Abstract
R-spondin (RSPO) proteins amplify Wnt signaling and stimulate regeneration in a variety of tissues. To repair tissue in a tissue-specific manner, tissue-targeted RSPO mimetic molecules are desired. Here, we mutated RSPO (RSPO2 F105R/F109A) to eliminate LGR binding while preserving ZNRF3/RNF43 binding and targeted the mutated RSPO to a liver specific receptor, ASGR1. The resulting bi-specific molecule (αASGR1-RSPO2-RA) enhanced Wnt signaling effectively in vitro, and its activity was limited to ASGR1 expressing cells. Systemic administration of αASGR1-RSPO2-RA in mice specifically upregulated Wnt target genes and stimulated cell proliferation in liver but not intestine (which is more responsive to non-targeted RSPO2) in healthy mice, and improved liver function in diseased mice. These results not only suggest that a tissue-specific RSPO mimetic protein can stimulate regeneration in a cell-specific manner, but also provide a blueprint of how a tissue-specific molecule might be constructed for applications in a broader context.
Highlights
R-spondin (RSPO) proteins amplify Wnt signaling and stimulate regeneration in a variety of tissues
We investigated whether we could engineer a bi-specific RSPO by fusing an antibody that would bind to tissue-specific receptors to the module of RSPOs responsible for binding E3 ligases
We introduced mutations in RSPO Fu2 domains to abolish LGR binding without perturbing with zinc and ring finger 3 (ZNRF3)/ring finger protein 43 (RNF43) binding
Summary
R-spondin (RSPO) proteins amplify Wnt signaling and stimulate regeneration in a variety of tissues. The resulting bi-specific molecule (αASGR1-RSPO2-RA) enhanced Wnt signaling effectively in vitro, and its activity was limited to ASGR1 expressing cells. Systemic administration of αASGR1-RSPO2-RA in mice upregulated Wnt target genes and stimulated cell proliferation in liver but not intestine (which is more responsive to non-targeted RSPO2) in healthy mice, and improved liver function in diseased mice These results suggest that a tissue-specific RSPO mimetic protein can stimulate regeneration in a cell-specific manner, and provide a blueprint of how a tissue-specific molecule might be constructed for applications in a broader context. We fused the mutant RSPO to single-chain variable fragments (scFv) or full-length IgG of an antigen-binding moiety targeting liver-specific receptors that potentially undergo rapid endocytosis This coupling targets the Wnt enhancing activity of RSPOs to tissue-specific receptors, rather than to LGR. The engineered molecules upregulated Wnt signaling and stimulated cell
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