Chronic inflammatory diseases such as arthritis are characterized by dysregulated responses to pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α). Pharmacologic anti-cytokine therapies are often effective at diminishing this inflammatory response but have significant side effects and are used at high, constant doses that do not reflect the dynamic nature of disease activity. Using the CRISPR/Cas9 genome-engineering system, we created stem cells that antagonize IL-1- or TNF-α-mediated inflammation in an autoregulated, feedback-controlled manner. Our results show that genome engineering can be used successfully to rewire endogenous cell circuits to allow for prescribed input/output relationships between inflammatory mediators and their antagonists, providing a foundation for cell-based drug delivery or cell-based vaccines via a rapidly responsive, autoregulated system. The customization of intrinsic cellular signaling pathways in stem cells, as demonstrated here, opens innovative possibilities for safer and more effective therapeutic approaches for a wide variety of diseases.

Here, we propose a regenerative medicine approach to the treatment of chronic inflammatory diseases by engineering cells that execute real-time, programmed responses to environmental cues, including pro-inflammatory cytokines. We used genome editing with the CRISPR/Cas9 system to create stem cells that antagonize IL-1- and TNF-α-mediated inflammation in an autoregulated manner. To achieve this, we selected to overtake the chemokine (C-C) ligand 2 (Ccl2) gene, which is also known as macrophage chemoattractant protein-1 (Mcp-1). The Ccl2 gene product regulates trafficking of monocytes/macrophages, basophils, and T lymphocytes (). TNF-α and IL-1 serve as two of the most potent stimulators of Ccl2 expression (); however, the persistence of Ccl2 expression depends on continued exposure to inflammatory cues (), so resolution of inflammation results in rapid decay of Ccl2 transcripts. Thus, we performed targeted gene addition of IL-1 and TNF-α antagonists at the Ccl2 locus to confer cytokine-activated and feedback-controlled expression of biologic therapies. These programmed stem cells were then used to engineer articular cartilage tissue to establish the efficacy of self-regulated therapy toward protection of tissues against cytokine-induced degeneration. We hypothesized that this approach of repurposing normally inflammatory signaling pathways would allow for transient, autoregulated production of cytokine antagonists in direct response to cytokine stimulation. This type of approach could provide an effective “vaccine” for the treatment of chronic diseases while overcoming limitations associated with delivery of large drug doses or constitutive overexpression of biologic therapies.

A number of exogenous anti-cytokine therapies have been shown to effectively counteract the negative sequelae of TNF-α and IL-1 dysregulation. In particular, anti-TNF therapies such as the soluble type 2 TNF receptor (etanercept) and monoclonal antibodies to TNF-α (adalimumab, infliximab) have demonstrated efficacy toward offsetting pain associated with chronic and rheumatic diseases, including arthritis (). The soluble type 1 TNFR receptor (sTNFR1) has also been investigated as a gene therapy for treatment of chronic diseases (). More recently, competitive antagonists of IL-1 such as IL-1 receptor antagonist (IL-1Ra, anakinra) have been shown to alleviate symptoms of rheumatoid arthritis () and the onset of post-traumatic arthritis (). Although they are effective, these therapies are administered at very high and generally unregulated doses. Due to the pleiotropic roles of TNF-α and IL-1 and their involvement in tissue homeostasis, the use of such therapies may have significant side effects, including increased susceptibility to infection as well as to autoimmune diseases such as lupus, interstitial lung disease, and vasculitis (). Moreover, excess inhibition of these cytokines can interfere with tissue regeneration and repair (). Therefore, methods to dynamically deliver precisely calibrated doses of anti-inflammatory biologic therapies could improve treatments by combating cytokine-mediated pain and degeneration while spatially and temporally regulating the production of anti-cytokine drugs.

Chronic inflammatory and autoimmune diseases such as arthritis are characterized by aberrant activity of cytokines such as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1). These pro-inflammatory mediators are expressed by a variety of cells, including synovial cells, osteoblasts, myotubes, satellite cells, chondrocytes, and innate immune cells. These cell types are also capable of responding to TNF-α and IL-1 through canonical signaling via cognate cell surface receptors. Under normal physiologic conditions, appropriate signaling of TNF-α and IL-1 contributes to organ and tissue homeostasis by promoting tissue remodeling (), orchestrating phagocytosis of cellular debris and immunogenic substrates (), and coordinating transitions between niche stem cell quiescence and proliferation/differentiation programs (). However, in chronic diseases, elevated levels of these pro-inflammatory cytokines can lead directly to pain (), cytotoxicity (), accelerated tissue catabolism (), and exhaustion of resident stem cell niches ().

