The use of an assay to mimic adhesion qualities of mammalian cells

Abstract

A synthetic is disclosed. The synthetic tissue comprises a colony of modified single celled organisms; and a protein produced by the modified single cell organism. The modified single cell organism may be modified by a plasmid. The protein may resemble at least one protein of the collection of proteins known as tight junction or another cell-to-cell adhesion protein. A region of the protein that resembles a specific region of a cell-to-cell adhesion protein may link to the same region of other proteins using linker regions. The region of the protein that resembles the specific region of a cell-to-cell adhesion protein may resemble a protein of the outer cell membrane of the single cell organisms in the colony of single cell organisms.

Description

THE USE OF AN ASSAY TO MIMIC ADHESION QUALITIES OF MAMMALIAN CELLSTECHNICAL FIELD

[0001] This disclosure relates to cell adhesion and modifications to single cell organisms to represent mammalian tissues.BACKGROUND

[0002] Multicellular organisms, like humans, require cellular interactions for function. Cell-cell interactions are mostly achieved by adhesion or interaction of proteins between cells. There is a family of cell-adhesion proteins that establish contact points rather than a comprehensive cell adhesion with its neighbors. A membrane protein structure, named Tight Junction (TJ), provides a cell-cell interaction that spreads throughout the circumference of the cell. Cell-adhesion molecules (CAM) are membrane proteins responsible for cell-cell interactions or cell-extracellular matrix interactions.SUMMARY

[0003] In a first aspect, the disclosure provides a synthetic tissue. The synthetic tissue comprises a colony of modified single celled organisms; and a protein produced by the modified single cell organism.

[0004] In certain aspects the modified single cell organism may be modified by a plasmid. The protein may resemble at least one protein of the collection of proteins known as tight junction or another cell-to-cell adhesion protein. A region of the protein that resembles a specific region of a cell-to-cell adhesion protein may link to the same region of other proteins using linker regions. The region of the protein that resembles the specific region of a cell-to-cell adhesion protein may resemble a protein of the outer cell membrane of the single cell organisms in the colony of single cell organisms.

[0005] The single cell organisms may be E. coli. The single cell organisms may be yeast.

[0006] The cell-to-cell adhesion protein may establish the tight junction. The plasmid may incorporate one or more of a JAM protein gene, a claudin protein gene, or an occludin protein gene into the single cell organism. The JAM protein may be a JAM-A protein. The claudin protein may be one of CLDN 1, CLDN 2, or CLDN 10.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The following drawings are provided to illustrate certain embodiments described herein. The drawings are merely illustrative and are not intended to limit the scope of claimed inventions and are not intended to show every potential feature or embodiment of the claimed inventions. The drawings are not necessarily drawn to scale; in some instances, certain elements of the drawing may be enlarged with respect to other elements of the drawing for purposes of illustration.

[0008] Figure 1 is a schematic of E. coli showing the relevant structures.

[0009] Figure 2 is a plasmid map showing the integration of Claudin 3 gene and an OmpW gene into a plasmid.

[0010] Figure 3 is a photo of a gel electrophoresis comparison of two plasmids, one with an OmpW gene and one with an OmpW gene and a CLDN3 gene.

[0011] Figure 4 is an electron micrograph of two E. coli colonies. One wild type colony which does not aggregate and one colony in which the claudin (CLD3) protein is expressed on the outer membrane of the E. coli. The E. coli with the claudin expression aggregates. The aggregates form tight junction -like assemblies.

[0012] Figure 5 is a photo of growth plates. The expression of claudins in the outer membrane of E. coli prevents the E. coli from colonizing the entire plate.

[0013] Figures 6A-6F show data analysis using FlowJo and R Studio software. Flow cytometry data was gated in FlowJo software (Fig. 6A and Fig. 6B) and R Studio converted the data into Hexbin graphs (Figs. 6C and Fig. 6D) and determined the slope of the line (Fig. 6E). Fig. 6F, graphical representation of slopes of cells not expressing claudins (OmpW) or expressing a variety of members of the claudin family.

[0014] Figure 7A is a graph demonstrating dose-dependent changes to permeability caused by Caprate. Our data suggests iCLASP is capable of identifying changes to CLDN-CLDN and also OCLN-OCLN interactions and can be used for discovery of small molecules that affect paracellular permeability controlled by TJs.

[0015] Figure 7B is a graph demonstrating dose-dependent changes to permeability caused by Ethanol. Our data suggests iCLASP is capable of identifying changes to CLDN-CLDN and also OCLN-OCLN interactions and can be used for discovery of small molecules that affect paracellular permeability controlled by TJs.

[0016] Figure 8A shows the effect of Ion permeable human CLDN2 (cations) exposed to the influence of different salts. For CLDN2 Chloride salts were used. Flow Cytometry analysis enables the determination of Experimental Slopes that represent adhesive properties of CAMs in thepresence of 100 mM salts. Data presented here was statistically analyzed. All points are expressed as the average value of 12-replicates in 4 different experiments (n=4) + SE. All points are statistically significant and significantly different from each other.

[0017] Figure 8B shows the effect of human CLDN10 (anions) exposed to the influence of different salts. For CLDN10 Sodium salts were used. Flow Cytometry analysis enables the determination of Experimental Slopes that represent adhesive properties of CAMs in the presence of 100 mM salts. Data presented here was statistically analyzed. All points are expressed as the average value of 12-replicates in 4 different experiments (n=4) + SE. All points are statistically significant and significantly different from each other. Asterisks are omitted for display purposes.

