Engineering Human Induced Pluripotent Stem Cells (HIPSCS) to Investigate the Contribution of the APOE Risk Allele to the Onset and Progression of Alzheimer’s Disease

Abstract

Developing therapies for the treatment of Alzheimer’s disease (AD) requires an in-depth understanding of disease etiology. Although the majority of AD patients are sporadic, multiple genetic risk variants have been identified, the most powerful and prevalent of which is polymorphism in the Apolipoprotein E (APOE) gene. Amyloid-dependent and -independent mechanisms have been postulated to explain the effects of ApoE, but currently how ApoE modulates AD disease risk, especially during aging, remains unclear. The advent of human pluripotent stem cell technology (hPSC) has allowed for the development of in vitro ‘disease-in-a-dish’ models that allow for the modeling of complex genetics associated with idiopathic disease as well as a platform for the screening of potential therapeutic compounds. Additionally, the development of designer nucleases that allow for genome manipulation at single base pair resolution has greatly expanded the utility of cellular disease models, however genome modification in hPSCs has proven to be extremely inefficient. To this end, we have developed a novel tool using Cas9 Base Editors, called Transient Reporter for Editing Enrichment (TREE), which allows for a real-time readout of base editing activity within individual cells. We demonstrate that TREE allows for efficient isolation of edited cell populations in human cells, including hPSCs, with single or multiple simultaneous genomic edits, without the need for integrated selection markers. As proof of principle, we compare TREE to traditional editing enrichment strategies, demonstrating higher editing efficiencies across all tested target sites. Next, we adapted TREE to facilitate facile generation of isogenic hPSC lines using Cas9 Base Editors and show the utility of the technology for generating clonal lines with single base edits, multiplex edits, and gene knockouts. Finally, using isogenic ApoE hPSC lines generated using TREE, we characterize a disease model that recapitulates disease phenotypes and allows for the interrogation of mechanisms by which ApoE contributes to Alzheimer’s disease progression. This system will be useful to the broader stem cell biology field as a tool to efficiently generate edited hPSC lines for a wide variety of applications in developmental biology, disease modeling, drug discovery, and cell-based therapies.