However, Ccl2-driven expression of IL-1Ra was not sufficient to protect against the suppression of the extracellular matrix constituents Col2a1 and Acan by 1 ng/mL IL-1. Coupled with the increased expression of degradative enzymes, this resulted in loss of a significant fraction of sGAG in the engineered tissue ( Figure 6 B). At the 0.1 ng/mL IL-1 level, cartilage derived from engineered Ccl2-Il1ra cells was less susceptible to degradation than tissue derived from control Ccl2-Luc cells, although sGAG loss normalized to total DNA content was still statistically significant after cytokine treatment in both tissue types ( Figures 6 C and 6D). This protection, in comparison with cartilage derived from Ccl2-Luc cells, was imparted by the cytokine-induced expression of 20.50 ± 0.67 ng/mL IL-1Ra, which was higher than the basal expression of 1.82 ± 0.24 ng/mL observed in the engineered cells or 0.88 ± 0.25 ng/mL observed in Ccl2-Luc cells.

Cartilage derived from Ccl2-Il1ra or Ccl2-sTNFR1 cells displayed a markedly different response to cytokine treatment at the gene expression level. Tissue generated from both the Ccl2-Il1ra and Ccl2-sTNFR1 cell lines demonstrated lower induction levels of inflammatory and degradative gene products compared with cartilage engineered from WT or Ccl2-Luc cell lines ( Figures 4 and 5 , respectively). Although haploinsufficiency of the Ccl2 gene affected basal levels of Ccl2 transcripts in the Ccl2-Luc, Ccl2-sTNFR1, and Ccl2-Il1ra cells compared with WT cells, cytokine stimulation rendered more marked upregulation of Ccl2 in cartilage derived from the Luc cells than sTNFR1 or Il1ra cells at 72 hr, suggesting that these transgenes ameliorated the impact of cytokine on Ccl2 gene expression, as expected. It is noteworthy, however, that in some cases these genes were still significantly upregulated relative to tissues treated with 0 ng/mL cytokine. In the case of Ccl2-sTNFR1, cartilage aggregates displayed resilience after 72 hr of treatment with TNF-α, with no suppression of Col2a1 or Acan. The preservation of a more homeostatic gene expression profile was consistent with the biochemical composition of cartilage aggregates engineered from the Ccl2-sTNFR1 cell line, which demonstrated preservation of sGAG in the tissue even after treatment with 20 ng/mL TNF-α, as determined by both biochemical and histologic analyses ( Figures 6 A and 6E). TNF-α induced secretion of sTNFR1, as specimens treated with 20 ng/mL TNF-α produced 18.45 ± 0.17 ng/mL sTNFR1 and those cultured in the absence of TNF-α produced only 3.31 ± 0.17 ng/mL sTNFR1.

Engineered cartilage specimens derived from WT and Luc cell lines exhibited a significant degradative response to this 72-hr cytokine treatment. We measured the changes in gene expression induced by 1 ng/mL IL-1 or 20 ng/mL TNF-α by qRT-PCR ( Figures 4 and 5 , respectively) and observed significant upregulation of markers of inflammation, such as Ccl2 and Il6, as well as degradative enzymes, such as matrix metalloproteinases and aggrecanases. Furthermore, significant suppression of expression of matrix components of cartilage, including collagen type 2 α1 (Col2a1) and aggrecan (Acan), was noted in cartilage engineered from either WT or Ccl2-Luc cells. The cartilage derived from these control cell lines also displayed a loss of sulfated glycosaminoglycan (sGAG), a major component of articular cartilage critical to proper tissue function, in response to both concentrations of IL-1 and to 20 ng/mL TNF-α ( Figures 6 A–6D ).

(C) sGAG/DNA in cartilage aggregates engineered from either Ccl2-Luc or Ccl2-Il1ra cells and maintained in control medium or medium supplemented with 0.1 ng/mL IL-1α for 3 days after maturation.

