Therapeutic agents and methods

Abstract

The present invention relates to modified stem cell-like cells for use in organ regeneration or repair, wherein the modified stem cell-like cells exhibit reduced ion flow through a sodium leak channel (NALCN). The invention also relates to ion channel modulators for organ regeneration or repair. The invention also relates to related methods, such as methods of repairing or regenerating an organ.

Description

Technical Field

[0001] The present invention relates to modified stem cell-like cells for organ regeneration or repair. The present invention also relates to ion channel modulators for organ regeneration or repair. The present invention also relates to related methods, such as methods for repairing or regenerating organs. Background Art

[0002] Regenerative medicine is a field that seeks to repair or replace damaged or diseased human cells or tissues to restore normal function. Loss and damage to organs and tissues can occur in a variety of ways, including through, for example, disease, injury, or congenital abnormalities. The field of regenerative medicine is generally considered an interdisciplinary field that applies developments in both engineering and biological sciences to promote the regeneration of damaged or diseased human cells or tissues. Methods generally include tissue engineering and cell therapies, such as the use of stem cells to regenerate damaged tissues, and there are many available regenerative medicine therapies. However, there are also some problems associated with the development of regenerative medicine. Regarding cell therapy, one challenge includes how to effectively and consistently deliver such therapeutic agents, such as stem cells, to tissues or organs that need to be regenerated or repaired, such as the kidneys. One such example is the kidney, where human patients with renal fibrosis will further progress to renal disease and damage, resulting in higher mortality. Identifying and treating patients at high risk for kidney disease or those in the early stages of kidney disease to prevent further disease progression, or even potentially reverse tissue damage, will have a huge impact.

[0003] The object of the present invention is to develop therapeutic agents for regenerating or repairing organs and / or tissues, ie for use in regenerative medicine. Summary of the invention

[0004] The inventors have identified that the single ion channel NALCN is a key regulator of epithelial cell shedding. The inventors have demonstrated that stem cell-like epithelial cells in which Nalcn is missing or treated with chemical entities to reduce the ion flow through NALCN cause cell mobilization to different tissues, such as the lungs, liver, pancreas, kidneys, and peritoneum, and form normal structures in these organs, including glomeruli, renal tubules, and pulmonary bronchioles. The transcriptomes of these circulating cells in tumor-bearing and non-tumor-bearing mice are indistinguishable and are closely related to the transcriptomes of human circulating tumor cells (CTCs). The inventors have shown that NALCN is independent of cancer regulation and cell shedding from solid tissues. Therefore, cells modified to display reduced ion flow through NALCN can be used to repair or regenerate organs or tissues.

[0005] Accordingly, the present invention provides a modified stem-like cell for use in organ regeneration or repair, wherein the modified stem-like cell exhibits reduced ion flux through a sodium leak channel (NALCN).

[0006] Also provided is an ion channel modulator for organ regeneration or repair, wherein the ion channel modulator reduces ion flow through the sodium leak channel (NALCN).

[0007] The ion channel modulator can target NALCN and / or one of the proteins associated with NALCN, wherein the protein associated with NALCN is selected from: GPCR (M3 muscarinic receptor M3R, TACR1, CaSR), UNC80, UNC79, FAM155A (NLF1A), Fam155B, SLO2.1.

[0008] Ion channel modulators can target the pore turret domain, voltage sensing domain, or linker domain of NALCN.

[0009] The organ to be regenerated may be selected from the group consisting of: thymus, adrenal glands, thyroid gland, intestine, lung, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidney and bladder, skin, hair follicles, pancreas, bone and cartilage.

[0010] The modified stem-like cells or ion channel modulators can be used to treat chronic kidney diseases, such as glomerulonephritis and / or renal failure, lung diseases, such as COPD, or liver fibrosis.

[0011] The reduction in ion flow through the NALCN may be temporary.

[0012] Also provided are pharmaceutical compositions comprising the modified stem-like cells or ion channel modulators.

[0013] The present invention also provides a method of repairing or regenerating an organ in a subject, comprising administering to the subject a therapeutically effective amount of the modified stem-like cells, ion channel modulators, or pharmaceutical compositions described herein.

[0014] The method may further comprise obtaining stem-like cells from the subject to be treated.

[0015] The method may also include modifying the stem-like cell such that the stem-like cell exhibits reduced ion flux through NALCN.

[0016] Stem-like cells can be modified in the following ways:

[0017] contacting the stem-like cells with an ion channel modulator, wherein the ion channel modulator inhibits ion flux through NALCN; and / or

[0018] Introducing a Nalcn mutation and / or a Nalcn deletion and / or reducing the expression of Nalcn in the stem-like cells, wherein the mutation and / or the deletion and / or the reduced expression results in reduced ion flux through NALCN.

[0019] The modified stem-like cells may be epithelial stem cells.

[0020] The modified stem-like cells can:

[0021] i) comprises a mutation in Nalcn that reduces ion flux through NALCN; or

[0022] ii) with knockout of Nalcn;

[0023] iii) exhibit reduced expression of Nalcn; or

[0024] iv) have been treated with an ion channel modulator to reduce ion flux through NALCN.

[0025] The reduced ion flow through the NALCN may be temporary.

[0026] Also provided is a method for preparing a modified stem-like cell, the method comprising:

[0027] i) contacting the stem-like cells with an ion channel modulator, wherein the ion channel modulator inhibits ion flux through NALCN; or

[0028] ii) introducing a Nalcn mutation and / or a Nalcn deletion and / or reducing the expression of Nalcn in stem-like cells, wherein the mutation and / or the deletion and / or the reduced expression results in a reduced ion flux through NALCN.

[0029] The stem-like cell may be an epithelial stem cell.

[0030] Contacting can be performed in vitro or ex vivo.

