CENTER for GENOME ENGINEERING

Project 1. GPCRs

1-1. Structural and functional changes in arrestin-mediated GPCR signaling

G protein-coupled receptors (GPCRs) are important regulators for physiologic and pathologic functions. GPCRs bind with agonists to activate either G protein-dependent or G protein-independent signaling pathways, resulting in different functional outcomes. With the latter, GPCR kinases (GRKs) phosphorylate the GPCR to recruit arrestins, which in turn suppresses GPCRs and G protein binding. This G protein-independent pathway can induce GPCR internalization and mediates downstream signals, such as mitogen-activated protein kinases.

Selective and specific regulation of GPCRs can improve therapeutics, making them more effective while resulting in fewer complications. Thus, the development of G protein- or arrestin-specific biased ligands is crucial for therapeutic advancement, yet details on the GPCR-arrestin complex are very much lacking. Even with regard to class A GPCRs, the largest family of this kind, little is known about the structural transformation that affects their distinct downstream signals. However, having precise structural information about the GPCR-arrestin complex is fundamental for biased ligand drug development, which is customized for each GPCR status.

1-2. Structural and functional studies of Wnt/Frizzled receptors

Wnt plays a profound role in cell fate, proliferation, migration, polarity, and apoptosis. As a fundamental pathway in stem cells, Wnt signals regulate homeostasis, embryonic development, and tissue regeneration. The majority of cancers have alterations throughout the Wnt pathway. Twenty Wnts and ten Frizzled receptors have been identified in mammals. While the canonical and non-canonical Wnt pathway molecules and their diverse interaction combinations have been studied widely, there is still rather little known about the structural properties of each interactive complex. To be able to specify the concrete molecular basis and downstream signaling of Wnt/Frizzled receptor combinations, knowing their structural status is essential. This structural information is expected to provide a basis for novel and specific therapeutic designs and will also expand our understanding of developmental biology.

Project 2. Gene editors

2-1. Molecular engineering and 3D structure validation of gene editors

The field of gene editing has gradually evolved from ZFNs to TALENs and CRISPR/Cas9. A variety of adenine and cytosine deaminases have been developed to be fused with Cas9 nickase to generate CRISPR-mediated base editors. Furthermore, a prime editor with an enhanced gene editing function has been developed by the fusion of reverse transcriptase on Cas9 nickase. Nevertheless, many challenges remain in gene editing as in vivo application is delayed due to immunogenicity, molecular size decreasing practicality, and off-target effects.

To address these issues we are working on the following: First, we are investigating strategies to reduce off-target effects during gene editing. Our approach involves applying an advanced molecular modeling method to optimize domain interface in sgRNA or intermolecular binding of Cas9, Cas protein subtypes and ZFNs. Second, to solve the challenges related to immunogenicity and in vivo delivery, we are trying to reduce the overall molecule size and leave only an essential function domain. To achieve this objective, we are developing novel types of gene editors with small-size Cas molecules designed by the methods mentioned above. Our overall goal is to advance gene editors by structure determination and integration of predictive and experimental data.