(A) Sulfated glycosaminoglycan (sGAG) per double-stranded DNA as measured via the dimethylmethylene blue assay in cartilage aggregates engineered from either WT, Ccl2-Luc, or Ccl2-sTNFR1 cells and maintained in control medium or medium supplemented with 20 ng/mL TNF-α for 3 days after maturation.

Fold changes were determined relative to a reference group cultured without IL-1α and by using 18S rRNA as a reference gene. Bars represent group means of fold change ± SEM (n = 3 independent experiments). Groups not sharing the same letter are statistically different (p < 0.05). Notation of n.s. implies no significance for the evaluated gene. Primer sequences are available in the table appended to Figure S2

Fold changes were determined relative to a reference group cultured without IL-1α and by using 18S rRNA as a reference gene. Error bars represent group means of fold change ± SEM (n = 3 independent experiments). Groups not sharing the same letter are statistically different (p < 0.05). Notation of n.s. implies no significance for the evaluated gene. Primer sequences are available in the table appended to Figure S2

After establishing that engineered cells express transgenes in a cytokine-inducible manner and that Ccl2-driven sTNFR1 provides a tunable and effective response to even a high dose of TNF-α in monolayer experiments, we assessed whether tissues engineered from engineered stem cells could overcome the degenerative effects of TNF-α and IL-1. To this end, we further differentiated the WT, Ccl2-Luc, Ccl2-Il1ra, and Ccl2-sTNFR1 cells toward the chondrocyte lineage for the production of engineered cartilage tissue (). Engineered tissues from WT and Ccl2-Luc cell lines were treated with 0 ng/mL cytokine, 0.1–1 ng/mL IL-1, or 20 ng/mL TNF-α. Engineered tissues from Ccl2-Il1ra and Ccl2-sTNFR1 cell lines were treated with only IL-1 or TNF-α, respectively, at the same concentrations as WT and Ccl2-Luc tissues.

When treated with 0.1 ng/mL IL-1, which sTNFR1 should not antagonize, there was approximately 300-fold stimulation of sTNFR1 production ( Figure 3 B) to ∼630 ng/mL. When treated with 20 ng/mL TNF-α, production of sTNFR1 increased only approximately 50-fold over basal levels to ∼90 ng/mL. Similarly, treatment of Ccl2-Il1ra cells with IL-1 resulted in an increase of IL-1Ra protein in the medium of approximately 30-fold over basal levels of expression to ∼180 ng/mL, whereas treatment with TNF-α resulted in an increase of approximately 88-fold to ∼570 ng/mL ( Figure 3 C). In WT cells, IL-1Ra production after a single pulse was 1.65 ± 0.35, 1.74 ± 0.11, and 1.84 ± 0.16 ng/mL after treatment with no cytokine, 0.1 ng/mL IL-1, or 20 ng/mL TNF-α, respectively ( Figure S2 ). Thus, in the case of both Ccl2-Il1ra and Ccl2-sTNFR1 cells, either IL-1 or TNF-α was capable of potently inducing transgene expression. However, a lower level of induction was achieved when an antagonizing therapy was produced in response to the stimulatory cytokine.

We then performed iterative stimulation of Ccl2-driven sTNFR1 and IL-1Ra cells in monolayer with either 0.1 ng/mL IL-1α or 20 ng/mL TNF-α. After 24 hr, the cytokine-containing medium was exchanged for cytokine-free medium, and specimens were collected. Three days later, cells were stimulated with cytokine again to establish the capacity of the cells to respond to recurrent stimulation with cytokine. Control specimens without cytokine stimulation were maintained in parallel. sTNFR1-engineered cells displayed a basal level of production of less than 3 ng/mL ( Figures 3 A and 3B ). Engineered cells rapidly produced sTNFR1 after either IL-1 or TNF-α stimulation ( Figures 3 A and 3B). Withdrawal of cytokine-containing medium resulted in a decline in sTNFR1 accumulation over subsequent collection periods, irrespective of whether IL-1 or TNF-α served as the stimulant. In both cases, production of sTNFR1 decreased to basal levels within 48 hr of removing cytokines ( Figure 3 A).

(C) The same experiment as described in (B) was performed using Ccl2-Il1ra-engineered cells, and ELISA was performed on samples to determine protein levels of IL-1Ra secreted into the culture media. Error bars represent the mean ± SEM (n = 3 independent experiments).

