Abstract The Center for Synthetic Regulatory Genomics (SyRGe) is tasked with development and application of revolutionary technology for making dramatic, coordinated changes to extensive gene loci, which enables broad investigations into the function of regulatory sequences such as GWAS (genome wide association study) “hits” and foster translational applications to biotechnology, personalized medicine and gene therapy. The Center dramatically supersedes present and predicted technologies for manipulation and assessment of regulatory genome function through its focus on Big DNA. This supplement request focuses on one of the top “hits” in multiple GWAS performed on Alzheimer’s disease patients, namely the clusterin (CLU) gene on human chromosome 8. The CLU gene, largely expressed by astrocytes in the central nervous system, encodes clusterin protein (apolipoprotein J), a 75 - 80 kDa disulfide-linked heterodimeric protein associated with the clearance of cellular debris and apoptosis. CLU protein is a molecular chaperone responsible for aiding folding of secreted proteins, and its three isoforms have been differentially implicated in pro- or antiapoptotic processes. Most interestingly, CLU is implicated in multiple diseases related to oxidative stress, including neurodegenerative diseases, cancers, inflammatory diseases, and aging. We will build a ~100 kb human CLU locus, including both the risk and protective haplotypes, and introduce them into mouse embryonic stem cells (mESCs), deleted for the endogenous murine Clu locus. With our collaborator Dr. Liddelow, we will differentiate these into astrocytes using well established protocols, and evaluate both the CLU transcript isoforms and abundance in both “developing” and “mature” astrocytes, as well as performing a battery of tests of astrocyte function. A library of variants will be constructed to pinpoint the causative variant(s) in the risk haplotype.

Narrative The big-DNA technology developed in our Center will be used to pinpoint causative variants in human genome sequence that underlie susceptibility to Alzheimer’s disease. Specifically, the causative variants in a gene called Clusterin, identified by multiple previous studies as associated with Alzheimer’s disease will be identified.