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A device is described for an extracorporeal bioreactor comprising cells selected for their ability to produce cytokines and/or cytokine inhibitors for controlling inflammation in a patient.

One current goal of medicine is to facilitate the intrinsic self-renewing ability by relieving damaged tissues from their functional burden and facilitating tissue healing. In order to achieve this goal, it is necessary to acknowledge, understand, and control acute inflammation. We have developed mathematical models of inflammation that inter-related inflammation and tissue damage/dysfunction. Our therapeutic goal is not to abolish inflammation per se but to reduce damage/dysfunction (i.e. promote healing) by modulating inflammation in a rational fashion based on these computational models.

To do so, a self-regulating device and related methods are described herein for individualized regulation of inflammation. The basic concept of the proposed device is to create negative feedback proportional to the exact degree of inflammatory stimulus. More precisely, several inflammatory mediators known as cytokines (protein hormones that induce, modulate, and augment inflammation) are in turn regulated by endogenous inhibitors. In the proposed device, the device would produce or release one or more units of the neutralizing protein for every one or more unit of a given inflammatory cytokine. The device would be a biohybrid device, in which gene-modified cells are housed in a bioreactor or matrix. The genetic modification(s) of the cells housed in this device would serve to 1) sense the levels of a given inflammatory cytokine or cytokines; 2) produce the appropriate levels of the appropriate cytokine inhibitor(s); and 3) possibly also release diagnostic markers that would serve to either delineate the degree of inflammatory cytokines produced by the patient, to delineate the degree of production of the cytokine inhibitor(s), or both (i.e. a “theranostic” device). These genetic modifications could also include other diagnostic, cytotoxic, or therapeutic proteins stimulated by the genetic elements that would sense the presence of inflammatory cytokines in the patient. This device will, in theory, solve the current need for a personalized (yet standardized) inflammation-modulating therapy. The device will be standardized since, for a given disease, a single bioreactor or release matrix would be used. The device would be personalized since a given patient's individual production of cytokines (i.e. the quality and quantity of overall cytokine production) would be counteracted in a precise fashion and only as required and guided by the mathematical model of a given inflammatory disease. Moreover, this device would both obviate the need for a diagnostic method prior to determination of treatment of a given inflammatory disease, since the device would essentially serve as a diagnostic through either the genetic elements that would sense the levels of relevant inflammatory cytokines in the patient, or through the production of indicator proteins at levels proportional to the inflammatory cytokines produced by the patient. Finally, since this device would greatly reduce the time from diagnosis (including possibly the calibration of patient-specific computational simulations of an inflammatory disease) to treatment, since both diagnosis and treatment would be carried by the device in a simultaneous fashion. The reduction in time to treatment is especially important in inflammatory settings such as sepsis, trauma, traumatic brain injury, and wound healing, in which individual trajectories (e.g. length of stay in the hospital) and outcomes (e.g. survival, death, or long-term scarring) may be determined after a short period of inflammation.

The device described herein could be used to modulate inflammation in, for example: 1) acute, systemic inflammatory diseases (e.g. sepsis, trauma); 2) chronic, systemic inflammatory diseases (e.g. rheumatoid arthritis); and 3) cancer with an inflammatory etiology. The device could also be used to reprogram inflammation in a local context (e.g. a skin wound or psoriatic lesion) if interfaced appropriately with such a wound or lesion. The device could be used to influence the early-, mid-, or late-stage inflammatory response to any trauma (accidental or iatrogenic [e.g. surgical]) so as to bring about improved healing and rehabilitation. The device could serve solely as a therapeutic device, solely as a diagnostic device, or both. The device could be fine-tuned to each of these applications based on specifications for optimal outcome given by mathematical and other computer simulations of the interrelated inflammatory and damage/healing responses. This device could be used both in civilian and military settings. It could be applied rapidly to start monitoring and/or modulating inflammation in a personalized fashion, or could be applied at any point in time of the inflammatory response if its properties are tuned properly.

Current inflammation therapy can only be individualized to a small extent, given that FDA approval requires that a device or drug be utilized in a standard fashion. This device would be 1) a standardized device that would not require harvesting and culturing of a patient's cells, and yet 2) personalized, in that it would elaborate molecules that specifically neutralize only those inflammatory agents made by a given patient, and at levels driven by the levels of these inflammatory agents in each patient. This device would obviate the need for a time-consuming and expensive cycle of blood sampling, analysis of inflammation biomarkers, calibration of patient-specific mathematical models of inflammation, determination of individual-specific therapy, and implementation of that therapy. Instead, the device's general operating characteristics would be tuned to a given disease by a generalized model, for example a mathematical model, of the disease (based on population data), and yet the specific degree of elaboration of molecules that antagonize each inflammatory agent would be driven by the characteristics of a given patient. Thus, the device would offer the benefit of personalized, rational inflammatory modulation that would take much less time and resources to implement.