(B) Ccl2-sTNFR1-engineered cells were treated with cytokine, and 24 hr later media were collected. Cytokine was then withdrawn for 3 days prior to a second and then third stimulation to probe the kinetics of 24-hr sTNFR1 secretion after iterative stimulations.

(A) sTNFR1 levels measured in culture media conditioned for 24 hr prior to (D0) and after (D1) cytokine treatment. On D1, cytokine was withdrawn from all samples, and media were collected at 24-hr intervals for the subsequent 3 days.

To ascertain whether this attenuation was mediated by cytokine-inducible production of the TNF-α antagonist sTNFR1 from the engineered cells, we measured the expression of sTNFR1 transgene in parallel with Il6 expression. We found that sTNFR1 expression was rapidly upregulated at the 4-hr time point ( Figure 2 C). In the groups treated with 0.2 and 2 ng/mL of TNF-α, transgene expression began to decline between the 4- and 12-hr time points, in accordance with the decreased Il6 expression ( Figures 2 A and 2B). This transition likely reflects an attenuated state of inflammation after low and medium treatment of TNF-α. Ccl2-driven sTNFR1 expression continued to increase through 24 hr in response to the high TNF-α treatment, but this level declined rapidly toward baseline values at 72 hr, consistent with an inflammatory signal at 24 hr that largely resolved by 72 hr, as suggested by the Il6 and NF-κB transcription data ( Figures 2 A and 2B). In accordance with these qRT-PCR data, we measured increased accumulation of sTNFR1 protein in culture media over time in a dose-dependent fashion ( Figure 2 C).

To evaluate whether the observations of Il6 gene expression reflect the general state of inflammation in these cells, we transduced WT and Ccl2-sTNFR1 cells with a lentiviral vector delivering a nuclear factor κB (NF-κB) luminescence reporter. We then treated these cells with 0 or 20 ng/mL TNF-α and after 24, 48, and 72 hr measured luminescence as a surrogate for activity of the NF-κB transcription factor, which is activated in response to various inflammatory signals. At 24 hr, the NF-κB transcriptional activity was upregulated in both WT and Ccl2-sTNFR1 cells. However, at the 48- and 72-hr time points, a sharp decline in NF-κB transcriptional activity was observed in engineered cells expressing sTNFR1 under control of the Ccl2 locus ( Figure 2 B). Taken together, the Il6 gene expression and NF-κB transcriptional assays further support that the Ccl2-sTNFR1 cells are capable of attenuating the TNF-α-induced regulation of Il6 as well as a more general inflammatory state. Furthermore, these results suggest that, after 3 days of TNF-α treatment, the cells are capable of antagonizing even a high (20 ng/mL) concentration of TNF-α, while control WT cells remain in a state of inflammation even after treatment with only 0.2 ng/mL TNF-α.

As early as 4 hr after TNF-α treatment, the 2- and 20-ng/mL treatments significantly upregulated Il6 transcription in both the WT and Ccl2-sTNFR1 cells, while 0.2 ng/mL did not significantly upregulate Il6 ( Figure 2 A). At the 12-hr time point, Il6 expression was significantly elevated at all TNF-α concentrations in WT cells; however, Il6 was only significantly upregulated in the Ccl2-sTNFR1-engineered cells at the 20-ng/mL level of TNF-α treatment ( Figure 2 A). Even at the 20-ng/mL level of treatment, the engineered cells showed a significantly lower level of Il6 induction than WT cells. At the 24-hr time point, the medium and high concentrations of TNF-α drove an upregulation of Il6 in WT cells, but only the high 20-ng/mL concentration resulted in significant upregulation of Il6 in the sTNFR1-engineered cells ( Figure 2 A). By the 72-hr time point, all three doses of TNF-α resulted in significant upregulation of Il6 in the WT cells, while TNF-α treatment only induced an upregulation of Il6 in the Ccl2-sTNFR1 cells at the 20-ng/mL treatment level ( Figure 2 A). These results show reduced inflammatory response as a result of cytokine-mediated induction of sTNFR1 from the Ccl2 locus.

(D) ELISA data showing the concentration of sTNFR1 protein in culture media in samples treated with the indicated concentrations of TNF-α. Samples were collected at the indicated time. Values represent mean ± SEM (n = 3 independent experiments).

