A programmable arthritis-specific receptor for guided articular cartilage regenerative medicine

Abstract

Objective. Investigational cell therapies have been developed as disease-modifying agents for the treatment of osteoarthritis (OA), including those that inducibly respond to inflammatory factors driving OA progression. However, dysregulated inflammatory cascades do not specifically signify the presence of OA. Here, we deploy a synthetic receptor platform that regulates cell behaviors in an arthritis-specific fashion to confine transgene expression to sites characterized by cartilage degeneration. Methods. An scFv specific for type II collagen (CII) was used to produce a synthetic Notch (synNotch) receptor that enables CII-synNotch mesenchymal stromal cells (MSCs) to recognize CII fibers exposed in damaged cartilage. Engineered cell activation by both CII-treated culture surfaces and on primary tissue samples was measured via inducible reporter transgene expression. TGFbeta3-expressing cells were assessed for cartilage anabolic gene expression via qRT-PCR. In a co-culture with CII-synNotch MSCs engineered to express IL-1Ra, ATDC5 chondrocytes were stimulated with IL-1α, and inflammatory responses of ATDC5s were profiled via qRT-PCR and an NF-kB reporter assay. Results. CII-synNotch MSCs are highly responsive to CII, displaying activation ranges over 40-fold in response to physiologic CII inputs. CII-synNotch cells exhibit the capacity to distinguish between healthy and damaged cartilage tissue and constrain transgene expression to regions of exposed CII fibers. Receptor-regulated TGFbeta3 expression resulted in upregulation of Acan and Col2a1 in MSCs, and inducible IL-1Ra expression by engineered CII-synNotch MSCs reduced pro-inflammatory gene expression in chondrocytes. Conclusion. This work demonstrates proof-of-concept that the synNotch platform guides MSCs for spatially regulated, disease-dependent delivery of OA-relevant biologic drugs.

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

“…For example, immobilization of the Delta-like 1 (DLL1) ligand has been applied to activate Notch in cells embedded within photopatterned fibrin-based hydrogels 24 , and a bone mineral-binding ligand has been used to selectively stimulate Notch in cells surrounding skeletal growth plates in vivo 25 . From a synthetic biology perspective, SynNotch receptors recognizing native ECM components have recently been developed using a collagen-II binding antibody fragment 26 , and strategies involving microcontact printed ligands have been exploited to define SynNotch activities with micro-scale spatial precision 27,28 .…”