[0018] Figure 9 is a graph depicting Flow Cytometry analysis for the recombinant expression of cpOmpW-JAM-A protein. Experimental slopes of empty plasmid (pET28a), cpOmpW protein alone and the fusion with JAM-A are plotted in the graph. The higher Experimental Slope indicates the presence of BL21 DE3 cells aggregates, which represent the adhesive properties of JAM-A. Data presented here was statistically analyzed. All points are expressed as the average value of 12-replicates in 4 different experiments (n=4) + SE. All points are statistically significant and significantly different from each other. Asterisks are omitted for display purposes.

[0019] Figure 10 is a depiction of an experimental model. The iCLASP experimental model is presented here. Unicellular BL21 DE3 cells are transformed with plasmids hosting a fusion of OmpW and CAMs.

[0020] Figure 11 is a flow chart showing Protein Engineering strategies for the expression of single membrane proteins of the Tight Junction or complex mixtures. A) A native E. coli protein that normally is located in the outer membrane can be used to shuttle CLDNs or OCLN since their topology matches that of OmpW. B) Circular permutated OmpW changes the positions of the bland C-terminal amino acids to be extracellular, enabling the fusion of JAMs that ultimately can display their cell-adhesion properties extracellularly. C) Using cpOmpW JAMs can be concatenated and include CLDNs and OCLN to the single chain polypeptide that ensures identical expression of all proteins. These proteins when fused correspond to a mixture that represents a full Tight Junction.

[0021] Figure 12A is a depiction of a first round of screening to analyze potential drug compounds. Each well contained 10 compounds in the first Round, 10 pM each. This approach reduced the 50,000 challenges in quadruplicates to only 55 days. Based on a twice-a-week schedule availability of our Core Facility. The time to complete Round 1 was 6 months.

[0022] Figure 12B is a graph depicting the compounds that show increased aggregation of cells as compared to the control.

[0023] Figure 12C is a graph depicting the compounds that show decreased aggregation of cells as compared to the control.

[0024] Figure 12D is a depiction of the second round of screening to analyze potential drug compounds. Each well contained a single compound, 10 pM. This approach identified which compound among the 10 in the Round 1 screen had the most activity. Based on a twice a week schedule availability of our Core Facility, the time to complete Round 2 was 2 weeks.

[0025] Figure 12E is a depiction of the third round of screening to analyze potential drug compounds. Each well contained a single compound. Dilutions were 10, 5, 2, 1, 0.8, 0.4, 0.2, 0.1 pM. This approach identified which compound among the ones selected in Round 2 had a dose response. Based on a twice a week schedule availability of our Core Facility, the time to complete Round 3 was 2 weeks.

[0026] Figure 12F is a Graph of one compound (G04:6502939) which demonstrated dose dependence.DETAILED DESCRIPTION

[0027] The following description recites various aspects and embodiments of the inventions disclosed herein. No particular embodiment is intended to define the scope of the invention. Rather, the embodiments provide non-limiting examples of various compositions, and methods that are included within the scope of the claimed inventions. The description is to be read from the perspective of one of ordinary skill in the art. Therefore, information that is well known to the ordinarily skilled artisan is not necessarily included.Definitions

[0028] The following terms and phrases have the meanings indicated below, unless otherwise provided herein. This disclosure may employ other terms and phrases not expressly defined herein. Such other terms and phrases shall have the meanings that they would possess within the context of this disclosure to those of ordinary skill in the art. In some instances, a term or phrase may be defined in the singular or plural. In such instances, it is understood that any term in the singular may include its plural counterpart and vice versa, unless expressly indicated to the contrary.

[0029] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a substituent” encompasses a single substituent as well as two or more substituents, and the like.

[0030] As used herein, “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. Unless otherwise expresslyindicated, such examples are provided only as an aid for understanding embodiments illustrated in the present disclosure and are not meant to be limiting in any fashion. Nor do these phrases indicate any kind of preference for the disclosed embodiment.

[0031] Mammalian tissues are composed of cells that possess specific adhesion characteristics. These adhesion characteristics are unique to each cell type and in each type of tissue. The adhesion characteristics of each cell type play an important role in the health and functionality of the cell and the tissue. Obvious examples of how the adhesion characteristics of a cell affect its function arise in the form of barrier permeability, and tissue structure. However, these are not the only reasons that cell adhesion characteristics are important. Research has shown that even elements of cell health such as rate of proliferation can be modulated by cell adhesion proteins.

[0032] The most significant group of proteins that modulate cell-cell adhesion is the tight junction (TJ). The tight junction is made up of at least 3 significant parts, namely the JAM (junctional adhesion molecule) protein, the claudin protein, and the occludin protein. Different combinations of these proteins allow for different qualities in the TJ. Each TJ has a JAM protein (JAM A, JAM B, JAM C, or JAM 4), at least one claudin protein (of 24 varieties), and one occludin protein (only one variety exists).

[0033] Replicating the function of the TJ has been an important field of research in the past. One specific tissue of interest is the blood-brain barrier (BBB). Many attempts have been staged to try and replicate the TJ function of the BBB, and some have made impressive strides, but all remain far from the in vivo characteristics of the BBB. Furthermore, all are very expensive, or very time consuming. More often than not they are both expensive and time-consuming. There is a dire need for high throughput, cheap, and efficient ways to model TJ function in mammalian cells.

[0034] In a previous patent disclosure to the BYU technology transfer office (TTO) Dr. Dario Mizrachi outlines a novel method for using bacteria to mimic adhesion qualities of animal cells. This disclosure can be found under the title "Flow Cytometry to evaluate adhesion proteins for pharmacological interventions in cell-cell interactions". The first is titled "Expression of celladhesion molecules in E. coli: a high-throughput method to identify paracellular modulators" (https: / / www.biorxiv.org / content / 10.1101 / 2021.04.08.439041vl) and the second is titled "Expression of Cell-Adhesion Molecules in E. coli: A High Throughput Screening to Identify Paracellular Modulators" (https: / / www.mdpi.com / 1422-0067 / 24 / 12 / 9784). These articles outline a technology that enables the use of E. coli bacteria to mimic cell-cell adhesion. Let me explain in brief how it works. A bacterium is transformed with a plasmid containing the sequence for one specific membrane protein fused to a protein native to the outer membrane of E. coli called OMPW. The protein is subsequently expressed in the outer membrane of E. coli.