(C) Changes in Ccl2-driven expression of the sTNFR1 transgene over time as measured by qRT-PCR. Values plotted represent the mean fold change in expression ± SEM (n = 3 independent experiments) compared with matched cells of the same genotype treated with 0 ng/mL TNF-α and as normalized by the r18S reference gene. The 0-hr time point (shaded) was not measured and is shown for illustration purposes only, as all samples at 0 hr measure 1 by definition.

(B) Fold change in NF-κB transcriptional activity as measured by the luminescence signal from NF-κB-dependent luciferase expression. Bars represent the mean fold change in relative luminescence units (RLU) ± SEM of cells treated with 20 ng/mL TNF-α for the indicated time compared with controls cultured with 0 ng/mL TNF-α (n = 4–6 independent experiments).

(A) The profile of Il6 expression in response to various doses (x axes) of TNF and across indicated time points. Values plotted represent the mean fold change in expression ± SEM (n = 3 independent experiments) compared with matched cells of the same genotype treated with 0 ng/mL TNF and as normalized by the r18S reference gene. Hash indicates value greater than treatment with 0 ng/mL TNF ( # p < 0.05). ∗ p < 0.05 for WT versus sTNFR1-engineered cells.

Initially, we performed a time-course and dose-response experiment, in which Ccl2-sTNFR1 and WT cells were treated with a range of TNF-α concentrations (0.2–20 ng/mL) for a variety of times (4, 12, 24, and 72 hr). We measured the expression of the sTNFR1 transgene at both the mRNA and protein levels by qRT-PCR and ELISA, respectively. We also measured the expression of Il6, a pro-inflammatory cytokine whose expression serves as a sentinel marker of inflammation, at the mRNA level by qRT-PCR in order to additionally characterize the inflammatory response of the WT and engineered Ccl2-sTNFR1 cells.

We then probed the responsiveness of our engineered cells endowed with Ccl2-driven anti-cytokine transgenes. We performed these experiments primarily by evaluating gene expression and transgene production in the Ccl2-sTNFR1 group, as the inability of these murine cells to otherwise produce this human transcript and protein allows for direct conclusions regarding transgene production from the Ccl2 locus.

Next, using two Ccl2-luciferase cell lines, we induced luciferase expression by stimulating cells with 20 ng/mL TNF-α to evaluate whether transgene expression reflected endogenous Ccl2 expression in WT cells. Relative luminescence measurements indicated that transgene expression in both clones was indeed stimulated by cytokine and increased across the 72-hr TNF-α treatment period (p < 8.5e-10, Figure 1 C), consistent with findings from TNF-induced Ccl2 expression in WT cells.

Clones for each transgene with targeted gene addition on one allele were selected for further analysis (referred to as Ccl2-Luc, Ccl2-Il1ra, or Ccl2-sTNFR1) and expanded on murine embryonic fibroblasts (MEFs) followed by pre-differentiation in micromass culture (). First, we evaluated whether targeted transgene integration at the Ccl2 start codon would enable cytokine-inducible transgene expression. As a point of reference, wild-type (WT) cells were treated with a range of TNF-α concentrations (0.2–20 ng/mL), and mRNA was collected at 4, 12, 24, and 72 hr ( Figure 1 B). Ccl2 gene expression was evaluated by qRT-PCR. At all TNF-α concentrations tested, Ccl2 gene expression was elevated at each time point compared with cells cultured in the absence of TNF-α (p < 0.016). In the 2-ng/mL and 20-ng/mL groups, Ccl2 gene expression continued to increase throughout the 72-hr period of TNF-α treatment (p < 1.8e-10).

The primary goal of this work was to program induced pluripotent stem cells (iPSCs) with the capacity to respond to an inflammatory stimulus with potent and autonomously regulated anti-cytokine production ( Figure 1 A). As such, we aimed to perform targeted gene addition to the locus of the pro-inflammatory chemokine Ccl2, which is potently activated in response to cytokine-mediated signaling and of which disruption of a single allele would not be expected to compromise overall cellular function. Thus, transgenes encoding a firefly luciferase transcriptional reporter or a cytokine antagonist, either murine IL-1Ra or a chimeric human sTNFR1-murine immunoglobulin G (), were targeted to the Ccl2 start codon in murine iPSCs () using the CRISPR/Cas9 gene-editing platform. After hygromycin selection, clonal isolation, and screening by PCR of the junctions of the transgene and target locus, multiple clones were identified that possessed targeted integration events at the Ccl2 locus ( Figure S1 ).

