A novel phagocytosis assay combining a synthetic cell death switch and a phagocytosis reporter system

Abstract

The present invention relates to recombinant expression vectors encoding an inducible cell death switch, a pH-stable fluorophore, and a pH-sensitive fluorophore. Furthermore, the present invention relates to cells containing said recombinant expression vectors and their use in in vitro phagocytosis assays.

Description

[Technical Field]

[0001] The present invention relates to recombinant expression vectors encoding an inducible cell death switch, a pH-stable fluorophore, and a pH-sensitive fluorophore. Furthermore, the present invention relates to cells containing said recombinant expression vectors and their use in in vitro phagocytosis assays. [Background technology]

[0002] Maintaining any organ's functional state requires rapid and efficient clearance of pathogens and cellular debris. Macrophages, as part of the immune system, maintain tissue homeostasis by sequestering, engulfing, and digesting such particles through a process called phagocytosis. In the brain, microglia perform the majority of apoptotic cell clearance. Dysfunction of macrophages and microglia, resulting in inappropriate clearance, has been linked to multiple autoimmune and neurodegenerative diseases, including systemic lupus erythematosus, Alzheimer's disease, and Parkinson's disease.

[0003] Given this important role of macrophages and microglia, various phagocytosis assays are currently in use. Commonly used substrates are opsonized red blood cells, yeast particles, E. coli bioparticles, amyloid beta plaques, myelin, or cellular debris. Substrates are often labeled with the pH-sensitive dye pHrodo, which increases its fluorescence intensity upon the decrease in pH that occurs in phagolysosomes, thus indicating phagocytosis. The substrate is typically co-incubated with phagocytes for a set period of time. Phagocytic cells are then isolated, and the internalized substrate is measured, for example, by ELISA (if the substrate has a specific epitope), in a plate reader (e.g., for RBCs), or by flow cytometry (e.g., for fluorescently labeled debris). Time-lapse imaging during co-incubation with phagocytes is used to observe and quantitate phagocytosis.

[0004] Current phagocytosis assays have two major limitations. First, there is high experimental variability due to variations in efficiency in generating apoptotic cells and alteration of apoptotic cells by labeling, making quantification and comparison of results difficult. Second, there is a lack of timing flexibility. The assay begins when all components are mixed, which is undesirable in more complex multicellular in vitro models. Therefore, we identified the following needs for an improved phagocytosis assay:

[0005] The need for precise and specific generation of apoptotic cells Because cell clearance constitutes a major part of macrophage and microglial activity in both physiological and pathological conditions, cellular debris from apoptotic cells is one of the most important substrates for phagocytosis. However, protocols for obtaining apoptotic cells (e.g., by incubating with the bacterial product staurosporine) can result in varying proportions of dead cells. Another concern is that this product may contain other forms of cellular debris (e.g., necrotic cells, leaked DNA, and other components).

[0006] Furthermore, if the substrate is labeled, its surface may be modified (eg, by adding a fluorescent dye) to affect substrate recognition by phagocytes.

[0007] To obtain consistent and physiologically relevant apoptotic cell substrates, cellular apoptosis should ideally be induced with high efficiency, specificity, reproducibility, and without subsequent modification / external labeling.

[0008] The need for intrinsically labeled apoptotic cells Importantly, to clearly identify phagocytic events, it is necessary to differentially label phagocytosed and non-phagocytosed apoptotic cells. The commonly used pHrodo dye fluoresces weakly in non-phagocytosed cells and produces a strong signal in labeled cells that are internalized during phagocytosis and reside within the low-pH lysosomes. Due to variable labeling efficiency and occasional desorption of the dye from the surface, the pHrodo signal varies between experiments, and the threshold between high and low signals must be adjusted for each experiment.

[0009] The need for an on-demand apoptosis gene switch for assay flexibility Finally, current assays require the generation and labeling of apoptotic cells (or other substrates) prior to contact with phagocytes. Therefore, phagocytosis begins immediately upon mixing of the two components. However, some complex assays (spheroids, organoids, organ-like structural models) often require seeding multiple cell types together within a 3D matrix, requiring multiple days for the cells to align and self-organize. Therefore, if the phagocytosis assay needs to be performed several days after seeding all components, conventional assays cannot be used. In this case, it is necessary to have an apoptosis switch (on system) that can generate phagocytic substrates at the desired time point.

[0010] Novelty and Advantages of the Present Invention Over Established Methods We have developed a novel phagocytosis assay that combines two features: 1) on-demand cell-specific apoptosis and 2) a phagocytosis-specific fluorescent reporter. First, we use a method that can generate apoptotic cells of any cell type on demand at any desired time point with high efficiency and reproducibility. We utilize an inducible caspase-9 construct, first described in Straathof, KC et al. An inducible caspase-9 safety switch for T-cell therapy. Blood. 2005;105(11):4247-54 and disclosed in WO 2011 / 146862, in which the caspase-9 dimerization domain is replaced with the FKBP12-F36V dimerization domain. This substitution results in caspase-9 dimerization only upon addition of the small molecule AP20187, which subsequently triggers the apoptotic cascade.

[0011] Second, we constitutively coexpressed a fusion construct consisting of mCherry and the GFP-based pH-sensitive Superecliptic pHluorin (SEP; Sankaranarayanan, S et al. The Use of pHluorins for Optical Measurements of Presynaptic Activity. Biophys. J. 2000;79(4):2199-208) in the same cells. At pH 7.4, this reporter emits both green (SEP) and red (mCherry) fluorescence upon excitation at 488 nm and 580 nm, respectively. At acidic pH, SEP fluorescence is quenched, and the reporter emits only red fluorescence. This genetically encoded reporter allows us to distinguish phagocytosed from non-phagocytosed cells with high precision without externally modifying the cells. This novel construct, iCaspase9-SEP-mCherry (Figure 1), can be transfected or transduced into mammalian cells (cell lines, primary cells, iPSCs) and expressed transiently or stably.

[0012] The present phagocytosis assay can be performed in 2D or 3D several days after seeding multiple cell types, including inducible stromal cells (e.g., H4-iCaspase9-SEP-mCherry or Jurkat-iCaspase9-SEP-mCherry cells) and phagocytic cells (e.g., iPSC-derived microglia or macrophages). Summary of the Invention

[0013] In one aspect, the present invention provides recombinant expression vectors encoding an inducible cell death switch, a pH-stable fluorophore and a pH-sensitive fluorophore.

[0014] In one embodiment, the recombinant expression vector is a viral vector. In one embodiment, the viral vector is a lentiviral vector.

[0015] In one embodiment, the inducible cell death switch induces apoptosis, necroptosis, pyroptosis, or ferroptosis, preferably apoptosis. In another embodiment, the inducible cell death switch comprises an inducer binding domain and a signaling protein of a cell death pathway. In one embodiment, the signaling protein of the cell death pathway is a pro-apoptotic protein, preferably caspase 9 or a functional fragment thereof. In one embodiment, the inducer binding domain comprises a dimerization domain, preferably FKBP12-F36V. In one embodiment, the inducer binding domain can bind an inducer, preferably a chemical inducer of dimerization, most preferably AP20187. In one embodiment, the inducible cell death switch is inducible caspase 9.

