Pathology of the intervertebral disc (IVD) and associated back pain is a major healthcare concern, which ranks third in disease burden (disability adjusted life years). Inflammation and related pro-inflammatory cytokines (e.g. TNF-a, Il-1, Il-6, Il-8) have been implicated as active components in many events and processes associated with back pain, including disc degeneration, IVD herniation, and the sensitization of nociceptive neurons both in radiculopathy and in the degenerative disc. As a result, this network of cytokines is a primary target for low back pain therapeutics. Due to significant redundancy in these cytokines, there is a need for therapeutics that are capable of targeting multiple inflammatory cytokines simultaneously in the IVD. Strategies to inhibit and activate multiple genes using the CRISPR system will provide a powerful tool to antagonize the redundant pro-inflammatory cytokine network active in IVD pathology. The CRISPR regulation method is capable of providing high specificity, sustained effects, and multiplex capabilities in a single system. The proposed plan has been designed to develop the novel utility of CRISPR based regulation of inflammatory signaling in the degenerative disc, and test its potential as a therapeutic strategy.
In aim 1, we will test the hypothesis that CRISPR gene regulation systems can functionally modulate inflammatory response in cell types found in the degenerative disc.
In Aim 1 A, we will develop CRISPR-based sequence-specific down regulation (CRISPRi) of gene expression of key inflammatory cytokine receptors involved in disc degeneration. Lentiviral vectors expressing dCAS-KRAB and guide RNAs targeting receptors for TNF-?, Il-1? and Il-6 will be developed (i.e. TNFR1i, IL1R1i, Il6Ri, Il6STi) and screened for efficacy in regulating receptor presentation in human NP cells, human DRG neurons, and human macrophages.
In Aim 1 B, we will develop CRISPR-based sequence-specific up regulation (CRISPRa) of gene expression of anti-inflammatory receptors. Lentiviral vectors expressing dCAS-VP64 and guide RNAs targeting Il1R2 and TNFR2 will be developed and evaluated for efficacy in NP cells, human DRG neurons, and human macrophages. In both aim 1A and 1B, the cells response to inflammatory cytokine exposure after CRISPR-based receptor modulation will be measured using qPCR and an NF-?B reporter.
In Aim 2, we will test the hypothesis that CRISPR gene regulation systems can modulate the effects of inflammation on IVD cells and promote functional IVD tissue development in an inflammatory environment. We will use CRISPR-based sequence-specific multiplex regulation of inflammatory receptors to antagonize deleterious degenerative effects of inflammation in IVD cells and tissue. Functional IVD tissue development and cell response under inflammatory conditions will be assessed via qPCR, an NF- ?B luminescent reporter, mechanical testing and histology/biochemistry for collagen and proteoglycan.
Intervertebral disc disorders and back pain are mediated by inflammatory cytokines that directly influence a number of cell types in the intervertebral disc, including intervertebral disc cells, immune cells, and pain sensing neurons. This project will evaluate if two novel techniques of gene regulation, CRISPRi and CRISPRa, can mediate and antagonize these interactions in these cell types. The CRISPR systems are unique and provide high specificity gene regulation, sustained effects, and multiplex capabilities in a single system that may provide dramatically improved treatments for back pain.
|Farhang, Niloofar; Brunger, Jonathan M; Stover, Joshua D et al. (2017) * CRISPR-Based Epigenome Editing of Cytokine Receptors for the Promotion of Cell Survival and Tissue Deposition in Inflammatory Environments. Tissue Eng Part A 23:738-749|
|Bowles, Robert D; Setton, Lori A (2017) Biomaterials for intervertebral disc regeneration and repair. Biomaterials 129:54-67|
|Stover, Joshua D; Farhang, Niloofar; Berrett, Kristofer C et al. (2017) CRISPR Epigenome Editing of AKAP150 in DRG Neurons Abolishes Degenerative IVD-Induced Neuronal Activation. Mol Ther 25:2014-2027|