(C) Two cell lines were engineered to express luciferase from the endogenous Ccl2 locus and were then stimulated with 20 ng/mL TNF-α. Cells were lysed at the indicated time after TNF treatment and luminescence was measured as a readout for Ccl2-driven transgene expression (n = 6 independent experiments). Values plotted represent the mean fold change in relative luminescence units (RLU) ± SEM compared with untreated controls of each cell line.p < 0.05 between each time point for each clone, and alsop < 0.05 between clones for each time point. See also Figure S1 and the appended table.

(B) qRT-PCR data showing the expression profile of Ccl2 after treatment of WT cells with various concentrations of TNF-α (n = 3 independent experiments). Values plotted represent the mean fold change in expression ± SEM compared with untreated controls.

(A) Top left: in wild-type (WT) cells, TNF-α signaling through its type 1 receptor initiates a cascade leading to nuclear translocation and increased transcriptional activity of NF-κB, activating an inflammatory transcriptional program. One gene rapidly and highly upregulated by cytokine-induced NF-κB activity is Ccl2 (shown in orange). Top right: a CRISPR/Cas9 RNA-guided nuclease (not depicted) generates a double-strand break in the endogenous chromosomal locus near the start codon for Ccl2. Provision of a targeting vector with a transgene flanked by regions homologous to the Ccl2 locus promotes the use of this template for repair of the damaged allele in a subset of cells. Bottom left: such alleles would then be activated by TNF-α, which would now induce expression of the soluble TNF type 1 receptor (sTNFR1). Bottom right: upon antagonism of TNF-α in the microenvironment, signal transduction through the membrane receptor would halt, NF-κB would remain sequestered in the cytoplasm, and expression of the sTNFR1 transgene would autonomously decay upon resolution of the local inflammation.

Discussion

Overcoming aberrant pro-inflammatory signals in chronic diseases while preserving critical homeostatic signaling nodes represents a significant challenge for regenerative medicine. This work demonstrates the utility of genome editing for the development of “designer” stem cells that sense levels of inflammation and respond according to the degree of the pathology. Using CRISPR/Cas9, we engineered pluripotent stem cells with the prescribed feature of inflammatory cytokine resistance by performing targeted addition of therapeutic transgenes to the cytokine-responsive Ccl2 locus. Transgene expression from engineered cells was feedback-controlled with rapid on/off dynamics and was adequate to mitigate the inflammatory effects of physiologic concentrations of both IL-1 and TNF-α in the context of precursor cells cultured in monolayer as well as in engineered tissues such as cartilage. These cells provide the foundation for a cell-based vaccine for the treatment of a variety of autoimmune or inflammatory diseases.

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Baltimore D. The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory molecules. In this work, we sought to commandeer an endogenous gene promoter to engineer custom-designed stem cells with the ability to regulate anti-cytokine therapy in an autonomous, real-time fashion. Critical to our selection of Ccl2 as the target locus for controlling transgene expression is the temporal pattern associated with its cytokine-inducible expression profile. By targeting our transgenes to the Ccl2 start codon, we preserved many of the endogenous regulatory features associated with Ccl2 expression, including distal and proximal regulatory regions encompassing two NF-κB regulatory elements as well as SP1 and AP-1 binding sites (). As such, the repurposed Ccl2 promoter did indeed endow engineered cells with the capacity to substantially upregulate transgene expression in an inflammation-inducible manner. Importantly, this upregulation was both dose- and time-dependent and was transient in nature. Treatment with a range of TNF-α concentrations spanning three orders of magnitude resulted in differential induction of transgene transcription. The concomitant decay in transgene expression and transcription of markers of inflammation such as Il6 suggests that cells were capable of autonomously tuning expression of the transgene. Importantly, our experiments also demonstrated that cells continue to respond to cytokines by robustly producing additional therapy after iterative exposure. Insertion of our transgene cassette did, however, uncouple regulation of our system from the endogenous AU-rich elements in the 3′ UTR of Ccl2, which are thought to play a role in driving transcript levels back toward a basal state after inflammation is resolved (). Despite this, expression of our transgenes did decay after resolution of cytokine stimulation, which came about by transgene therapy or simple withdrawal of cytokine. In future iterations of this work, preservation of the AU-rich elements in the transgene cassette may provide a means whereby even more rapid declines in transgene expression may be achieved.