[0035] Previous technology possesses a fatal flaw that prevents it from being more widely useful in the world of biotechnology. That flaw is the fact that it is limited to one membrane protein at a time. For example, using the bacterial technology you could see expression of claudin (CLDN) 5, or of occludin (OCLDN), but not both at once. This is a problem mainly because in mammalian tissues the TJ characteristics depend on the combination of several TJ proteins. One protein on its own produces different characteristics than those produced by the tissue specific combination of several TJ proteins.

[0036] A new technology consists of E. coli bacteria that express several TJ proteins all at the same time, and in a way that more strongly mimics mammalian TJ function.

[0037] It is significant that use of this technology allows the person in possession of it to create a model for a specific TJ in a specific human tissue (dependent on their understanding of the proteins involved in the tight junctions of the tissue in question.) This technology will certainly provide for a system that mimics mammalian cell behavior in a way that allows us to use it to make predictions about how a mammalian cell or tissue would behave under certain conditions.

[0038] This technology could be used for countless applications. One example could be to create a series of cultures that mimic a series of human tissues for the purpose of testing drugs against these tissues. It is high throughput and closely mimics mammalian cell behavior. Another example of how this technology could be used is to figure out what the targets of existing drugs are. Another example of how this technology could be used is to explore why diseases progress by testing the effect of different substances (biological or synthetic) on a model of the effected tissue.

[0039] This technology is not limited to being used in E. coli. It can be used in any model organism that one chooses. One example of another model organism that we want to use is yeast. More details on the application of this technology in yeast can be found in the detailed description section.

[0040] This technology is also not limited to tight junction proteins only. It can reasonably be applied to any cell adhesion proteins. You would just have to input the desired proteins in the plasmid in the desired order and orientation.

[0041] II. Advantages: Describe the advantages of the Invention(s) over previous work.

[0042] As noted previously, the largest advantage over previous work is the fact that is efficient, fast, and low cost. Furthermore, it allows for a closer representation of a mammalian cell than the technology that has previously been disclosed. This makes for more reliable predictions about the effects of drugs, sources of disease, etc.

[0043] Additionally, if yeast is chosen as the organism in which to use this technology, there is even more similarity between yeast and a mammalian system. This would provide aeukaryotic system that can mimic mammalian cell-cell adhesion behavior in an extremely high throughput way. This could potentially help this technology compete even with immortalized mammalian cell cultures in efficacy of testing.

[0044] III. Detailed Description: Provide a detailed description of the Invention(s) and how it functions — include drawings, sketches, and flow charts if appropriate.

[0045] This newly developed technology is executed in a similar way to how the previous technology is executed, but with key improvements. The single celled organism used is BL21 E-coli DE3 (commercially available). The plasmid that the Bactria are transformed with consists of several regions. The first is a T7 promoter region, which has been described previously in public works. Second comes Circularized Protein OMPW (cpOMPW). cpOMPW is different from regular OMPW because the N and C terminus of the protein are moved from the inside of the cell to the outside of the cell. This is significant because it allows us to use a protein next that starts with an extracellular region. So, next comes a region called Tobacco Etch Virus (TEV) Digestible Region. This region allows us to disconnect the model TJ from the cpOMPW after it has been delivered to the outer membrane of the E. coli, helping to ensure that the cpOMPW will not interfere with the movement or function of the model TJ. To the C-terminus of the TEV digestible region to which the desired JAM protein is added. This can be any of the four verities of the JAM protein. After the JAM, one or more CLDN proteins is added in the desired combination. Then the OCLDN is added. An antibiotic resistance sequence is added into the plasmid.

[0046] A key to the function of this technology is that a linker region is added in-between each TJ protein. This is very important because it allows for the TJ proteins to organize spatially in the membrane and adopt conformations that are currently unknown to science but are required for the proper function of these tight junctions. For example, the sequence may have CLDN-5, CLDN- 2, and then CLDN-17 in that order, but in nature the cells in question have the proteins arranged in the order CLDN-17, CLDN-5, CLDN-2. Without the linker regions, there may be inappropriate interactions between proteins that don't happen in nature, or perhaps interactions that would have happened in nature don't happen because of lack of proximity between two proteins. These linker regions are one of many key parts of what makes the technology successful, novel, and unique.

[0047] This technology allows the creation of a replica of any tight junction that exists in nature, or any that does not exist in nature. All that is needed is to rearrange the plasmid so that the correct combination of membrane proteins is modeled. One plasmid may include the genes PET28a cpompW-TEV-JAMA-CLDN5-OCLN.

[0048] The above-mentioned process allows this technology to be used in E. coli bacteria. However, let the protection of this technology not be limited to just E. coli. Other organisms couldbe induced to express a similar protein chain in their outer membrane, likely using a very similar plasmid (with some changes to make it suited to expression in the organism of choice).