In its most general form, the device comprises a bioreactor seeded with cells, such as hepatocytes, genetically engineered to respond to a cytokine with that cytokine's own inhibitor. An additional benefit is that many different types of such genetically engineered cells could be made and stored indefinitely in liquid nitrogen. The stored cells could be thawed and combined in the proportions prescribed by a computational model of a given inflammatory disease, such as a mathematical model described by ordinary or partial differential equations or an agent-based model. Thus, another benefit of this device would be the theoretical capability of a nearly infinite spectrum of operating characteristics. Various embodiments of such a device could be conceived, including reservoirs or biomaterials sensitive to a given cytokine that release the inhibitor, implantation of cells genetically modified to express the inhibitor upon exposure to the cytokine, or bioreactors seeded with these genetically modified cells.

One exemplary procedure for creating and using the bioreactor version of this device is:

Utilize mathematical models of inflammation in a given systemic inflammatory disease to determine the optimal modulation of inflammation that would result in reduced tissue damage/dysfunction. See, e.g., United States Patent Publication Nos. 20030087285 and 20080228456 for examples of modeling methods for, e.g., sepsis, wound healing, vocal fold damage, and, generally inflammation using both object-oriented (agent-based) and equation-based modeling. Currently, factors that enhance damage/dysfunction in our existing mathematical models of inflammation include circulating cytokines that activate macrophage, neutrophils, and TH1 cells (e.g. TNF, IL-6, IL-12, IFN-λ, IL-2) as well as effector products such as nitric oxide, superoxide, and peroxynitrite. A computerized algorithm can search the parameter space of the mathematical model of acute inflammation, in order to determine what changes to the circulating cytokines characteristic of the inflamed state (in which damage/dysfunction is high) will result in reducing damage/dysfunction to levels characteristic of health.

Recombinant DNA constructs are made that consist of a promoter region sensitive to a given cytokine and that cytokines endogenous inhibitor, based on the predictions of the mathematical model in Step 1. Examples include: 1) tumor necrosis factor (TNF) and its endogenous inhibitor, soluble TNF receptor, 2) interleukin-1 (IL-1) and IL-1 receptor antagonist, 3) transforming growth factor-β1 (TGF-(β1) and TGF-β1 latency-associated peptide (LAP). Many additional examples also exist. The device could be tuned for more rapid or slower response to cytokines by incorporating multiple copies of a given promoter element, or by using promoter elements of various inherent sensitivities to cytokines. The device could be tuned for various degrees of suppression of a given cytokine by incorporating multiple copies of the gene for the cytokines endogenous neutralizer.

Stable transfection in cells such as a hepatocyte cell line, hepatocytes, or other suitable cells, are made with the gene constructs described in Step 2. This step may take place by first creating viruses that contain the DNA constructs and subsequently infecting the cells described above, or by means of stable transfection methods. The cell lines could be of human or non-human origin, although the likeliest embodiment would utilize human cells in order to reduce the likelihood of immune reactions to nonhuman proteins.

The transfected cells are seeded into vessels that allow for nutrients, oxygen, etc. to be delivered to the cells in order to maintain their viability. Bioreactors containing hepatocytes have been maintained stably for a month or more.

The device is connected to a patient's circulation via, e.g., catheters. Alternatively, the device is connected in some fashion to a skin wound or other local site of inflammation.

Blood or another relevant bodily fluid from the patient is circulated through the device. Inflammatory cytokines in the patient's bodily fluid would stimulate the release of the cytokine-specific neutralizing proteins, with a rate and magnitude driven both by the device's characteristics and the patient's own characteristics of inflammation. With time, lower levels of the patient's inflammatory cytokines would be made as the device's inflammation dampening process proceeds. As this happens, less of the neutralizing proteins would be made, since the stimulus for their production would be lower. During this process, small samples of the inflow and outflow from the device could be removed for analysis of cytokines and comparison to the predictions of the mathematical model, to determine that the device is operating as predicted and that this operation is predicted to result in reduced damage. Alternatively, if the device is constructed so as to allow for the direct or indirect detection of either the patient's inflammatory cytokines or of the inhibitor(s) produced by the bioreactor, then diagnosis could be carried in this manner. Other blood parameters (e.g. liver transferases, bilirubin, etc.) could also be measured as adjunct measures of the function of the device and the patient's health status. Eventually, an inflammatory steady state compatible with improved outcome would be reached. The device may then be either disconnected or replaced with another, similar device that modulates a related or subsequent inflammatory process, or a device that modulates a known co-morbidity or consequence of the inflammatory response (e.g. cancer).