[0016] In one embodiment, the pH-stable fluorophore and the pH-sensitive fluorophore have different excitation and emission spectra. In a further embodiment, the pH-stable fluorophore is from the RFP family, Alexa Fluor dye, protein-based fluorophore, simple organic fluorophore or organic polymer, preferably mCherry. In one embodiment, the fluorescence of the pH-sensitive fluorophore changes in response to a decrease in pH, preferably the fluorescence is quenched or the excitation / emission spectrum is shifted. In another embodiment, the pH-sensitive fluorophore is Superecliptic pHluorin (SEP), pHLemon, pHmScarlet, pHTomato, pHuji, LysoSensor or pH nanosensor, preferably Superecliptic pHluorin (SEP).

[0017] In one embodiment, the recombinant expression vector comprises a promoter, particularly a ubiquitous promoter, a cell-specific promoter, a constitutive promoter, or an inducible promoter. In one embodiment, the recombinant expression vector comprises a CMV promoter. In one embodiment, the recombinant expression vector comprises the polynucleotide sequence set forth in SEQ ID NO: 1.

[0018] According to a further aspect of the present invention, there is provided a cell comprising the recombinant expression vector of the present invention. In one aspect, the cell is a mammalian cell, particularly a human cell. In one aspect, the cell is a stem cell. In a further aspect, the cell is a nerve cell, a neuron cell, a glial cell, a mesenchymal cell or a hematopoietic cell, preferably a glioma cell or a T cell. In one aspect, the cell is an H4 cell or a Jurkat cell. In one aspect, the expression of the recombinant expression vector in the cell is transient or stable.

[0019] Also encompassed by the present invention is an in vitro method for assessing phagocytosis, comprising the steps of: a) providing inducible stromal cells according to the present invention; b) co-culturing the inducible stromal cells in combination with phagocytes; c) inducing cell death in the inducible stromal cells; and d) detecting a fluorescent signal from the inducible stromal cells, wherein a change in the fluorescent signal of a pH-sensitive fluorophore indicates phagocytosis of the inducible stromal cells.

[0020] In one embodiment, the phagocytes in step b) are macrophages or tissue-resident macrophages. In one embodiment, the tissue-resident macrophages are microglia.

[0021] In one embodiment, inductive substrate cells and phagocytes are co-cultured in an in vitro model that includes additional cell types.In one embodiment, the in vitro model is a 2D or 3D culture system.In another embodiment, the in vitro model is an organ chip, spheroid or organoid.In one embodiment, the in vitro model is a neurovascular unit or blood-brain barrier spheroid.

[0022] In one embodiment, cell death of the inducible stromal cells in step c) is induced after the inducible stromal cells are combined with the phagocytes in step b), hi a further embodiment, cell death of the inducible stromal cells in step c) is induced by an inducer, preferably a chemical inducer of dimerization, most preferably AP20187.

[0023] In another embodiment, the fluorescent signal in step d) is detected by fluorescent imaging, flow cytometry or by a fluorescent plate reader. In one embodiment, the fluorescent signal in step d) is detected at several time points after induction of cell death. [Brief explanation of the drawings]

[0024] [Figure 1]Lentiviral vector map showing iCaspase 9 linked to the SEP-mCherry fluorophore with a cleavable P2A linker, placed under the control of the CMV promoter. The construct also contains a puromycin selection cassette under the control of the mPGK promoter. [Figure 2] iCaspase-9 transfected cells rapidly underwent apoptosis upon exposure to AP20187, whereas staurosporine only caused less apoptosis after 3 hours of incubation. [Figure 3] H4-i caspase-SEP-mCherry cells imaged in the green channel (top) and red channel (bottom). SEP is quenched at lower pH, whereas mCherry is present in all environments tested. [Figure 4] H4-iCaspase-SEP-mCherry cells co-cultured with labeled HMC3 microglia in a 2D phagocytosis assay. Apoptosis of transduced H4 cells was triggered at time t0 by adding 10 nM AP20187, and the cells were imaged for 8 hours. [Figure 5] Phagocytosis of H4-iCaspase-SEP-mCherry cells co-cultured with iPSC-derived microglia in 2D, imaged with an Incucyte® instrument. [Figure 6] Efferocytosis of Jurkat-i caspase-SEP-mCherry cells by THP-1 macrophages, quantified by flow cytometry. [Figure 7] Phagocytosis of H4-iCaspase-SEP-mCherry cells by HMC3 microglia in 3D blood-brain barrier (BBB) ​​spheroids at day 2 after apoptosis was specifically induced in transduced H4 cells. [Figure 8] Phagocytosis of H4-iCaspase-SEP-mCherry cells by HMC3 microglia in a 3D neurovascular unit model after 7 days of vascular self-assembly. Apoptosis was specifically induced in transduced H4 cells. DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless otherwise defined below, terms are used herein as commonly used in the art.

[0026] As used herein, the term "recombinant expression vector" refers to a polynucleotide molecule capable of directing the expression of a polypeptide encoded therein by a polynucleotide sequence. A recombinant expression vector comprises regulatory sequences that result in efficient transcription of the encoding polynucleotide sequence. In the context of the present invention, the term "recombinant expression vector encoding an inducible cell death switch, a pH-stable fluorophore, and a pH-sensitive fluorophore" includes (i) a single vector encoding all of the elements (i.e., the inducible cell death switch, the pH-stable fluorophore, and the pH-sensitive fluorophore), or (ii) multiple vectors, each encoding one or more of the elements and collectively encoding all of the elements. Thus, in some embodiments according to the present invention, the inducible cell death switch, the pH-stable fluorophore, and the pH-sensitive fluorophore are encoded by a single vector, while in other embodiments, the inducible cell death switch, the pH-stable fluorophore, and the pH-sensitive fluorophore are encoded by multiple vectors.

[0027] As used herein, the term "inducible cell death switch" refers to a molecule that, when activated, can induce the death of a cell expressing the molecule. The cell death switch comprises an inducer-binding domain and a signaling protein of a cell death pathway. The signaling protein is added to the cellular environment and activated via an inducer that binds to the inducer-binding domain of the inducible cell death switch. This specifically induces cell death in cells expressing the inducible cell death switch. In the absence of an inducer, cells expressing the inducible cell death switch exhibit a physiological rate of cell death, i.e., a rate equivalent to that of cells that do not express the inducible cell death switch. Thus, cell death can be induced at a specific time point.

[0028] As used herein, the term "pH-stable fluorophore" refers to a fluorescent protein that emits fluorescence independently of the pH of the environment, meaning that the fluorophore emits fluorescence with the same fluorescence spectrum at various pH values.For example, even if the pH of the environment decreases, the fluorescence of the fluorophore is maintained.Therefore, the pH-stable fluorophore expressed by phagocytosed cells maintains its fluorescence during the maturation of phagolysosomes, which is accompanied by a decrease in pH.