[0049] Multicellular organisms, like humans, require cellular interactions for function.Cell-cell interactions are mostly achieved by adhesion or interaction of proteins between cells. There is a family of cell-adhesion proteins that establish contact points rather than a comprehensive cell adhesion with its neighbors. A membrane protein structure, named Tight Junction (TJ), provides a cell-cell interaction that spreads throughout the circumference of the cell. Cell-adhesion molecules (CAM) are membrane proteins responsible for cell-cell interactions or cell-extracellular matrix interactions. In the membrane, the TJ is composed of claudins (CLDNs), occludin (OCLN), and junctional adhesion molecules (JAMs). The CLDN family is composed of 25 members, OCLN is a single protein, and JAMs are four members. TJ protein expression is tissue specific and can be very complex, from a single CLDN, OCLN and JAM to include several CLDNs and more than one JAM. At the Tight Junction, CLDNs are responsible for the tightness between cells to establish a barrier. Thus, CLDNs are responsible for the isolation of the brain from the blood components (blood-brain barrier), lung-air barrier, and others. The native environment of the TJ provides many limitations to isolate the contribution of each membrane protein associated with its function.

[0050] The tight junction (TJ) is a single proteic structure which safeguards the paracellular space. The TJ is responsible for controlled permeability of blood-tissue barriers, regulating the passage of molecule passage by size and charge. Currently there is no translational solution to manipulate the TJ with the exception of Focused Ultra-sound (FUS) and Micro bubbling (MB) techniques, still in clinical trials.

[0051] The expression of TJ proteins in the outer membrane of E. coli is described. When expression is induced, the unicellular behavior of E. coli is replaced with multicellular aggregations that can be quantified using Flow Cytometry (FC). The adhesion properties of the aggregates are representative of the individual membrane proteins expressed.

[0052] This method, called iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols), allows the high-throughput interrogation of small-molecules influence on paracellular permeability, enabling for the first time the discovery of its modulators for therapeutic strategies.

[0053] Currently, there are no therapeutic solution to control the permeability of the paracellular space. CAMs like the TJ’s integral membrane proteins are responsible for paracellular permeability. A high throughput E. coli- a cd method that recombinantly expresses CAMs has been created. This expression results in bacterial cell aggregates that can be inspected using Flow Cytometry. The method, named iCLASP, can examine small molecule libraries and identify candidates that increase or decrease permeability. Compared to classical methods, iCLASP is faster,only 4 days to set up (from transformation to experiment), and Flow Cytometry is performed at speeds of one 96-well plate every 45 minutes. CAMs are responsible for a number of other cell-cell, cell-extracellular matrix, cell-pathogen interactions. The iCLASP method offers a model to examine CAMs behavior in a cellular environment that is often challenging in mammalian cells.

[0054] Cell adhesion molecules (CAMs) are proteins located on the cell surface involved in binding with other cells or with the extracellular matrix (ECM) in a process called cell adhesion. CAMs are complex membrane proteins. In the classical sense, CAMs are grouped into four major families: selectins, immunoglobulin superfamily (IgSF), integrins, and cadherins.

[0055] Novel integral membrane proteins, claudins (CLDN) and occludin (OCLN), have been identified as major cell adhesion molecules working at the tight junction (TJ). Claudins comprise a multigene family, and each member of approximately 23 kDa bears 4-a-helix transmembrane domains. OCLN is a unique protein of the TJ with structural homology to CLDN’s 4-a-helix transmembrane domains. Junctional adhesion molecules (JAM), another membrane component of the TJ, is a member of the IgSF. CAMs involvement in developmental and physiological processes, or pathophysiological events such as tumorigenesis and metastasis, enhances their relevance in translational solutions.

[0056] Epithelial and endothelial cell-cell contacts are needed for homeostasis as intercellular junctional complexes are key for their maintenance. Among them, the adherens junctions (AJs) provide essential adhesive and mechanical properties. The TJs occupy the most apical region of the cell and create an almost impenetrable barrier that forms without interruption in the entire perimeter of the cell. AJs and TJs play essential roles in vascular permeability, but only the TJ controls the paracellular permeability.

[0057] As an example of the relevance of TJs the blood-brain barrier (BBB) will be discussed. The BBB is a cellular barrier that maintains the homeostasis of the neural microenvironment. The TJs between brain capillary endothelial cells greatly limit molecules to traffic across the paracellular route, with the exception of small molecules (<500 Da) and gaseous molecules. Additionally, brain transcytosis occurs at low rates as compared with peripheral tissues, restraining the vesicle-mediated transcellular transport of macromolecules. The paracellular and transcellular barrier properties of BBB also set up challenges for drug delivery to the central nervous system (CNS). Current solutions to overcome the BBB in the clinical setting are extremely limited. The BBB prevents approximately 98% of small molecule drugs from entering the brain. Focused ultrasound (FUS) combined with intravenous microbubbles (MBs) is a promising technique, being both temporary and reversible, that increases BBB permeability. FUS induced BBB permeability is reestablished within 24 h. Side effects caused by FUS and MBs can be detected by MRI. FUS- induced edema has been reported. MRI images have detected extravasation of red blood cells andare used to evaluate vascular damages following FUS treatment. A review of FUS and MBs was published recently. Data demonstrates that with these treatments the degree of BBB permeability is increased while the risk of tissue damage also rises, in some cases with months-long side effects.

[0058] TJ’s dysfunction in the BBB is responsible for increased permeability. Thus, controlling the TJ in the BBB can result in advantageous manipulation of the paracellular permeability. The development and delivery of small molecule drugs is relatively straightforward. Drug discovery of small molecules from target selection through to clinical evaluation is a very complex are challenging areas of drug discovery. The main obstacle is the initial hit-finding. For example, the combination of so little effort in developing solutions to the BBB permeability leads directly to the present situation in neurotherapeutics with few effective treatments for the majority of brain- related disorders. This situation can be reversed by an accelerated effort, knowledge base in the fundamental transport properties of the BBB, and the molecular and cellular biology of the brain capillary endothelium. Currently, dynamics of the BBB and cytotoxicity testing and drug permeation are carried out in vitro using Trans epithelial electrical resistance (TEER). Some challenges encountered in TEER are lengthy set-up, up to three weeks prior to obtaining measurements, and the availability of suitable cell lines that represent the desired system of study. Finally, identified cellular systems may fail to produce adequate and measurable TEER values.

