Topol and Levy, the research team includes Sarah S. Murray, Ph.D., Director of Genetics at STSI; Andrew Carson, Ph.D., Research Scientist III; Glenn Oliveira, Janel K. Lee and Rebecca Tisch, all Research Associates; Kai Post, M.S., Senior Bioinformatics Programmer; and Sharon Haaser, R.N., Clinical Trials Coordinator.