[0029] As used herein, the term "pH-sensitive fluorophore" refers to a fluorescent protein that emits a fluorescent signal that depends on the pH of the environment, in the sense that its fluorescence emission changes under varying pH values. Under varying pH values, the fluorophore may emit a different fluorescence spectrum, or the fluorophore may exhibit increased or decreased fluorescence. A pH-sensitive fluorophore may emit a strong fluorescent signal at neutral pH, and when the environment is acidified, the fluorescent signal may be quenched. For example, a pH-sensitive fluorophore expressed by a cell undergoing phagocytosis may exhibit a decrease in its fluorescence during phagolysosome maturation accompanied by a decrease in pH.

[0030] As used herein, the term "promoter," as commonly understood by those skilled in the art, is defined as a polynucleotide sequence in a recombinant expression vector that regulates the expression of an encoded polypeptide. A promoter recruits the cell's transcriptional machinery to the expression vector and controls when and / or where the encoded polypeptide is expressed. An expression vector may contain several independent promoters that control the expression of different polypeptides. Thus, different polypeptides may be under the control of different promoters, i.e., the expression of a polypeptide is controlled by a separate promoter. A promoter may be a "constitutive promoter," which is thought to confer stable expression levels over varying conditions, or an "inducible promoter," which drives expression in response to a specific stimulus. Furthermore, a promoter may be a "ubiquitous promoter," which is active in a wide range of cell types and / or developmental stages, or a "cell-type-specific promoter," which is active only in one or more specific cell types.

[0031] The terms "transfection" or "transfecting," as used herein, as commonly understood by those skilled in the art, are defined as the process of introducing an exogenous polynucleotide sequence into a cell by non-viral methods. Common transfection methods include calcium phosphate, cationic polymers (such as polyethyleneimine (PEI)), magnetic beads, electroporation, and commercially available lipid-based reagents such as Lipofectamine® and FuGENE®.

[0032] The term "transduction" or "transducing," as used herein, is defined as the process of introducing an exogenous polynucleotide sequence into a cell via a viral vector, as commonly understood by those skilled in the art. Transduction generally results in stable expression of the encoded polypeptide.

[0033] As used herein, the term "in vitro model" refers to a cell culture system designed to reproduce certain aspects of cellular behavior seen in vivo, thereby facilitating the study of cellular processes. In vitro models may contain a single cell type or may include two or more cell types and extracellular matrix and / or form-giving elements (e.g., microfabricated devices). Thus, in vitro models may be simplified representations of various tissues or organs, or may mimic the in vivo structure and organization of tissues or organs. Thus, cells in an in vitro model may be cultured in 2D or 3D culture systems. A "2D culture system" refers to an in vitro model in which cells are cultured essentially as a monolayer (i.e., in a two-dimensional structure). In contrast, a "3D culture system" refers to an in vitro model in which cells are organized into a three-dimensional structure (e.g., an organ chip, spheroid, or organoid, as described herein below).

[0034] As used herein, the term "organ-on-a-chip" refers to a culture system on a microfluidic chip that simulates the activity, mechanics, and physiological responses of an organ or organ system.

[0035] As used herein, the term "spheroid" or "spheroid culture system" refers to a 3D in vitro model of cells grown in suspension, in which cells aggregate to form a spheroid shape. Spheroids can provide cells with a physicochemical environment similar to that in vivo by promoting cell-cell and cell-matrix interactions to overcome the limitations of traditional monolayer cell culture.

[0036] As used herein, the term "organoid" refers to a 3D in vitro model that mimics its corresponding in vivo tissue or organ so that it can be used to study aspects of that organ within a tissue culture dish.

[0037] As used herein, the term "phagocytosis" refers to the cellular process of ingesting and removing particles, such as microorganisms, foreign substances, cells, or cellular debris. The term "phagocytosis" encompasses the term "efferocytosis," which refers to a specialized phagocytic process. During the phagocytic process, particles to be eliminated are engulfed by the plasma membrane, forming specialized intracellular vacuoles called phagosomes. The phagosomes mature into phagolysosomes, within which the engulfed particles are degraded and eliminated. The maturation of the phagosome into a phagolysosome is characterized by the acidification of the vacuole, i.e., a decrease in pH.

[0038] As used herein, the term "phagocytosis assay" refers to a cellular assay in which phagocytic activity is assessed. Phagocytosis assays generally include at least one cell type that acts as a phagocyte and a particle or substrate to be phagocytosed.

[0039] As used herein, the term "phagocyte" or "phagocyte" refers to a cell type that exhibits phagocytic activity, i.e., is capable of phagocytosis.

[0040] As used herein, the term "inducible substrate cells" refers to cells that contain an inducible cell death switch that can be phagocytosed, i.e., become a substrate for phagocytes, following induction of cell death.

[0041] The present invention provides a recombinant expression vector encoding an inducible cell death switch, a pH-stable fluorophore, and a pH-sensitive fluorophore.The recombinant expression vector of the present invention can be a viral vector.The recombinant expression vector can be a lentiviral vector, an adenoviral vector, or a retroviral vector.In one embodiment, the recombinant expression vector is a lentiviral vector.

[0042] The inducible cell death switch encoded by the recombinant expression vector of the present invention can include an inducer-binding domain and a signaling protein of a cell death pathway. When an inducer, particularly a chemical inducer, binds to the inducer-binding domain, the signaling protein is activated, which initiates the cell death pathway. Activation of the signaling protein can be achieved by dimerization. Thus, the inducer-binding domain can be a dimerization domain. Upon binding of the inducer, the dimerization domain dimerizes with another dimerization domain, resulting in activation of the cell death signaling protein. The inducer-binding domain can be FKBP12-F36V, as described in Straathof, KC et al. An inducible caspase 9 safety switch for T-cell therapy. Blood. 2005;105(11):4247-54. FKBP12-F36V is a human FK506-binding protein (FKBP12; GenBank AH002 818) containing the F36V mutation. The polynucleotide sequence of FKBP12-F36V is set forth in SEQ ID NO: 2. The amino acid sequence is set forth in SEQ ID NO: 11. In one embodiment, the inducer-binding domain is a dimerization domain. In another embodiment, the inducer-binding domain is FKBP12-F36V. In one embodiment, the inducer-binding domain is encoded by the polynucleotide sequence of SEQ ID NO: 2. In one embodiment, the inducer-binding domain comprises the amino acid sequence of SEQ ID NO: 11. In one embodiment, the inducer is a chemical inducer. In a specific embodiment, the inducer is AP20187. In one embodiment, the inducer-binding domain is FKBP12-F36V and the inducer is AP20187.