[0059] Prodrug methods used to improve drug penetration via the transcellular pathway have been successfully developed, and some prodrugs have been used to treat patients. The use of transporters to improve absorption of some drugs (e.g., antiviral agents) has also been successful in treating patients. Other methods, including (a) blocking the efflux pumps to improve transcellular delivery and (b) modulation of cell-cell adhesion in the intercellular junctions to improve paracellular delivery across biological barriers are still in the investigational stage.

[0060] Using a synthetic biology approach, E. coli was designed to recombinantly express TJ membrane proteins. Our method, named iCLASP (inspection of cell-adhesion molecules aggregation through FC protocols) is a high throughput solution to identify paracellular modifiers to potentiate drug delivery of hydrophilic molecules.

[0061] Bacterial cells are transformed with plasmids hosting CAMs, regardless of their secondary structure or orientation in the mammalian plasma membrane. CAMs induce multicellular behavior in E. coli (aggregates or clumps of cells) that can be measured by Flow Cytometry protocols. A typical experiment will contain cells expressing CAMs untreated, as internal control accounting for growth and protein expression variability; and treated. The power displayed by iCLASP is two-fold, the screen of a library of small compounds may identify, in the same experiment, compounds that decrease or increase the size of aggregates. The first will represent cases in which the compound induces hyperpermeability of the paracellular space, while the secondrepresents a hypopermeability outcome. Hyperpermeability may be desirable when trying to overcome, as an example, the BBB for drug delivery. TJ dysfunction may contribute to epithelial permeation disorder and multiple intestinal diseases like inflammatory bowel diseases (IBD)42. In such cases, hypopermeability may be required to foster cell-cell interactions to prevent progression of the disease.

[0062] Expression system design.

[0063] The initial goal was to recombinantly express adhesion molecules in the outer membrane of E. coli. A fusion protein between OmpW (accession number P0A915) and a human CLDN was created. The N- and C-terminus of outer membrane proteins are located in the periplasm thus a fusion between OmpW and CLDN will result in an exposure of the adhesive domains (extracellular loops) to the exterior of the cell (Figure LA). A challenge to this design was the recombinant expression of JAM-A. JAM proteins have an N-terminus that contains the adhesive immunoglobulin domains, followed by the transmembrane domain. To overcome this bottleneck, OmpW was converted to a circularly permutated protein (cpOmpW) after the design offered for OmpX, another E. coli protein. The cpOmpX protein and our cpOmpW create new N- and C- terminus that are now located outside of the cell (Figure l.B). This strategy enabled the fusion of cpOmpW and JAM-A. The linker between the fused proteins contains a TEV protease cleavage site. Treatment of cells for 2-hours at room temperature releases cpOmpW from JAM-A and enables the interpretation of the adhesive properties of JAM-A.

[0064] Strategy for CAM expression in E. coli. A) OmpW is a native outer membrane protein of E. coli that populates the outer membrane, its N- and C-terminus are located in the extracellular space. These features make OmpW a suitable fusion partner for 4-a-helix CAMs like CLDN, OCLN, and others. Plasmids are synthesized with a C-terminal His-tag for Western blot detection with anti-His antibody (Abeam, product number abl l87, Cambridge, MA, USA): 1- OmpW (24 kDa),2-OmpW-CLDN (47 kDa). B) Circularly permutated OmpW (Supplementary file) is a suitable fusion partner for CAM proteins, which have an extracellular N-terminus. This strategy was employed to recombinantly express JAM-A. A linker region containing TEV protease sequence enables separation of cpOmpW and JAM-A post expression. The Plasmid contains a His-tag C- terminal to JAM-A. A Western blot with anti-His antibody is shown: 1-cpOmpW, 2-cpOmpW- JAM-A, 3- cpOmpW-JAM-A after TEV protease cleavage (only JAM-A is observed). C) Consequences of overexpressing CLDN in E. coli. BL21 DE3cells are captured in Electron Microscopy (http: / / www.ihcworld.com / _protocols / em / em_negative2.htm) displaying their natural behavior (unicellular) in a Negative Staining experiment (insert). In the background photo, BL21 DE3 after 16 hours of protein expression of OmpW -CLDN, in the images the large aggregates are evident. D) NG1655, a swimming variant of E. coli, growing on plates of LB+0.25% agar. Top image contains NG1655 cells after 24 hours of growth, from a single drop cell at OD600=1, placedat the center of the plate. In contrast, and under identical conditions, when protein expression (OmpW-CLDN) is induced (1 mM IPTG in the LB+ agar plate), cells growth is reduced, after 24 hours. The plate demonstrates that aggregation of the cells, induced by expression of OmpW- CLDN, prevents them from displaying their typical swimming behavior, and cannot advance beyond a small radius from the center.

[0065] Flow Cytometry of bacterial cells expressing TJ membrane proteins.

