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Authorship note: JAT and MR are co-first authors and contributed equally to this work. Conflict of interest: JT and TW are listed inventors on a provisional patent application (62818538) by the University of California Santa Barbara (UCSB) related to discoveries reported in this paper. TW is an inventor on a patent application (15/326296) by UCSB. BC reports consulting fees from Otsuka Pharmaceuticals. MM reports grants and consulting fees from Otsuka Pharmaceuticals, Sanofi, and Chinook Therapeutics. BH is a consultant for Oxthera AB, Alexion Pharmaceuticals, Dicerna Pharmaceuticals, Alnylam Pharmaceuticals, and Allena Pharmaceuticals. RH is on the Advisory Board of Chinook Therapeutics and is an inventor on 2 pending patents (15/545818 and 62/849564) filed by Wake Forest University and UAB. VET reports grant funding from

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. The Wnt ("Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Int") signaling pathway plays a key role in the development, homeostasis and regeneration of many essential organs and tissues 1. Timely activation, modulation, or enhancement of Wnt signaling holds potential for the treatment of various degenerative diseases and pathologies in which tissue regeneration could confer a therapeutic benefit. R-spondins 1-4 (RSPO1-4) are a family of ligands that amplify Wnt signals through a receptor complex containing the zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43) proteins and the coreceptor leucine-rich repeat-containing G-protein coupled receptors 4-6 (LGR4-6) 2-4. RSPOs contain two furin (Fu) repeats, Fu1 and Fu2, which in combination are sufficient to recapitulate Wnt signaling enhancing activity 5. Fu1 primarily interacts with ZNRF3/RNF43 and Fu2 interacts with LGR4-6 5-7. ZNRF3 and RNF43 are membranebound E3 ligases that specifically target Wnt receptors (FZD1-10 and LRP5 or LRP6) for degradation 8,9. Binding of RSPOs to ZNRF3/RNF43 and LGR4-6 causes clearance or sequestration of the ternary complex, which stabilizes Wnt receptors and amplifies Wnt signaling. In addition, R-spondins might work through mechanisms that are independent of LGRs 10,11. RSPOs may be beneficial in adult tissue repair 12,13 , particularly in situations where the expression of endogenous Wnt ligands is upregulated but signaling (presumably limited by receptor stability) is insufficient to overcome tissue damage. In liver, RSPO function is important for metabolic zonation and for hepatocyte proliferation and regeneration 12,14. Therefore, RSPO may provide therapeutic benefit for various acute and chronic liver injury and diseases. One major challenge to exploring RSPO for tissue repair and regeneration is limiting RSPO effects to specific tissue of interest such as liver, as LGR4-6 and ZNRF3/RNF43 are widely expressed in various tissues. The mucosa of the gastrointestinal tract h...

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