[0043] The signaling protein of a cell death pathway may be, among others, a signaling protein of the apoptosis pathway, necroptosis pathway, pyroptosis pathway, or ferroptosis pathway. Thus, the inducible cell death switch may induce, among others, apoptosis, necroptosis, pyroptosis, or ferroptosis. Preferably, the inducible cell death switch induces apoptosis. The signaling protein may be a pro-apoptotic protein, such as a protein of the caspase signaling cascade. Sequential activation of caspases plays a central role in the execution of cell apoptosis. The signaling protein may be caspase 9 (Casp9; UniProtKB:P55211) or a functional fragment thereof. As used herein, the term "functional fragment" refers to a portion of a protein that retains the biological function of the full-length protein, i.e., a functional fragment of a protein of a cell death pathway also induces cell death. A functional fragment of caspase 9 may have the amino acid sequence shown in SEQ ID NO: 12 or may be encoded by SEQ ID NO: 3. In their monomeric form, caspase 9 or a functional fragment of caspase 9 is inactive. Through dimerization, caspase 9 or a functional fragment of caspase 9 is activated and functions as an initiating caspase, activating downstream executioner caspases. In one embodiment, the cell death pathway signaling protein is caspase 9, particularly human caspase 9, or a functional fragment thereof. In one embodiment, the cell death pathway protein is encoded by the sequence of SEQ ID NO: 3. In one embodiment, the cell death pathway protein comprises the amino acid sequence of SEQ ID NO: 12.

[0044] As used herein, the term "inducible caspase 9" or "iCasp9" refers to iCasp9, as described by Straathof, KC et al., including functional fragments of FKBP12-F36V and caspase 9. MThis refers to the construct F-Casp9, also referred to as F-Casp9. The amino acid sequence of inducible caspase 9 is set forth in SEQ ID NO: 13. The polynucleotide sequence of inducible caspase 9 is set forth in SEQ ID NO: 4. In one embodiment, the inducible cell death switch comprises a dimerization domain and a pro-apoptotic protein. In one embodiment, the inducible cell death switch comprises FKBP12-F36V and caspase 9. In one embodiment, the inducible cell death switch is inducible caspase 9. In one embodiment, the inducible cell death switch is encoded by the sequence of SEQ ID NO: 4. In one embodiment, the inducible cell death switch comprises the sequence of SEQ ID NO: 13.

[0045] The recombinant expression vectors of the present invention encode pH-stable and pH-sensitive fluorophores. pH-stable and pH-sensitive fluorophores have different excitation and emission spectra; that is, their fluorescent signals can be distinguished from each other using a fluorescence detection system. This allows phagocytosed cells to be distinguished from non-phagocytosed cells in phagocytosis assays. The pH-stable fluorophore can be mCherry, other fluorophores from the RFP family, Alexa Fluor dyes, protein-based fluorophores (e.g., PE), simple organic fluorophores, or organic polymers. Generally, any pH-stable fluorophore with an excitation and emission spectrum different from the pH-sensitive fluorophore can be used. mCherry is a member of the mFruits family of monomeric red fluorescent proteins (mRFPs). mCherry absorbs light in the 540-590 nm range and emits light in the 550-650 nm range. The polynucleotide sequence of mCherry is shown in SEQ ID NO: 5. The amino acid sequence of mCherry is shown in SEQ ID NO: 14. The pH-sensitive fluorophore can be Superecliptic pHluorin (SEP) or pHLemon in the green spectrum, pHmScarlet, pHTomato, or pHuji in the red spectrum, LysoSensor, a pH nanosensor (e.g., a QD-protein FRET-based pH sensor), or a (molecular) fluorescent switch. SEP is a pH-sensitive green fluorescent protein that emits a strong green fluorescent signal at neutral pH. Upon acidification of the environment, the fluorescent signal gradually decreases, i.e., is quenched, with a pKa of 7.2 and an apparent Hill coefficient of 1.9. The polynucleotide sequence of SEP is shown in SEQ ID NO:6. The amino acid sequence of SEP is shown in SEQ ID NO:15. Alternatively, the excitation / emission spectrum of the pH-sensitive fluorophore can change depending on the pH of the environment. Thus, the excitation / emission spectrum of the pH-sensitive fluorophore can shift during the phagocytic process. In one embodiment, the pH-stable fluorophore is mCherry. In one embodiment, the pH-sensitive fluorophore is SEP.In one embodiment, the pH-stable fluorophore is mCherry and the pH-sensitive fluorophore is SEP. In one embodiment, the pH-stable fluorophore and the pH-sensitive fluorophore are linked via a linker, thereby generating a fluorophore fusion protein. The linker can be encoded by the polynucleotide sequence of SEQ ID NO:9. In one embodiment, mCherry and SEP are linked by the amino acid sequence of SEQ ID NO:16.

[0046] The cell death switch and fluorophore fusion protein can be linked by a cleavable P2A linker. In one embodiment, the cleavable P2A linker is encoded by the sequence of SEQ ID NO: 10. In one embodiment, the iCaspase 9 and SEP-mCherry fusion protein are linked by the amino acid sequence of SEQ ID NO: 17.

[0047] The inducible cell death switch, pH-stable fluorophore, and pH-sensitive fluorophore encoded by the recombinant expression vector of the present invention can be under the control of a promoter. Depending on the cell type to be transfected or transduced and / or the particular assay to be performed, the promoter can be a ubiquitous promoter, a cell-type-specific promoter, a constitutive promoter, or an inducible promoter. For example, the immediate-early gene of human cytomegalovirus (CMV), also known as the CMV promoter, can be used. The sequence of the CMV promoter is set forth in SEQ ID NO: 7. This promoter is believed to confer stable constitutive expression of the encoded protein in a wide range of cell types. A cell-type-specific promoter can be beneficial when the cell type is difficult to transfect or transduce. For example, a neuron-specific promoter can be used for expression in neurons. Furthermore, a cell-type-specific promoter can be useful when undifferentiated stem cells are to be transduced or transfected with a recombinant expression vector. This allows the encoded protein to be expressed only in specific cell types during or after differentiation. Furthermore, an inducible promoter can be used to initiate expression of the encoded protein at a specific time point. In one embodiment, the cell death switch, pH stability, and pH-sensitive fluorophore are under the control of a promoter. In one embodiment, the expression vector comprises a CMV promoter. In one embodiment, the cell death switch, pH stability, and pH-sensitive fluorophore are under the control of a CMV promoter.

[0048] The recombinant expression vector of the present invention may further comprise a selectable marker gene. A selectable marker gene is useful for selecting successfully transfected or transduced cells. The selectable marker gene may be an antibiotic resistance gene, such as the puromycin resistance gene (Puro). In this case, the selectable marker gene may be under the control of a promoter that is active in the transfected or transduced cell type. This promoter may be different from the promoters driving the expression of the inducible cell death switch, pH-stable fluorophore, and pH-sensitive fluorophore. The promoter may be mPGK (mouse phosphoglycerate kinase), which is efficient for driving high expression in various cell types. The sequence of the mPGK promoter is shown in SEQ ID NO: 8. Alternatively, successfully transfected or transduced cells may be selected by fluorescence. For example, cells expressing a pH-stable fluorophore may be sorted by fluorescence-activated cell sorting (FACS). In one embodiment, the recombinant expression vector of the present invention comprises a selectable marker gene. In one embodiment, the recombinant expression vector of the present invention comprises a puromycin resistance gene. In one embodiment, the selectable marker gene is under the control of the mPGK promoter.