[0066] BL21 DE3 cells are transformed with the corresponding plasmids (CLDNs, OCLN, or JAM- A). Cells grow to an OD600 of 1, induced with 1 mM IPTG and allowed to continue to grow for 18 hours at Room Temperature. Cells are prepared for 96-well plates setup. Samples were prepared in suspension (50 pL cells per well and 150 pL of PBS) and run through a Beckman Coulter Cytoflex flow cytometer (Beckman Coulter, Indianapolis, IN, USA). Readings were collected using the side scatter (SSC) data from the 405 nm (violet) laser for excitation and a 405 / 10 bandpass filter for emission detection. The violet laser SSC has a greater sensitivity than the forward scatter (FSC) or SSC detection of the 488 nm (blue) laser for detecting alterations in cell shape as reported by othersand according to our assessment of flow data results in this study. SSC area versus height readings were plotted for data analysis. Cytoflex-generated FCS flow data files were analyzed using FlowJo 10 software (BD Biosciences, Ashland, OR, USA). The violet SSC-area by SSC-height data was gated for data analysis set to exclude upper and lower extremes that would interfere with the calculation of the slope of the line (Figure 2). Structural cell changes are detected as the area readings move away from the height readings where area increases at a lower rate than height and the slope of the line decreases (Figure 3). Further manipulation of the data using R Studio software, generously prepared by Stephen Picollo, Biology Department, Brigham Young University, resulted in Experimental Slopes calculated for each sample (Materials and Methods) and used to plot the results.

[0067] The adhesive properties of TJ proteins were studied with a newly developed tool. CLDNs are a family of 27 proteins in mammals. OmpW was used as a fusion partner to express CLDN1 through 10, OCLN, and tricellulin (TRCL). The latter is the first integral membrane protein found to concentrate at the vertically oriented TJs of tricellular contacts.. We included in the analysis stargazing (STRGZ), an AMPA receptor 30 believed to have some structural and functional homology to CLDN was included in the analysis. Figure 4 correlates the adhesive properties of the above- mentioned proteins by plotting the Experimental Slopes calculated as described. The control for these experiments is BL21 DE3 cells with plasmid pET28a, and BL21 DE3 cells recombinantly expressing OmpW alone in a pET28a backbone.

[0068] Experimental slopes of 4-a-helical CAMs. Protein expression of 4-a-helical CAMs is presented here. Flow Cytometry analysis, described in this article, enables the determination ofExperimental Slopes that represent adhesive properties of CAMs. Data presented here was statistically analyzed. All points are expressed as the average value of 12-replicates in 4 different experiments (n=4) + SE. All points are statistically significant and significantly different from each other. Asterisks are omitted for display purposes. Additionally, all values presented are the result of subtracting the value of BL21 DE3 cells with pET28a (0.92+0.039).

[0069] iCLASP can be used to determine changes in TJ- induced paracellular permeability which was verified through a study of a dose response of molecules known to non-specifically alter TEER. Sodium Caprate is a known detergent that disrupts CLDN-CLDN interactions resulting in increased permeability 31 . On the other hand, ethanol has been described as an agent that increases CLDN-CLDN interactions, resulting in a decreased permeability.

[0070] Experimental slopes of 4-a-helical CAMs in the presence of paracellular permeability influencers. Protein expression of 4-a-helical CAMs or OmpW alone, is presented here. Flow Cytometry analysis is plotted here to demonstrate the dose-dependent effect of Caprate (left panel), an agent knows to disrupt paracellular permeability. The right panel displays the dosedependent effect of ethanol, a known agent to increase tightness of the paracellular space. The effects of Caprate and ethanol are observed in CLDN 1 , CLDN5 and OCLN but not on OmpW alone. Data presented here was statistically analyzed. All points are expressed as the average value of 12-replicates in 4 different experiments (n=4) + SE. All points are statistically significant and significantly different from each other, at all concentrations of the influencer.Ion permeable CLDNs and the Hofmeister effect.

[0071] CLDNs are key regulators of barrier properties of the TJ. CLDNs are better recognized for their primary responsibility to tighten the paracellular pathway. A few CLDNs, and therefore the TJs they regulate, serve as paracellular channels for water and ions. Among CLDNs with these properties’ cation-selective human CLDN2 and anion-selective human CLDN 10 were selected.

[0072] The Hofmeister series characterizes the ions as to their ability to “salt-in” or “salt- out” proteins. Experimental results show that ion cooperativity may play an important role in affecting water properties. In Figure 6, both CLDN2 and CLDN10 are examined under the effects of slats that have been well characterized in the Hofmeister Series. The same salts for CLDN1, CLDN2 and CLDN10 were employed (see Materials and Methods). CLDN1 did not display any trends under conditions of any of the salts employed. Chloride salts (Al+^, Mg+^, Na+, and Rb+) only show a trend with CLDN2 (Figure 5). Sodium salts in the series representing different negative groups(HPO4", CT, NO3", and C1O4") only show a trend with CLDN10 and no other CLDNs tested (Figure 5).

[0073] The Hofmeister series and ion permeable CLDNs. Ion permeable human CLDN2 (cations) and human CLDN10 (anions) were exposed to the influence of different salts. For CLDN2 Chloride salts were used. For CLDN10 Sodium salts were used. The left panel indicates the Hofmeister disruptive effects of cations. Similarly, the right panel displays the Hofmeister ranking of disruptive effects of the negative ions. Flow Cytometry analysis enables the determination of Experimental Slopes that represent adhesive properties of CAMs in the presence of 100 mM salts. Data presented here was statistically analyzed. All points are expressed as the average value of 12- replicates in 4 different experiments (n=4) + SE. All points are statistically significant and significantly different from each other. Asterisks are omitted for display purposes.

[0074] For CLDN2, the Hofmeister series for cations has a trend of increasing cell-cell adhesion (Figure 5). Al+^ having a more relaxing effect on CLDN2-CLDN2 interactions, while Rb+fosters increased CLDN2-CLDN2 interaction. The Hofmeister series ranks the salts as stabilizing or the degree of destabilization. These rankings are denoted in Figure 5. In the case of CLDN2, losing stability drives further CLDN2-CLDN2 interactions. This effect could be detrimental to the cation permeability function, as it will tighten the TJ but reduce ability to sort cations. CLDN10, on the other hand, has a trend that seems to indicate that at a lower stability the permeability of the TJ will increase. Taken together, when salts decrease stability of the microenvironment, CLDN2 loses function but tightens the TJ (hypopermeability), while CLDN10 loses function and the TJ’s organization that can lead to hyperpermeability.

