title: *CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments.
abstract: Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-α and IL-1β. These inflammatory cytokines promote apoptosis and senescence of cells in diseased tissue and extracellular matrix breakdown. Stem cell-based therapies are being considered for the treatment of musculoskeletal diseases, but the presence of these inflammatory cytokines will have similar deleterious action on therapeutic cells delivered to these environments. Methods that prevent inflammatory-induced apoptosis and proinflammatory signaling, in cell and pathway-specific manners are needed. In this study we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)-based epigenome editing to alter cell response to inflammatory environments by repressing inflammatory cytokine cell receptors, specifically TNFR1 and IL1R1. We targeted CRISPR/Cas9-based repressors to TNFR1 and IL1R1 gene regulatory elements in human adipose-derived stem cells (hADSCs) and investigated the functional outcomes of repression of these genes. Efficient signaling regulation was demonstrated in engineered hADSCs, as activity of the downstream transcription factor NF-κB was significantly reduced or maintained at baseline levels in the presence of TNF-α or IL-1β. Pellet culture of undifferentiated hADSCs demonstrated improved survival in engineered hADSCs treated with TNF-α or IL-1β, while having little effect on their immunomodulatory properties. Furthermore, engineered hADSCs demonstrated improved chondrogenic differentiation capacity in the presence of TNF-α or IL-1β, as shown by superior production of glycosaminglycans in this inflammatory environment. Overall this work demonstrates a novel method for modulating cell response to inflammatory signaling that has applications in engineering cells delivered to inflammatory environments, and as a direct gene therapy to protect endogenous cells exposed to chronic inflammation, as observed in a broad spectrum of degenerative musculoskeletal pathology.
authors.affiliation: 1 Department of Bioengineering, University of Utah , Salt Lake City, Utah.
authors.forename: Niloofar
authors.initials: N
authors.lastname: Farhang
authors.affiliation: 2 Department of Biomedical Engineering, Duke University , Durham, North Carolina.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Brunger
authors.affiliation: 1 Department of Bioengineering, University of Utah , Salt Lake City, Utah.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Stover
authors.affiliation: 2 Department of Biomedical Engineering, Duke University , Durham, North Carolina.
authors.forename: Pratiksha I
authors.initials: PI
authors.lastname: Thakore
authors.affiliation: 3 Department of Orthopaedics, University of Utah , Salt Lake City, Utah.
authors.forename: Brandon
authors.initials: B
authors.lastname: Lawrence
authors.affiliation: 4 Department of Biomedical Engineering, Washington University in St. Louis , Saint Louis, Missouri.|5 Department of Orthopaedic Surgery, Washington University in St. Louis and Shriners Hospitals for Children-St. Louis , Saint Louis, Missouri.
authors.forename: Farshid
authors.initials: F
authors.lastname: Guilak
authors.affiliation: 2 Department of Biomedical Engineering, Duke University , Durham, North Carolina.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Gersbach
authors.affiliation: 4 Department of Biomedical Engineering, Washington University in St. Louis , Saint Louis, Missouri.|5 Department of Orthopaedic Surgery, Washington University in St. Louis and Shriners Hospitals for Children-St. Louis , Saint Louis, Missouri.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Setton
authors.affiliation: 1 Department of Bioengineering, University of Utah , Salt Lake City, Utah.|3 Department of Orthopaedics, University of Utah , Salt Lake City, Utah.
authors.forename: Robby D
authors.initials: RD
authors.lastname: Bowles
mesh_terms: D000273:Adipose Tissue / Q000473:pathology; D002454:Cell Differentiation; D002470:Cell Survival / Q000235:genetics; D020219:Chondrogenesis; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D044127:Epigenesis, Genetic; D005109:Extracellular Matrix / Q000378:metabolism; D000072669:Gene Editing; D006025:Glycosaminoglycans / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D056747:Immunomodulation; D007249:Inflammation / Q000473:pathology*; D016086:Lentivirus / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D018121:Receptors, Cytokine / Q000235:genetics* / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism; D023822:Tissue Engineering; D014161:Transduction, Genetic
chemical_list: D006025:Glycosaminoglycans; D016328:NF-kappa B; D018121:Receptors, Cytokine; D018124:Receptors, Tumor Necrosis Factor; D004247:DNA
keywords: CRISPR; adipose derived stem cells; epigenome editing; inflammation; intervertebral disc; receptor