[0049] The recombinant expression vector of the present invention may contain an antibiotic resistance gene for selecting bacterial clones producing viral vectors during vector production. In this case, the antibiotic resistance gene is under the control of a bacterial promoter. In one embodiment, the recombinant expression vector contains an antibiotic resistance gene. In one embodiment, the recombinant expression vector contains an ampicillin resistance gene.

[0050] In one embodiment, a recombinant expression vector of the invention comprises the polynucleotide sequence set forth in SEQ ID NO:1.

[0051] The present invention further relates to cells comprising the recombinant expression vectors of the invention, which are particularly suitable as inducible substrate cells in phagocytosis assays, since cell death of such cells can be initiated at specific time points and their phagocytic uptake visualized by the expressed fluorophore.

[0052] In principle, the recombinant expression vector of the present invention can be expressed in any cell type. Thus, cells containing the recombinant expression vector of the present invention can be any cell type. The cells can be cell lines derived from induced pluripotent stem cells (iPSC-derived), embryonic stem cells (ESC-derived), or patient-derived cell lines. The cells can be, for example, nerve cells, neuronal cells, glial cells, mesenchymal cells, or hematopoietic cells. The cells can be healthy or diseased cells. Diseased cells can be, for example, cancer cells, such as glioma cells. This can, for example, assess the phagocytosis of apoptotic glioma cells by microglia.

[0053] The cell type can be selected depending on the phagocytosis assay to be performed. For example, when studying phagocytosis of leukemic T cells, the cells containing the recombinant expression vector of the invention can be T cells.

[0054] Furthermore, cells containing the recombinant expression vector of the present invention can be stem cells. By selecting a cell-type specific promoter that regulates expression of the encoded polypeptide, only specific cell types express the encoded polypeptide after differentiation.

[0055] In one embodiment, the cell containing the recombinant expression vector of the present invention is a mammalian cell, particularly a human cell. In one embodiment, the cell containing the recombinant expression vector of the present invention is a stem cell. In one embodiment, the cell containing the recombinant expression vector of the present invention is a glioma cell or a T cell. In one embodiment, the cell containing the recombinant expression vector of the present invention is an H4 cell or a Jurkat cell.

[0056] Cells can be transfected or transduced with the recombinant expression vector of the present invention. Thus, expression of the encoded polypeptide can be transient or stable. In one embodiment, expression of the recombinant expression vector in the cell is stable. In a further embodiment, expression of the recombinant expression vector of the present invention in the cell is transient.

[0057] The present invention further relates to an in vitro method for assessing phagocytosis. The in vitro method described herein provides an improved method for generating stromal cells and assessing their phagocytosis. Thus, stromal cells according to the present invention are co-cultured with phagocytes, cell death of the stromal cells is induced, and phagocytosis of the stromal cells by the phagocytes is assessed. The stromal cells express both a pH-stable fluorophore and a pH-sensitive fluorophore. Because phagocytosis is accompanied by a decrease in pH within the phagolysosome, a change in the fluorescent signal of the pH-sensitive fluorophore indicates phagocytosis of the stromal cells. The pH-stable fluorophore allows for the precise location of the phagocytic event.

[0058] In one embodiment, an in vitro method for assessing phagocytosis comprises the steps of: a) providing inducible stromal cells according to the invention disclosed herein (i.e., cells comprising the recombinant expression vector of the invention as described herein above); b) co-culturing the inducible stromal cells in combination with phagocytes; c) inducing cell death of the inducible stromal cells; and d) detecting a fluorescent signal of the inducible stromal cells, wherein a change in the fluorescent signal of a pH-sensitive fluorophore indicates phagocytosis of the inducible stromal cells.

[0059] It is understood that steps b) to d) may be performed simultaneously or sequentially in any order. For example, cell death of substrate cells may be induced simultaneously when substrate cells and phagocytes are combined, i.e., steps b) and c) may be simultaneous. Alternatively, cell death of substrate cells may be induced before or after the cells are combined. Similarly, the fluorescent signal may be detected at any time point.

[0060] Phagocytic cells can be macrophages or tissue-resident macrophages. Tissue-resident macrophages can be microglia. Because macrophages and microglia arise from the same progenitor cells, their transcriptomes and proteomes overlap. In vitro, macrophages can be induced by differentiation of macrophage / microglial precursor cells for 7 days using M-CSF or GM-CSF. Microglial differentiation from macrophage / microglial precursor cells can be induced by M-CSF, TGF-β1, and IL-34. Microglial markers CX3CR1, P2RY12, or TMEM119 can be used to distinguish them from each other.

[0061] Inducible stromal cells and phagocytes can be co-cultured in an in vitro model containing additional cell types. This allows the in vitro method to evaluate phagocytosis in different types of in vitro models that mimic different tissues or organs. Therefore, depending on the in vitro model being studied, various additional cell types can be used. For example, when studying the phagocytosis of neuroblastoma cells by microglia, the in vitro model can include brain endothelial cells, pericytes, and astrocytes as additional cell types. In one embodiment, inducible stromal cells and phagocytes are co-cultured in an in vitro model containing additional cell types.

[0062] An advantage of the in vitro method described herein is that stromal cell phagocytosis does not necessarily begin at a specific time point when stromal cells and phagocytes are combined in an in vitro model. This allows for the assessment of phagocytosis in an in vitro model that requires different cell types to first organize (e.g., differentiate, migrate, adhere, connect) before the phagocytosis assay can begin, i.e., establish an in vitro model. Therefore, inducible stromal cell death and their phagocytosis can be induced after a period of time in which the in vitro model has been established. This is particularly true for 3D in vitro models such as organ chips, spheroids, or organoids. These culture systems may require several cell seeding steps and / or a period of time for the cells to organize.

[0063] It is further envisioned that the in vitro method for evaluating phagocytosis can be performed in an in vitro model established through stem cell differentiation. The recombinant expression vector of the present invention can be transduced into undifferentiated stem cells under the control of a cell type-specific promoter. After differentiation and establishment of a stem cell-derived in vitro model, the cell death switch, pH-stable and pH-sensitive fluorophore will be expressed only in the cell type in which the cell type-specific promoter is active. This allows the study of phagocytosis in stem cell-derived culture systems.

