^{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.

Designer Stem Cells: Genome Engineering and the Next Generation of Cell‐Based Therapies

“…These cells were capable of synthesizing a cartilaginous matrix that was protected against IL‐1‐mediated inflammation or tissue degradation, as measured by a decreased expression of proinflammatory genes and a reduced loss of proteoglycan content. In other studies, dead Cas9 (dCas9)‐KRAB was used for epigenome editing at loci encoding cytokine receptors IL1R1 and TNF receptor 1 ( TNFR1 ) to inactivate these receptors . This approach showed the ability to inhibit downstream activation of NF‐κB and to increase stem cell survival without changes in the gene sequence.…”

The potential of CRISPR/Cas9 genome editing for the study and treatment of intervertebral disc pathologies

“…Farhang et al recently used CRISPR/Cas9 epigenome editing to modulate inflammatory responses of human ASCs by repressing the expression of cytokine receptors TNFR1 and IL1R1. Using dCas9‐KRAB‐induced, site‐specific H3K9 methylation of TNFR1 and IL1R1 promoters, resistance of ASCs to inflammatory environments was achieved, thus demonstrating the ability of CRISPR/Cas9 epigenome editing to modulate inflammatory signaling in implantable cells (Figure ) . Although this method does not produce complete knockout of gene expression, these systems have the advantage of being inducible and reversible.…”

“…(A) Lentiviral transduction of primary human IVD cells (69 years, female) expressing CRISPR epigneome editing system . Protection of adipose tissue‐derived stem cells from TNF‐α after CRISPR epigenome editing of TNFR1, as demonstrated by (B) pellet size and (C) H&E staining of pellets…”

Genome Engineering for Personalized Arthritis Therapeutics

“…[92] In other studies, epigenome editing – using dCas9-KRAB at loci encoding the cytokine receptors IL1R1 and TNFR1 – was used attenuate the catabolic response of human adipose stem cell (hASC)-derived chondrocyte-like cells to inflammatory environments. [93] Specifically, epigenetic silencing of IL1R1 and TNFR1 to their promoters (dCas9-KRAB targeting), mitigated the downstream activation of NF-κB, a transcription factor that initiates catabolic pathways; this protected cartilage matrix integrity in the presence of IL-1 or TNF. [93] Moreover, CRISPR-based epigenome editing approaches have also been used to activate anti-inflammatory cytokines such as IL1RN , and these may hold promise for engineering inflammation-resistant cartilage tissue.…”

“…[93] Specifically, epigenetic silencing of IL1R1 and TNFR1 to their promoters (dCas9-KRAB targeting), mitigated the downstream activation of NF-κB, a transcription factor that initiates catabolic pathways; this protected cartilage matrix integrity in the presence of IL-1 or TNF. [93] Moreover, CRISPR-based epigenome editing approaches have also been used to activate anti-inflammatory cytokines such as IL1RN , and these may hold promise for engineering inflammation-resistant cartilage tissue. [66]…”