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Harris D.C. Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor-kappaB dependent. Li and Schwartz, 2001 Li Y.P.

Schwartz R.J. TNF-α regulates early differentiation of C2C12 myoblasts in an autocrine fashion. Li, 2003 Li Y.P. TNF-alpha is a mitogen in skeletal muscle. Basal levels of transgene product were detected in the absence of cytokine by ELISA. This observation is not surprising, as Ccl2 is detected at the protein level from certain tissues without activation from cytokines (). Despite this, even low levels of cytokine treatment were capable of inducing transgene expression and initiating the inflammatory transcriptional program, suggesting that basal levels of cytokine antagonists were insufficient to abolish signaling from low concentrations of cytokine. Concentrations as low as 2–6 pg/mL () of TNF-α are important for proper muscle regeneration and repair. Thus, since the engineered cells are capable of responding to low levels of cytokine, the basal levels of anti-cytokine therapy may not preclude detection and response of niche cells to low but critical levels of cytokine.

Our data reveal apparent differences in cell responses to inflammatory cytokines depending on their differentiation status or lineage commitment. We attribute this discrepancy to potential differences in cell number, differentiation state of the cells, and natural variation in gene expression profiles of cells adopting different phenotypes. These observations could also be strictly related to the physical features of the different culture systems. Specifically, the effective concentration of TNF-α or IL-1 could be reduced by one to two orders of magnitude due to partitioning within a tissue matrix. Moreover, secreted sTNFR1 and IL-1Ra may remain bound by the rich extracellular matrix in the engineered cartilage, whereas factors secreted by cells in monolayer remain more readily accessible to diffusion in culture media.

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Rodriguez A. IL10 released by a new inflammation-regulated lentiviral system efficiently attenuates zymosan-induced arthritis. Previous investigators have taken various approaches to confer inflammation-inducible, autoregulatory features to target cells. A prevailing strategy has involved cloning approximately 3 kb of characterized, cytokine-inducible promoters upstream of transgene coding sequences. One example of this strategy is the use of the E-selectin promoter (). In other studies, a self-limiting promoter construct was developed based on a truncated promoter sequence of cyclooxygenase-2 upstream of the IL-4 gene to express IL-4 only in the presence of inflammation (). Alternatively, tandem repeats of NF-κB response elements have been used to drive transgene expression (). These expression cassettes are typically delivered to cells by viral gene delivery. While these approaches have indeed been proved to be effective at generating cytokine-inducible expression, notable limitations do exist. Most of these relate to the reliance on viral vectors to deliver the expression cassette and include the limited packaging capacity of adeno-associated virus, which restricts the size of the cloned promoter sequence, and the potential for insertional mutagenesis by lentiviral vectors. Furthermore, reported basal levels of transgenes produced from these promoters have been high, and, in some cases, growth factors other than inflammatory mediators are needed to co-stimulate efficient induction of transgene expression (). While such gene delivery vehicles carrying inflammation-inducible cassettes could be delivered in vivo, this method lacks cell- and tissue-targeting specificity.

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ade Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Our work extends these efforts by directly targeting transgenes to inducible, endogenous loci using the efficient and highly specific CRISPR/Cas9 genome-engineering technology (). In this manner, our strategy forgoes limitations associated with predicting regulatory features in a genetic locus such as distal enhancers. In addition, this approach abrogates the need to consider limitations on packaging efficiency, as the entire regulatory region need not be packaged in a gene delivery vector. Moreover, by performing targeted integration, this strategy absolves concerns associated with random insertion of provirus within the host genome.

Our use of iPSCs in these studies also provides an important advance, as the base cell population can be precisely defined and potentially undergo additional genome modifications if needed. This approach may prove attractive for regenerative medicine strategies, as clones may be screened for function and then expanded and differentiated toward a variety of terminal cell types to treat multiple tissues from the same engineered cell population. To achieve the same end, prior approaches would require isolation and expansion of primary cells from multiple tissues, followed by treatment of each population with gene delivery vehicles, and finally delivery of engineered cells to the host. This approach lacks the specificity conferred by targeting pre-determined genomic sites for modification using gene-editing nucleases and requires that engineered, primary cells do not senesce prior to serving a therapeutic purpose.