[0064] Thus, in one embodiment, cell death of the inducible stromal cells is induced after a period during which the cells establish an in vitro model. In one embodiment, cell death of the inducible stromal cells is induced after the inducible stromal cells are combined with phagocytes (i.e., steps b and c of the in vitro method for assessing phagocytosis). In a further embodiment, cell death of the inducible stromal cells is induced about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or about 6 hours after combining and co-culturing the inducible stromal cells with the phagocytes. In a further embodiment, cell death of the inducible stromal cells is induced about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after combining and co-culturing the inducible stromal cells with the phagocytes.

[0065] As described above, cell death of inducible substrate cells can be induced at any specific time point selected depending on the in vitro model used or the preferences of the person performing the in vitro method. Cell death is induced by the presence of an inducer, such as a chemical inducer, in the environment of the inducible substrate cells. Thus, the inducer can be added to the cell culture medium at any selected time point. The inducer binds to the inducer-binding domain of the cell death switch and activates the signaling protein of the cell death pathway. The activated signaling protein initiates the respective cell death pathway in the inducible substrate cells, leading to their cell death. Depending on the cell death switch and its inducer-binding domain used in the inducible substrate cells, different inducers can be used. The inducer-binding domain can be a dimerization domain. Thus, the inducer can be a dimerization inducer. The inducible cell death switch can include FKBP12-F36V as the dimerization domain. Thus, the dimerization chemical inducer can be AP20187 (Formula I). [ka]

[0066] AP20187 (CAS number: 195514-80-8) is a non-toxic synthetic FK506 analogue modified to enhance its binding to FKBP12-F36V while reducing its interaction with endogenous FKBP. Binding of AP20187 to the inducer-binding domain of inducible caspase-9 causes the dimerization and activation of caspase-9, resulting in the initiation of apoptosis. In one embodiment, the cell death of the induced stromal cells in step c) is induced by a chemical inducer of dimerization. In one embodiment, the cell death of the induced stromal cells in step c) is induced by AP20187. In one embodiment, the cell death of the induced stromal cells in step c) is induced by 10 nM AP20187.

[0067] As described herein, changes in the fluorescent signal of pH-sensitive fluorophores indicate phagocytosis of induced substrate cells, whereas pH-stable fluorophores allow for cell localization. The fluorescent signal of induced substrate cells can be detected by fluorescent imaging, flow cytometry, or a fluorescent plate reader.

[0068] The fluorescent signal of the pH-stable and / or pH-sensitive fluorophore of the stromal cells can be detected at any time point during the in vitro methods described herein. The fluorescent signal can be detected before and after induction of cell death of the stromal cells. Furthermore, the fluorescent signal can be detected at several time points during the phagocytosis assay, thereby allowing for a time course study of phagocytosis. In one embodiment, the fluorescent signal of the pH-stable and / or pH-sensitive fluorophore is detected before induction of cell death of the stromal cells. In a further embodiment, the fluorescent signal of the pH-stable and / or pH-sensitive fluorophore is detected at several time points after induction of cell death of the stromal cells. In one embodiment, the fluorescent signal of the pH-stable and / or pH-sensitive fluorophore is detected every 5, 10, 15, 20, or 30 minutes after induction of cell death. In further embodiments, the fluorescent signal of the pH-stable and / or pH-sensitive fluorophore is detected about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours and / or about 8 hours after induction of cell death. [Example]

[0069] The iCaspase9-SEP-mCherry construct (SEQ ID NO: 1; Figure 1) was transduced into the cell lines H4 (neuroblastoma) and Jurkat (T lymphocytes), and the iCaspase9 construct was transfected into HEK293. H4 and Jurkat clones with stable expression of the construct were selected. The cell line HMC3 (microglia) or iPSC-derived macrophages or microglia (generated in-house as described in WO 2020 / 239714) were used as phagocytes.

[0070] Example 1: Efficient on-demand generation of apoptotic cells. HEK293 cells were transfected with 2 μg of the iCaspase 9 construct by nucleofection (Amaxa 4D nucleofector by Lonza, program CM-130) and plated in 24-well plates. The next day, 100 nM AP20187 was added to the cells and analyzed after 1, 2, or 3 hours. For control, cells were incubated with 2.5 μM staurosporine for 3 hours. For analysis, cells were harvested, labeled with Annexin V-BV421 and the viability dye APCeF780, and analyzed by flow cytometry. Apoptotic cells were defined as Annexin V-positive and viability dye-negative.

[0071] iCaspase-9-transfected HEK293 cells undergo rapid and sustained apoptosis when treated with 100 nM AP20187. After 1 hour, 88% of iCaspase-9-transduced cells were apoptotic, compared with 36% after 3 hours of staurosporine treatment (Figure 2). Non-transfected cells showed basal apoptosis levels of 10-35%.

[0072] In conclusion, induction of apoptosis with AP20187 in cells expressing iCaspase 9 is faster and more efficient than the standard method of inducing apoptosis with staurosporine.

[0073] Example 2: Accurate quantification of phagocytosis with increased dynamic range. 2.1. pH-sensitive dual fluorescent indicator H4 cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry and expanded in 1 μg / ml puromycin-containing medium to select for transduced cells. Cells were placed in different pH solutions (Intracellular pH Calibration Buffer Kit, Invitrogen) and imaged using an inverted fluorescence microscope (Leica).

[0074] At a physiological pH of 7.5, mCherry and SEP are co-expressed and co-localized in transduced cells (Figure 3). mCherry fluorescence is pH-independent (Figure 3, bottom panel), whereas the SEP fluorescence signal is strong at a physiological pH of 7.4 but is quenched in acidic conditions (Figure 3, top panel).

[0075] H4-iCaspase-SEP-mCherry constitutively expresses mCherry fused to SEP and exhibits quenching of the SEP fluorophore in acidic environments, making this construct a suitable fluorescent pH sensor.

[0076] 2.2.Assessment of phagocytosis of H4 cells by microglia in a 2D model H4 cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry, single-cell sorted for SEP and mCherry double-positive cells, and expanded in 1 μg / ml puromycin-containing medium to select for stably transduced cells. HMC3 or iPSC-derived microglia (Reich, M et al. Alzheimer's Risk Gene TREM2 Determines Functional Properties of a New Type of Human iPSC-Derived Microglia. 2021;11:617860) were seeded into 96-well imaging plates. To visualize microglial morphology during phagocytosis assays, cells were stained with far-red viability dye (eBioscience). H4-iCaspase-SEP-mCherry cells were added the next day and allowed to attach for 3 hours. For control wells, 1 μM cytochalasin D (a fungal toxin that binds to actin filaments, thereby inhibiting actin polymerization and phagocytosis) was added 30 minutes before the start of time-lapse imaging. Apoptosis was induced at t0 by adding 10 nM AP20187 to the culture medium, and time-lapse imaging was immediately initiated using an inverted fluorescence microscope (Leica) or Incucyte (Sartorius), with a 10x objective, and one image every 10 min for over 8 h.