As mentioned above, we have developed a series of mathematical models of inflammation and its interactions with tissue damage and healing, with the goal of understanding, predicting, and controlling inflammation (Kumar, R., et al., J. Theoretical Biol. 230, 145-155 (2004); Clermont, G. et al. Crit Care Med. 32, 2061-2070 (2004); Chow, C. C. et al. Shock 24, 74-84 (2005); Reynolds, A. et al. J. Theor. Biol. 242, 220-236 (2006); Day, J. et al. J. Theor. Biol. 242, 237-256 (2006); Prince, J. M. et al. Mol. Med. 12, 88-96 (2006); Lagoa, C. E. et al. Shock 26, 592-600 (2006); Constantine, G., et al. J. Pure Appl. Math. doi:10.1007/s10589-007-9118-9., (2007); and Upperman, J. S. et al. J. Pediatr. Surg. 42, 445-453 (2007); Li, N. Y. K., et al. PLoS ONE. 2008. 3:e2789; and Torres, A. et al. Shock. 2009. 32:172-178). The Translational Systems Biology models we have developed to date (An, G.; et al. J. Burn Care Res. 2008. 29:277-2; Vodovotz, Y., et al. PLoS Comput. Biol. 2008. 4:1-6; and Vodovotz, Y. et al. Math. Biosci. 2009. 217:1-10) have been based on multi-scale inter actions at the cell-tissue-organ-organism level and clinical trial simulations at the population level, constructing both equation-based and agent-based models of various degrees of granularity. These innovative models encompass the dynamics of relevant cells, cytokines, and the resulting global tissue dysfunction in order to begin to unravel these inflammatory interactions. “Global tissue damage/dysfunction” is conceptually equivalent to “alarm/danger signals” released from stressed or necrotic cells, and serves as a proxy for the overall health of the organism. Our published models describe and predict various features of septic shock (Redd, M. J., et al. Philos. Trans. R. Soc. Lond B Biol. Sci. 359, 777-784 (2004); Kumar, R., et al., J. Theoretical Biol. 230, 145-155 (2004); Clermont, G. et al. Crit Care Med. 32, 2061-2070 (2004); Chow, C. C. et al. Shock 24, 74-84 (2005); and Constantine, G., et al. J. Pure Appl. Math. doi:10.1007/s10589-007-9118-9., (2007)) and trauma/hemorrhage (Kumar, R., et al., J. Theoretical Biol. 230, 145-155 (2004); Reynolds, A. et al. J. Theor. Biol. 242, 220-236 (2006); and Day, J. et al. J. Theor. Biol. 242, 237-256 (2006)), including the simulation of anti-inflammatory strategies in clinical trials (Clermont, G., et al. Crit Care Med. 2004. 32:2061-2070; Mi, Q. et al. Wound Rep. Reg. 2007. 15:671-682; Kumar, R. et al. Shock. 2008. 29:104111.and An, G., et al. J. Crit. Care 22, 169-175 (2007)).

The basic concept of the proposed device is to create negative feedback proportional to the exact degree of inflammatory stimulus. More precisely, for every unit of a given inflammatory cytokine, the device would produce or release essentially one unit of the neutralizing protein. Examples include: 1) tumor necrosis factor (TNF) and its endogenous inhibitor, soluble TNF receptor (An, G. et al. J. Burn Care Res. 29, 277-285 (2008); Gallucci, S. et al. Curr. Opin. Immunol. 13, 114-119 (2001); Vodovotz, Y. Immunologic Res. 36, 237-246 (2006); and Aggarwal, B. B. et al. Ernst. Schering. Res. Found. Workshop161-186 (2006)); and, 2) interleukin-1 (IL-1) and IL-1 receptor antagonist (Hasegawa, A., et al. Takasaki, W., et al. Mini. Rev. Med. Chem. 1, 5-16 (2001)), 3) transforming growth factor-β1 (TGF-β1) and TGF-β1 latency-associated peptide (LAP, Fernandez-Botran, R., et al. Expert. Opin. Biol. Ther. 2, 585-605 (2002) and Bottinger, EP., et al.,Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5877-5882, June 1996). Many additional examples also exist. Various embodiments of such a bioreactor device could be conceived, including reservoirs or biomaterials sensitive to a given cytokine that release the inhibitor, implantation of cells genetically modified to express the inhibitor upon exposure to the cytokine, or bioreactors seeded with these genetically modified cells (FIG. 1). The proof of concept studies described here would take the latter approach.

