Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling

• Published in: PloS one Vol. 15; no. 1; p. e0226928
• Main Authors: Luca, Vincent C, Miao, Yi, Li, Xingnan, Hollander, Michael J, Kuo, Calvin J, Garcia, K Christopher
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 08.01.2020; Public Library of Science (PLoS)
• Subjects: Antibodies; Anticoagulants; Binding Sites; Biology; Biology and Life Sciences; CD25 antigen; Colon; Colon - growth & development; Colon - metabolism; Crosslinking; Drug development; Engineering and Technology; HEK293 Cells; Hematology; Heparan sulfate; Heparin; Homeostasis; Humans; Interleukin 2; Interleukin 2 receptors; Interleukin-2 Receptor alpha Subunit - metabolism; Intestine; Leucine; Ligands; Medicine; Medicine and Health Sciences; Organ Culture Techniques; Organ Specificity; Organoids; Physiology; Polymer crosslinking; Potentiation; Proteins; Proteoglycans; Receptors; Regenerative medicine; Regulators; Research and Analysis Methods; Signaling; Single-Chain Antibodies - metabolism; Stem cells; Sulfates; Surface markers; Technology; Therapeutics; Thrombospondins - metabolism; Tissue engineering; Tissues; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - immunology; Wnt protein; Wnt Signaling Pathway; Yeast; β-Catenin; United States > US; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0226928

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.