[0077] Apoptotic H4-iCaspase-SEP-mCherry was actively phagocytosed by cocultured HMC3 microglia (Figure 4). The mCherry signal indicates transduced H4 cells, and the SEP signal allows for the precise time point of phagocytosis by microglia to be determined (loss of SEP signal indicates phagocytosis). This example, highlighted in Figure 4, shows the colocalization of apoptotic H4 cells with microglia (arrows) at 2 hours and 40 minutes after the onset of apoptosis (top panel), and the phagocytosis of H4 cells at 3 hours and 10 minutes after the onset of apoptosis, as evidenced by the loss of SEP signal (bottom panel).

[0078] Figure 5 shows the phagocytosis of apoptotic H4-iCaspase-SEP-mCherry cells by iPSC-derived microglia. The number of phagocytosed cells per area was determined using IncuCyte software for automated image quantification by 1) dividing individual cells into segments, 2) identifying all H4-iCaspase-SEP-mCherry cells positive for mCherry (red fluorescence), and 3) identifying SEP-negative cells (green fluorescence) from those segments as phagocytosed cells. Apoptotic cells are phagocytosed by iPSC-derived microglia over a 12-hour period when a plateau is reached. Addition of cytochalasin D reduced both the occurrence and total amount of phagocytosed apoptotic cells; it did not induce apoptosis, and no phagocytosis was observed (Figure 5).

[0079] The rapid and efficient induction of apoptosis leads to rapid phagocytosis that can be accurately and reproducibly quantified.

[0080] 2.3.Assessment of Jurkat cell efferocytosis by macrophages in a 2D model Jurkat cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry, FACS-sorted for SEP and mCherry expression, and expanded in 1 μg / ml puromycin-containing medium to select for stably transduced cells. For pretreatment, Jurkat cells were incubated with the efferocytosis enhancer GAS6 or the efferocytosis inhibitor cytochalasin D, then exposed to 10 nM AP20187 and incubated with THP-1 macrophages. As a control, Jurkat cells were treated with staurosporine and labeled with pHrodo (a red pH-sensitive fluorophore). Efferocytosis was quantified using flow cytometry by gating on mCherry-positive and SEP-negative cells (apoptotic cells internalized by THP1 cells) and pHrodo-high cells in the control sample.

[0081] The range between maximal (pretreated with GAS6) and minimal (pretreated with cytochalasin D) uptake of apoptotic cells by macrophages using the iCaspase9-SEP-mCherry construct is 2-3 fold higher than in staurosporine-treated, pHrodo-labeled Jurkat cells (Figure 6). This increased range may be due to reduced fluorescence background and / or increased efferocytosis.

[0082] Apoptotic cells transduced with iCaspase-SEP-mCherry induce stronger phagocytosis / efferocytosis compared to conventional substrates, thus conferring a higher dynamic range. This allows for better ranking of distinct regulators of phagocytosis, enhancing the value of this assay compared to conventional approaches.

[0083] Example 3: Application of a novel phagocytosis assay to a complex 3D co-culture model 3.1.Use of a novel phagocytosis assay in 3D BBB spheroids H4 cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry, single-cell sorted for SEP and mCherry double-positive cells, and expanded in 1 μg / ml puromycin-containing medium to select for stably transduced cells. H4-iCaspase-SEP-mCherry and HMC3 microglia were co-seeded with primary human brain endothelial cells, pericytes, and astrocytes (Sciencell) to form blood-brain barrier (BBB) ​​spheroids, as described by Simonneau, C et al. Investigating receptor-mediated antibody transcytosis using blood-brain barrier organoid arrays. Fluids Barriers CNS. 2021;18(1):43. Briefly, cells were combined at a 1:1:1:1:1 ratio and seeded into glass-bottom GRI3D microwell plates (SUN Biosciences). After 2 days of culture in EGM2 medium (Lonza) containing astrocyte growth supplement (Sciencell), time-lapse experiments were performed as described above (for Figure 4): apoptosis was induced at t0 by adding 100 nM AP20187 to the medium, and time-lapse imaging was immediately initiated over 8 hours using an inverted fluorescence microscope (Leica) with autofocus, a 10x objective, and one image every 10 minutes.

[0084] Spheroids containing H4-iCaspase-SEP-mCherry gradually lost SEP signal upon AP20187 treatment, indicating active phagocytosis within the spheroid core (Figure 7). SEP and mCherry fluorescence remained stable over an 8-hour period in untreated spheroids.

[0085] H4 cells transduced with iCaspase 9-SEP-mCherry were successfully incorporated into a high-throughput 3D in vitro model. Two days after the 3D architecture of the complex cell model was established, apoptosis and subsequent phagocytosis were triggered. Because other known assays fail to trigger matrix generation (apoptotic cells) in a cell-specific manner after cell seeding, this assay cannot be compared with other phagocytosis assays.

[0086] 3.2. Use of a novel phagocytosis assay in a 3D neurovascular unit model. H4 cells were transduced with a lentiviral vector containing iCaspase-SEP-mCherry, single-cell sorted for SEP and mCherry double-positive cells, and expanded in 1 μg / ml puromycin-containing medium to select for stably transduced cells. H4-iCaspase-SEP-mCherry and HMC3 microglia were co-seeded with primary human brain endothelial cells, pericytes, and astrocytes (Sciencell) at a ratio of 1:1:4:0.5:1 and allowed to self-assemble into neurovascular units inside a fibrin gel matrix in a microfluidic chip (AIM Biotech). As described in Campisi, M et al., 3D Self-Organized Human Blood-Brain Barrier in a Microfluidic Chip. Methods Mol Biol. 2021;2258:205-219, cells were seeded into the microfluidic chip and vascular networks were grown for 7 days in EGM2 medium (Lonza) containing astrocyte growth supplements (Sciencell). Apoptosis was induced at time t0 by adding 10 nM AP20187 to the medium, and then after 30 minutes or 2 hours, the chips were fixed in 4% PFA, counterstained with DAPI, and imaged using an inverted fluorescence microscope.

[0087] H4-iCaspase-SEP-mCherry cells surrounded vasculature after 7 days in culture. Apoptosis was efficiently induced in transduced cells within 30 minutes inside the 3D matrix of the microfluidic chip. Single-cell resolution in this setting allowed us to identify single instances of SEP signal loss in apoptotic cells after 2 hours (Figure 8), indicating localized acidification due to phagocytosis.

[0088] H4 cells transduced with iCaspase 9-SEP-mCherry were successfully incorporated into a complex 3D in vitro model. Apoptosis and subsequent phagocytosis were triggered after 7 days of culture. Single events of phagocytosis were observed. This system also allows for the simultaneous localization of phagocytic events to individual microglia. We cannot compare our results with other phagocytosis assays because they do not offer the option of triggering substrate generation (apoptotic cells) on demand and in a cell-specific manner.