[0075] As described above (Figure l.B) the iCLASP method was slightly modified to accommodate JAM-A, a very important adhesion molecule where the N-terminus is located in the extracellular domain, opposite to CLDN, OCLN, and other 4-a-helical TJ membrane proteins where the N-terminus is intracellular. By circularly permutation of OmpW (cpOmpW) JAM-A was fused such that its adhesive domains are exposed to the extracellular space. A cleavable linker (TEV protease) was placed between cpOmpW and JAM-A to observe the adhesion properties of JAM-A as a fused protein or as a free molecule on the surface of E. coli. BL21 DE3 cells hosting the plasmids empty, cpOmpW or cpOmpW-JAM-A were grown over night after induction as described above. Each group is further divided in two: no treatment or TEV protease treatment (5 U / mL) for 2-hours at room temperature. Further, cells are diluted 1:4 with PBS and 200 pL are dispensed per well in a 96-well plate for Flow Cytometry. Four different experiments were carried out in 12 replicates each. The data displayed in Figure 7 describes the ability of iCLASP to successfully identify an increase of cell-cell interactions when BL21 DE3 recombinantly expressed cpOmpW- JAM-A. No significant difference was observed after TEV treatment. This could be a unique case of JAM-A and its quaternary structure to foster cell-cell adhesion. In Figure l.B a Western blot indicates that after 2 hours of TEV protease treatment the cleavage is successful. Other CAMs with a similar secondary structure (immunoglobulin domains) to that of JAM-A can also be studied usingiCLASP. It is expected that in other cases the treatment could be more significant and will need to be tailored for the corresponding CAM.

[0076] Flow Cytometry analysis of JAM-A fused to cpOmpW. Flow Cytometry analysis is resented here for the recombinantly expression of cpOmpW-JAM-A protein. Experimental slopes of empty plasmid (pET28a), cpOmpW protein alone and the fusion with JAM-A are plotted in the graph. The higher Experimental Slope indicates the presence of BL21 DE3 cells aggregates, which represent the adhesive properties of JAM-A. Data presented here was statistically analyzed. All points are expressed as the average value of 12-replicates in 4 different experiments (n=4) + SE. All points are statistically significant and significantly different from each other. Asterisks are omitted for display purposes.

[0077] Conceptual Model. The iCLASP experimental model is presented here

[0078] Bacterial cells are transformed with plasmids hosting CAMs, regardless of their secondary structure or orientation in the mammalian plasma membrane. CAMs induce multicellular behavior in E. coli (aggregates or clumps of cells) that can be measured by Flow Cytometry protocols. A typical experiment will contain cells expressing CAMs untreated, as internal control accounting for growth and protein expression variability; and treated. The power displayed by iCLASP is two-fold, the screen of a library of small compounds may identify, in the same experiment, compounds that decrease or increase the size of aggregates. The first will represent cases in which the compound induces hyperpermeability of the paracellular space, while the second represents a hypopermeability outcome. Hyperpermeability may be desirable when trying to overcome, as an example, the BBB for drug delivery. TJ dysfunction may contribute to epithelial permeation disorder and multiple intestinal diseases like inflammatory bowel diseases (IBD) 42. In such cases, hypopermeability may be required to foster cell-cell interactions to prevent progression of the disease.Example of Drug Discovery Workflow using iCLASP.Round 1

[0079] Fresh transformation of BL21 DE3 cells with the plasmid for 0mpW-CLDN2 was performed two days before. Colonies are selected from the plate and grown to OD600-1, IPTG is then added to a final concentration of 1 mM. Growth continues overnight (18 h) at room temperature with shaking. Colonies were selected from plates up to 3 weeks after transformation.

[0080] Cells are then transferred to the 96-well plates. The composition of the plates is 180 pL of PBS, 20 pL of compounds, and 50 pL of cells. Plates are prepared in quadruplicates. Each 96- well plate corresponds to 1 h of Flow Cytometry under our conditions and with our equipment and settings.

[0081] The 50,000-compound library was combined in 10-coumpounds per well, without particular order (to a final concentration oflO pM). Thus, each well contained 10 unique compounds, representing the challenge to cells overexpressing 0mpW-CLDN2. The Experimental Slope is determined for each challenge after combining the quadruplicate results from that day. Each plate preserves the first and last column for untreated cells overexpressing 0mpW-CLDN2 (a total of 16 wells). Experimental controls are compared to the unchallenged control. Decreased slopes represent HYPER-permeability. Increased slopes represent HYPO-permeability.

[0082] Each well contained 10 compounds in the first Round, 10 pM each. This approach reduced the 50,000 challenges in quadruplicates to only 55 days. Based on a twice-a-week schedule availability of our Core Facility. The time to complete Round 1 was 6 months.Round 2

[0083] Wells representing 10 unique compounds were identified as causing Hyper or hypopermeability (Round 1). The best challenges, altering permeability higher or lower are selected. Each challenge consisted of 10 compounds. These compounds are now used as challengers in a single-compound fashion. The best 4 challenges that lead to increased slope were selected and the best 4 leading to decrease in slope were selected. Corresponding to 40 compounds in each category.

[0084] From this second round the compounds that correspond to the changes in the slope, were identified and isolated from the others.

[0085] Each well contained a single compound, 10 pM. This approach identified which compound among the 10 in the Round 1 screen had the most activity. Based on a twice a week schedule availability of our Core Facility, the time to complete Round 2 was 2 weeks.Round 3

[0086] From Round 2 it was determined that a series of 10 compounds in each category correspond to the best single-compound challengers to cell-cell interactions leading to the greatest changes compared to the unchallenged cells.

