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[Preprint]. 2024 Feb 15:2024.01.31.578281.
doi: 10.1101/2024.01.31.578281.

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

Bonnie L Walton  1 , Rebecca Shattuck-Brandt  1 , Catherine A Hamann  1 , Victoria W Tung  1 , Juan M Colazo  1 , David D Brand  2 , Karen A Hasty  3 , Craig L Duvall  1   4 , Jonathan M Brunger  1   4   5
Affiliations

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

Bonnie L Walton et al. bioRxiv. .

Update in

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. TGFβ3-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-κB 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 TGFβ3 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.

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Figures

Figure 1:
Figure 1:. A mAbCII-programmed synNotch receptor licenses cells for potent and specific responses to type II collagen.
(A) The type II collagen (CII)-sensitive antibody mAbCII serves as a synNotch recognition motif, rendering a new synNotch receptor, CII-synNotch. CII-synNotch facilitates cell recognition of CII fibers, leading to transmembrane cleavage of a synthetic transcription factor for versatile output responses. (B) CII-synNotch MSCs were plated on surfaces treated with solutions of varying concentrations of collagen I (CI) and CII (t=72hr). Red dashed line indicates basal signal from untreated cells. (C) Luminescence values of engineered cells on either 25 μg/ml CI or CII (t=72hr). (D) Fluorescence microscopy of constitutively iRFP+ CII-synNotch MSCs with inducible mCherry expression (t=72hr). (E) Activation of synNotch MSCs with either a mAbCII receptor or non-cognate (anti-GFP) receptor, measured via luciferase detection (t=72hr). Scale = 200 μm. Data: n=3 replicates, plotted as mean ± SEM. Statistical analysis: one-way ANOVA with Tukey’s post-hoc analysis, ****p < 0.0001. NT: No treatment.
Figure 2:
Figure 2:. CII-synNotch MSCs detect degradation of cartilage tissue.
(A) 3 mm-diameter primary porcine cartilage explants were enzymatically treated or cryosectioned prior to enzymatic treatment. Enzyme treatment reveals CII epitopes detected by the mAbCII recognition motif. (B) SynNotch-inducible SEAP expression after plating engineered cells on enzymatically-damaged whole-plug explants (t=72 hr, Student’s t-test on n=3 replicates per group). (C) Inducible luciferase expression from engineered cells on cryosections treated with varying concentrations of trypsin-EDTA (t=48hr). (D) Representative images of synNotch activation on sagittal cryosection samples without treatment and with 0.25% trypsin-EDTA (t=48hr). iRFP is false-colored as blue to enable reporter discrimination in the merged image. Scale = 200 μm. Data plotted as mean ± SEM. *p<0.05, **p<0.01.
Figure 3:
Figure 3:. Transgene expression by CII-synNotch cells is spatially gated by direct contact with immobilized CII.
(A) Luminescence activity from engineered MSCs cultured in untreated conditions, in medium supplemented with CII (“Sol. CII”) or on cell culture surfaces pre-treated with CII (“Immob. CII”) (t=72 hr). (B) Representative image of cartilage explant (outlined in yellow dash) containing CII-synNotch cells (blue) that express mCherry (red) upon activation. Scale = 200 μm. iRFP is false-colored as blue to enable reporter discrimination in the merged image. (C) Comparison of CII-treated surfaces and trypsinized cryosectioned cartilage tissue, as measured with normalized luciferase (t=48hr). Scale = 200 μm. Data: n≥3 replicates, plotted as mean ± SEM. Statistical analysis: one-way ANOVA with Tukey’s post-hoc analysis, *p<005, ****p < 0.0001. NT: no treatment
Figure 4:
Figure 4:. CII-synNotch effectively regulates pro-anabolic TGFβ3 expression.
(A) A synNotch payload contains human TGFβ3 co-expressed with mCherry, connected through an internal ribosomal entry site (IRES), allowing for expression of both transgenes. Inducible mCherry measurements of TGFβ3-expressing cells with and without activation on CII as a readout of synNotch activation (t=72 hours, Student’s t-test on n=3 replicates per group). (B) ELISA measurements of TGFβ3 after plating on CII-adsorption to cell culture surfaces (t=72 hours, Student’s t-test on n=3 replicates per group). (C) qRT-PCR detection of transgene hTGFβ3 in experiments comparing TGFβ3-expressing cells to reporter-expressing cells. qRT-PCR data is normalized to no-treatment controls for each cell line. (D) qRT-PCR detection of pro-anabolic Acan and Col2a1 expression, as well as Col1a1 expression. All qRT-PCR: t=72 hrs, two-way ANOVA with Tukey’s post hoc analysis on n=5 replicates per group. *p<0.05, ****p<0.0001. n.d.: not detected.
Figure 5:
Figure 5:. CII-synNotch MSCs mitigate IL-1-induced inflammation in a CII-dependent manner.
(A) ELISA measurements of IL-1Ra after plating on CII-decorated surfaces (t=72 hours, Student’s t-test on n=3 replicates per group). (B) Schematic of trans-well co-culture for assessment of IL-1Ra synNotch circuits on inflammatory chondrocytes. Bottom: 25 μg/ml of CII is plated for activation of CII-synNotch MSCs containing inducible IL-1Ra. Top: ATDC5 chondrocytes are plated in 3 μm-porous inserts. After three days of synNotch activation by MSCs, 0.1 ng/ml IL-1α was added to the insert. Flow cytometry and qRT-PCR results were obtained two days after stimulation. (C) ATDC5s were transduced with an mKate2 reporter that is upregulated in response to NF-κB-driven inflammation. Flow cytometry of mKate2 reports for the effect of inducible IL-1Ra expression on the NF-κB signal in IL-1-stimulated co-cultures (t=5d from plating, two-way ANOVA on n=3 replicates per group). (D) qRT-PCR detection of pro-inflammatory genes Il6, Mmp13, Ccl5, Ccl2, and Adamts4 as well as ECM-related Acan and Col2a1. All qRT-PCR data is normalized to chondrocyte gene expression in co-cultures with neither CII-synNotch activation nor IL-1α stimulation (t=5d from plating, two-way ANOVA on n=five replicates per group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
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