[0089] [ka] [ka]

[0090] FKBP12-F36V sequence (SEQ ID NO: 2): GGAGTGCAGGTGGAGACTATCTCCCCAGGAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAAAGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGG AGGTGATCCGAGGCTGGGAAGAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTGGGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAA

[0091] Cleaved caspase 9 sequence (AA135-416 from Homo sapiens caspase 9, transcript variant alpha NM_001229.5) (SEQ ID NO: 3): GGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGG GCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTG GGAGGGAAGCCCAAGCTCTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACT CTACTTTCCCAGGTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCA

[0092] iCasp9 sequence(sequence number4):

[0093] mCherry sequence (SEQ ID NO: 5) ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA

[0094] SEP sequence (SEQ ID NO: 6): ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCTTGGCCAACACTTGTCACTACTTTAACTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACGATCACCAGGTGTACATCATGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTAGACACAACATTGAAGATGGAGGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGGCCCGTCCTTTTACCAGACAACCATTACCTGTTTACAACTTCTACTCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAA

[0095] CMV promoter sequence (SEQ ID NO: 7): TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATC

[0096] mPGK promoter sequence (SEQ ID NO: 8): TTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGC GCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACT AGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGG GCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCT

[0097] Linker between SEP and mCherry (GSSGSS linker) (SEQ ID NO: 9): GGCAGCAGCGGCAGCAGC

[0098] i Cleavable P2A linker between caspase 9 and SEP-mCherry (SEQ ID NO: 10): GGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT

[0099] FKBP12-F36V amino acid sequence (SEQ ID NO: 11): GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0100] Cleaved caspase 9 amino acid sequence (AA135-416 from Homo sapiens caspase 9, transcript variant alpha NM_001229.5) (SEQ ID NO: 12): GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSL GGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS

[0101] iCasp9 amino acid sequence (SEQ ID NO: 13): MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQ DHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRT FDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVDYPYDVPDYALD

[0102] mCherry amino acid sequence (SEQ ID NO: 14) MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSL QDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0103] SEP amino acid sequence (SEQ ID NO: 15): MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0104] Amino acid sequence of the linker between SEP and mCherry (SEQ ID NO: 16): GSSGSS

[0105] i Amino acid sequence of the cleavable P2A linker between caspase 9 and SEP-mCherry (SEQ ID NO: 17): GSGATNFSLLKQAGDVEENPGP

Claims

1. Recombinant expression vectors encoding an inducible cell death switch, pH-stable fluorophores, and pH-sensitive fluorophores.

2. The recombinant expression vector according to claim 1, wherein the recombinant expression vector is a viral vector.

3. The recombinant expression vector according to claim 2, wherein the viral vector is a lentiviral vector.

4. The recombinant expression vector according to claim 1, wherein the inducible cell death switch induces apoptosis, necrotosis, pyroptosis, or ferroptosis, preferably apoptosis.

5. The recombinant expression vector according to claim 1, wherein the inducible cell death switch comprises an inducible factor binding domain and a signaling protein of the cell death pathway.

6. The recombinant expression vector according to claim 5, wherein the signaling protein of the cell death pathway is an apoptosis-promoting protein, preferably caspase 9 or a functional fragment thereof.

7. The recombinant expression vector according to claim 5, wherein the inducing factor binding domain comprises a dimerization domain, preferably FKBP12-F36V.

8. The recombinant expression vector according to claim 5, wherein the inductor-binding domain can bind an inductor, preferably a dimerized chemical inductor, most preferably AP20187.

9. The recombinant expression vector according to claim 1, wherein the inducible cell death switch is inducible caspase 9.

10. The recombinant expression vector according to claim 1, wherein the pH-stable fluorophore and the pH-sensitive fluorophore have different excitation spectra and emission spectra.

11. The recombinant expression vector according to claim 1, wherein the pH-stable fluorophore is from the RFP family, Alexa Fluor dyes, protein-based fluorophores, simple organic fluorophores, or organic polymers, preferably from mCherry.

12. The recombinant expression vector according to claim 1, wherein the fluorescence signal of the pH-sensitive fluorophore changes in response to a decrease in pH, preferably the fluorescence signal is quenched or the excitation / emission spectrum is shifted.

13. The recombinant expression vector according to claim 1, wherein the pH-sensitive fluorophore is Superecliptic pHluorin (SEP), pHLemon, pHmScarlet, pHTomato, pHuji, LysoSensor, or pH nanosensor, preferably Superecliptic pHluorin (SEP).

14. The recombinant expression vector according to claim 1, wherein the recombinant expression vector comprises a promoter, particularly a ubiquitous promoter, a cell-specific promoter, a constitutive promoter, or an inducible promoter.

15. The recombinant expression vector according to claim 1, wherein the recombinant expression vector comprises a CMV promoter.

16. The recombinant expression vector according to any one of claims 1 to 15, wherein the recombinant expression vector comprises the polynucleotide sequence described in SEQ ID NO:

1.

17. A cell comprising the recombinant expression vector according to claim 1.

18. The cell according to claim 17, wherein the cell is a mammalian cell, particularly a human cell.

19. The cell according to claim 17, wherein the cell is a stem cell.

20. The cell according to claim 17, wherein the cell is a nerve cell, neuronal cell, glial cell, mesenchymal cell, or hematopoietic cell, preferably a glioma cell or T cell.

21. The cell according to claim 17, wherein the cell is an H4 cell or a Jurkat cell.

22. The cell according to any one of claims 17 to 21, wherein the expression of the recombinant expression vector in the cell is transient or stable.

23. An in vitro method for evaluating phagocytosis, a) A step of preparing the inducible substrate cells according to claim 17, b) A step of co-culturing the inducible substrate cells in combination with phagocytic cells, c) A step of inducing cell death of the inducible substrate cells, d) A step of detecting the fluorescence signal of the inducible substrate cells An in vitro method comprising a pH-sensitive fluorophore, wherein a change in the fluorescence signal of the inducible substrate cell indicates phagocytosis.

24. The method according to claim 23, wherein the phagocytic cell in step b) is a macrophage or a tissue-resident macrophage.

25. The method according to claim 24, wherein the tissue-resident macrophage is microglia.

26. The method according to claim 23, wherein the inducible substrate cells and the phagocytic cells are co-cultured in an in vitro model including further cell types.

27. The method according to claim 26, wherein the in vitro model is a two-dimensional or three-dimensional culture system.

28. The method according to claim 26, wherein the in vitro model is an organ-on-a-chip, spheroid, or organoid.

29. The method according to claim 26, wherein the in vitro model is a neurovascular unit or a blood-brain barrier spheroid.

30. The method according to claim 23, wherein the cell death of the inducible substrate cells in step c) is induced after the inducible substrate cells have been combined with the phagocytic cells in step b).

31. The method according to claim 23, wherein the cell death of the inducible substrate cells in step c) is induced by an inducing factor, preferably a dimerizing chemical inducing factor, most preferably AP20187.

32. The method according to claim 23, wherein the fluorescence signal in step d) is detected by fluorescence imaging, flow cytometry, or by a fluorescence plate reader.

33. The method according to any one of claims 23 to 32, wherein the fluorescence signal in step d) is detected at several time points after induction of cell death.

Images