[0087] In Round 3 we prepared dilutions of each compound to challenge cell- cell interactions: 10, 5, 2, 1, 0.8, 0.4, 0.2, 0.1 pM.

[0088] A graph of one compound (G04:6502939) that demonstrated a dose-dependence is presented in Figure. Each well contained a single compound. Dilutions were 10, 5, 2, 1, 0.8, 0.4, 0.2, 0.1 pM. This approach identified which compound among the ones selected in Round 2 had a dose response. Based on the twice-a-week schedule availability of our Core Facility, the time to complete Round 3 was 2 weeks.

[0089] ROUND 3. Each well contained a single compound. Dilutions were 10, 5, 2, 1 08.04.0.2, O.luM. This approach identified which compound among the ones selected in Round 2 had a response. Based on the twice-a-week schedule availability of our Core Facility, the time to complete Round 3 was 2 weeks.Materials and Methods.Reagents and Genes.

[0090] For Flow Cytometry, flat bottom 96-well cell culture plates were employed (GeneClone, https: / / geneseesci.com / ). All genes employed in this study were synthesized by TWIST biosciences (https: / / twistdna.com, San Francisco, CA, USA) and cloned in pET28a. All salts used for experimental performance of Figure 6 were obtained from Sigma Aldrich (https: / / www.sigmaaldrich.com / , St. Eouis, MO, USA). TEV protease was obtained from New England Biolabs (Ipswich, MA, USA).

[0091] Transformation, Cell growth, and Protein Expression (LB, fresh transformations, controls). Plasmids (pET28a, pET28a-OmpW-CEDN, pET28a-cpOmpW-JAM-A) are transformed in BE21 DE3 cells, plates are prepared EB, 2% agar, and 100 ug / mL of kanamycin. A single colony is grown over night in 5 mF of EB and kanamycin. A 1 : 1000 dilution of the overnight culture is started in the morning. Cells are grown at 30°C until OD600 is between 0.9- 1.0. IPTG (1 mM) is used to induce protein expression, cells are placed in shaker at 21 °C (room temperature) and allowed to continue growth for 18 hours. Samples can be analyzed by Western blot and anti-HIS antibody.Flow Cytometry data collection and analysis.

[0092] Samples were prepared in suspension for analysis and run through a Beckman Coulter Cytoflex flow cytometer (Beckman Coulter, Indianapolis, IN, USA). Readings were collected using the side scatter (SSC) data from the 405 nm (violet) laser for excitation and a 405 / 10 bandpass filter for emission detection. The violet laser SSC has a greater sensitivity than the forward scatter (FSC) or SSC detection of the 488 nm (blue) laser for detecting alterations in cell shape as reported by othersand according to our assessment of flow data results in this study. SSC area versus height readings were plotted for data analysis. Cytoflex-generated FCS flow data files were analyzed using FlowJo 10 software (BD Biosciences, Ashland, OR, USA). The violet SSC-area by SSC-height data was gated for data analysis sets to exclude upper and lower extremes that would interfere with the calculation of the slope of the line. Structural cell changes are detected as the area readings move away from the height readings where area increases at a lower rate than height and the slope of the line decreases. Below is the code used for R Studio software analysis, generously prepared by Stephen Picollo, Biology Department, Brigham Young University. Code lines with #indicate the functions of the code. The code uses a working file from FlowJo, generates hexbin plots to visualize analyzed data, and generates a spreadsheet of the calculated slopes as a .csv file.Statistical Analysis

[0093] Flow Cytometry data (Experimental Slope) were analyzed using SAS software (SAS Institute Inc., Cary, NC, United States) and the Mixed Procedure method to generate p-values, standard deviation, and standard error and to determine statistical significance (for Figure 5). For all experiments a = 0.05. Data was collected for each sample in four different experiments (n=4). Each condition was measured in 12-replicates. Thus, each data point corresponds to the average of 12- replicates and n=4. Statistical differences were identified for all samples in each graph. The final analysis concluded that all treatments are statistically significant (p<0.0001) and significantly different from each other and the control (asterisks omitted for display purposes).

[0094] The invention has been described with reference to various specific and preferred embodiments and techniques. Nevertheless, it is understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A synthetic tissue comprising: a colony of modified single celled organisms; and a protein produced by the modified single cell organism.

2. The synthetic tissue of claim 1 , wherein the modified single cell organism is modified by a plasmid.

3. The synthetic tissue of claim 1, wherein the protein resembles at least one of proteins of the collection of proteins known as tight junction or another cell-to-cell adhesion protein.

4. The synthetic tissue of claim 1 , wherein a region of the protein that resembles a specific region of a cell-to-cell adhesion protein links to the same region of other proteins using linker regions.

5. The synthetic tissue of claim 1, wherein the region of the protein that resembles the specific region of a cell-to-cell adhesion protein resembles a protein of the outer cell membrane of the single cell organisms in the colony of single cell organisms.

6. The synthetic tissue of claim 4, wherein the single cell organisms are E. coli.

7. The synthetic tissue of claim 4, wherein the single cell organisms are yeast.

8. The synthetic tissue of claim 1, wherein the cell-to-cell adhesion protein establishes the tight junction.

9. The synthetic tissue of claim 2, wherein the plasmid incorporates one or more of a JAM protein gene, a claudin protein gene, or an occludin protein gene into the single cell organism.

10. The synthetic tissue of claim 9, wherein the plasmid incorporates two or more of a JAM protein gene, a claudin protein gene, or an occludin protein gene into the single cell organism.

11. The synthetic tissue of claim 9, wherein the JAM protein is a JAM-A protein.

12. The synthetic tissue of claim 9, wherein the claudin protein is one of CLDN 1, CLDN 2, or CLDN 10.

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