Importantly, there must be a rational process by which to tailor the specific characteristics of such a device. For example, the specific cytokines to be antagonized and the timing and magnitude of such manipulation will vary depending on the nature of the inflammatory disease targeted. The mathematical models we have created are therefore useful for the rational construction and utilization of this device.

A bioreactor is therefore provided, for example, comprising a compartment comprising cells comprising a chimeric gene. The chimeric gene comprises a response element operably linked to a sequence encoding a cytokine or an inhibitor of a cytokine, in which the response element causes expression of the cytokine or causes expression of the inhibitor of the cytokine when the cells are contacted with the cytokine. The bioreactor comprising a selectively permeable membrane in contact with the cells. As disclosed herein, the gene can express a cytokine inhibitor of one of TNF, IL-1, TGFβ1 and IL-6, such as TNF receptor, IL-1 receptor agonist, TGF-β1 LAP (latency-associated peptide) and an IL-6Ralpha/gp130 fusion protein.

The selectively permeable membrane can be a selectively-permeable hollow fiber. Alternately, the compartment comprising the cells can comprise a vessel having a selectively permeable wall. The vessel may comprise a plurality of selectively permeable hollow fibers passing through the compartment through which one or both of a gas and a fluid comprising nutrients for the cells can be passed. In another embodiment, the compartment comprising the cells comprises a plurality of selectively permeable hollow fibers passing through the compartment in which the plurality of hollow fibers are fluidly connected to a plasma or blood circulation system in which blood or plasma from the patient can be circulated through the hollow fibers and into a patient.

The cells may be any cell that is effective in its use in the bioreactor, and may be xenogeneic, syngeneic, allogeneic, or autologous cells to a patient treated by use of the bioreactor. In one embodiment, the cells are transfected or transduced hepatocytes or a hepatocyte cell line, such as HepG2. The bioreactor may further comprise a cell comprising a (nucleotide) sequence encoding a fluorescent protein that either is: a) operably linked to the response element and the sequence encoding the cytokine or inhibitor of the cytokine is attached to and in frame with the sequence encoding the fluorescent protein and a self-cleaving polypeptide sequence between the sequence encoding the cytokine or inhibitor of the cytokine and the sequence encoding the fluorescent protein; or b) under control of a second response element (in the cell or a second cell) that causes expression of the fluorescent protein when the cells are contacted with the cytokine. Alternately, one or more of the cytokines or inhibitors of cytokines encoded by the one or more non-native inducible genes comprises a fluorescent tag that is contiguous with the one or more of the cytokines or inhibitors of cytokines.

Also described herein is a method of modulating (controlling, affecting) wound healing, sepsis, trauma, or traumatic brain injury (TBI), comprising, contacting a bodily fluid of a patient with the selectively permeable membrane of the bioreactor of claim1such that a cytokine in the bodily fluid can pass through the selectively permeable membrane and a cytokine or cytokine inhibitor produced by the cells can pass into the bodily fluid, and returning the bodily fluid to the patient. The bioreactor may be any bioreactor described herein. The cells, their quantity and chimeric genes they express, may be selected by use of a computer model of an inflammatory response characteristic of a disease or condition in the patient. The method may include modeling inflammation associated with wound healing, sepsis, trauma or TBI and determining one or more cytokines to inhibit or produce to control inflammation in the patient associated with sepsis, wound healing or trauma. In one embodiment, data obtained from a patient may be used to assist in modeling inflammation or in tailoring the treatment to a patient. For example, the method may comprise determining levels of one or more cytokines in the patient and modeling inflammation using the one or more levels of cytokines in the patient and determining a cytokine level to be controlled in the patient to determine a chimeric gene construct to place in the bioreactor based on an outcome of the modeling. The method may comprise determining levels of one or more cytokines in the patient and modeling inflammation using the one or more levels of cytokines in the patient and determining a cytokine level to be controlled in the patient to determine a chimeric gene construct to place in the bioreactor based on an outcome of the modeling. In one embodiment, the patient is a TBI patient, and for example, one or both of an inhibitor of TNF and an inhibitor of IL-6 are produced by the cells. In another embodiment, the cells comprise one or more genes that express an inhibitor of one or both of TNF and IL-1α or IL-1β. In another embodiment, the gene expresses an inhibitor of a cytokine selected from the group consisting of soluble TNF receptor, IL-1 receptor agonist, and TGF-β1 LAP (latency-associated peptide).

