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Lentiviral CRISPR Epigenome Editing of Inflammatory Receptors as a Gene Therapy Strategy for Disc Degeneration.

ABSTRACT: Degenerative disc disease (DDD) is a primary contributor to low-back pain, a leading cause of disability. Progression of DDD is aided by inflammatory cytokines in the intervertebral disc (IVD), particularly TNF-? and IL-1?, but current treatments fail to effectively target this mechanism. The objective of this study was to explore the feasibility of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) epigenome editing-based therapy for DDD, by modulation of TNFR1/IL1R1 signaling in pathological human IVD cells. Human IVD cells from the nucleus pulposus of patients receiving surgery for back pain were obtained and the regulation of TNFR1/IL1R1 signaling by a lentiviral CRISPR epigenome editing system was tested. These cells were tested for successful lentiviral transduction/expression of deactivated Cas9 fused to Krüppel Associated Box system and regulation of TNFR1/IL1R1 expression. TNFR1/IL1R1 signaling disruption was investigated through measurement of NF-?B activity, apoptosis, and anabolic/catabolic changes in gene expression postinflammatory challenge. CRISPR epigenome editing systems were effectively introduced into pathological human IVD cells and significantly downregulated TNFR1 and IL1R1. This downregulation significantly attenuated deleterious TNFR1 signaling but not IL1R1 signaling. This is attributed to less robust IL1R1 expression downregulation, and IL-1?-driven reversal of IL1R1 expression downregulation in a portion of patient IVD cells. In addition, RNAseq data indicated novel transcription factor targets, IRF1 and TFAP2C, as being primary regulators of inflammatory signaling in IVD cells. These results demonstrate the feasibility of CRISPR epigenome editing of inflammatory receptors in pathological IVD cells, but highlight a limitation in epigenome targeting of IL1R1. This method has potential application as a novel gene therapy for DDD, to attenuate the deleterious effect of inflammatory cytokines present in the degenerative IVD.

SUBMITTER: Farhang N

PROVIDER: S-EPMC6761595 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Project description:Musculoskeletal diseases have been associated with inflammatory cytokine action, particularly action by TNF-? and IL-1?. 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-?B was significantly reduced or maintained at baseline levels in the presence of TNF-? or IL-1?. Pellet culture of undifferentiated hADSCs demonstrated improved survival in engineered hADSCs treated with TNF-? or IL-1?, while having little effect on their immunomodulatory properties. Furthermore, engineered hADSCs demonstrated improved chondrogenic differentiation capacity in the presence of TNF-? or IL-1?, 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.

Project description:RationaleDSP (desmoplakin), the most abundant component of desmosomes, which maintain the mechanical integrity of epithelium, is a genome-wide association study-identified genetic risk locus in human idiopathic pulmonary fibrosis (IPF). Subjects with IPF express a significantly higher level of DSP than control subjects.ObjectivesDetermine potential mechanisms by which DSP is regulated in lung fibrosis.MethodsMatrigel-coated soft and stiff polyacrylamide gels were made to simulate the stiffness of normal and fibrotic lungs. Quantitative chromatin immunoprecipitation and electrophoretic mobility shift assay were used to evaluate transcription factor binding to the DSP promoter. Targeted DNA methylation was achieved by CRISPR (clustered regularly interspaced short palindromic repeats)/dCas9 (deactivated CRISPR-associated protein-9 nuclease)-mediated Dnmt3A (DNA methyltransferase 3A) expression under the guidance of sequence-specific single guide RNAs.Measurements and main resultsStiff matrix promotes DSP gene expression in both human and rodent lung epithelial cells as compared with soft matrix. A conserved region in the proximal DSP promoter is hypermethylated under soft matrix conditions and becomes hypomethylated/demethylated under stiff matrix conditions. Demethylation of this conserved DSP promoter region is associated with transactivation of transcription factor EGR1 (early growth response protein 1), resulting in EGR1-dependent DSP overexpression. Targeted DNA methylation by CRISPR/dCas9/Dnmt3A-mediated epigenome editing blocks EGR1 binding to the DSP promoter and inhibits stiff matrix-induced DSP overexpression.ConclusionsDSP is a matrix stiffness-regulated mechanosensitive gene. CRISPR/dCas9-Dnmt3A-mediated epigenome editing reverses DSP overexpression by reestablishment of the epigenetic control of DSP under the mechanically homeostatic environment. It provides a useful tool for investigations of the functional role of DSP in the pathogenesis of lung fibrosis.

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Lentiviral CRISPR Epigenome Editing of Inflammatory Receptors as a Gene Therapy Strategy for Disc Degeneration.

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