[0031] The method may further comprise expanding the modified stem-like cells.

[0032] Also provided is a method for increasing the level of solid tissue cell shedding of stem-like cells, comprising introducing a Nalcn mutation and / or a deletion of Nalcn and / or reducing the expression of Nalcn in the stem-like cells, wherein the mutation and / or deletion and / or reduced expression results in reduced ion flux through NALCN.

[0033] Also provided is a method of increasing the level of solid tissue cell shedding of stem-like cells, comprising contacting the cells with an ion channel modulator, wherein the ion channel modulator is capable of reducing ion flux through NALCN.

[0034] The cells can be contacted in vitro, ex vivo, or in vivo.

[0035] The solid tissue may be epithelial tissue.

[0036] Also provided are kits comprising modified stem-like cells and optionally instructions for use, wherein the modified stem-like cells exhibit reduced ion flux through NALCN.

[0037] The kit may further include one or more components selected from the group consisting of: a NALCN modulator, a cell culture medium, a buffer, an excipient, and a cell culture container. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Loss of NALCN function increases shedding of circulating non-tumor cells. (a) Circulating non-tumor cells (ntCZCs) identified in non-tumor-bearing mice (bars = median). The figure also shows that when mice were exposed to gadolinium chloride, the loss of NALCN function in mice with wild-type NALCN (labeled as Nalcn + / + (b) UMAP of 201,183 single-cell RNA sequencing profiles of PBMCs, tCZCs, and ntCZCs, as well as cells derived from the indicated normal and malignant mouse tissues. (c) P1 R Nalcn Flx / Flx Co-immunofluorescence of ntCZCs and PBMCs in mouse peripheral blood smears (ZsGreen [ZSG], scale bar = 10 μm). (d) ZSG in lungs and kidneys + Direct ZSG immunofluorescence micrographs of cells (scale bar = 50 μm) and counts in lung (e) and kidney (f). (g) ZSG was expressed in 1% of the rats injected with P1 R Nalcn + / + ntCZCs or P1 R Nalcn Flx / Flx Each mouse ZSG identified in the recipient mouse organs of ntCZCs + Organ heatmap of total number of cell clusters. (h) P1 incorporated into kidney of recipient mice R Nalcn Flx / Flx Co-immunofluorescence of ntCZCs (arrowheads indicate ZSG + cells, scale bar = 50 μm). (i) P1 R Nalcn Flx / Flx Confocal laser scanning microscopy images of CZCs (scale bar = 100 μm). In all figures, * = p < 0.05; *** = p < 0.0005, Mann-Whitney.

[0039] Figure 2 NALCN loss-of-function circulating non-tumor cells (ntCZCs) resemble human and mouse CTCs and engraft in distant organs. (a) Figure 1 Heatmap reporting gene set enrichment analysis in the UMAP clusters identified in b. Tested gene sets were derived from 2,086 different tissues and cell types, including bulk RNAseq of mouse normal tissues and tumors, huCTC signatures, and mouse and human intestinal stem and mature cell signatures (see Methods). (b) Aged Pdx1 R Nalcn + / + (upper left) and Pdx1 R Nalcn Flx / Flx (Bottom left) ZSG immunohistochemistry of mouse lung bronchioles (scale bar = 100 μm). Right, Pdx1 R Nalcn + / + (n=7) and Pdx1 R Nalcn Flx / Flx (n=15; **, p<0.005 Mann-Whitney) Number of ZSG+ cells / bronchiole in the lung. (c) In Pdx1 R Nalcn Flx / Fl Two-photon direct detection of ZSG+ cell clusters in whole lung sections of mice. (d) P1 injected into the tail vein of recipient mice R Nalcn Flx / Flx Example co-immunofluorescence of ntCZCs (arrowheads indicate ZSG + cells, scale bar = 50 μm).

[0040] Figure 3 .Implanted ZSG + ntCZCs express renal markers. (a) ntCZCs, ZSGs implanted into the kidneys of immunocompromised mice (NOD-scid-gamma-NSG) + UMAP of 166,878 SCS profiles of ntCZCs, control NSG kidney cortex and medulla. (bf) Signature plots of genes expressed in kidney projected onto the UMAP in (a): Bowman’s capsule (Ly6a), glomerulus (Clic5), proximal tubule (Lrp2), loop of Henle (Clcnkb), and distal tubule (Calb1).

[0041] Figure 4 .ZSG + The cells are present in the renal injury area in vivo. (a) Schematic diagram of the acute kidney injury model. (b) ZSG in Villin1-CreERT2; Rosa26ZSGreen (V1Z) mice on day 5 of renal injury induction. +Measurement of circulating cells (ntCZC). (c) Graph showing weight loss associated with disease progression and stage. (d) Example immunofluorescence images of kidneys from control (left) and folic acid treated (right) animals. Yellow indicates PDGFRb expression, a marker of inflammation and fibrosis in kidney disease. Light blue is the epithelial marker E-cadherin. (e) Indicates ZSG + Bright field image (4x) of a kidney where the cells are present. (fg) Exemplary immunofluorescence image of a kidney from a folate-treated mouse depicting ZSG + (h) Scatter plot showing the presence of 10 cells isolated from kidneys of two folate-treated animals. 6 ZSG sorted in cells + The number of cells.

[0042] Figure 5 .Schematic illustration of cell therapy applications of ntCZCs. DETAILED DESCRIPTION

[0043] Detailed description

[0044] Embodiments of the present invention will now be further described. In the following paragraphs, different embodiments are described. Unless there is a clear indication to the contrary, each aspect defined in this way can be combined with any other aspect or aspects.

[0045] Generally, the terms and techniques related to cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques disclosed herein are generally performed according to conventional methods described in various general and more specific references known in the art and cited and discussed in this specification. For example, see Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0046] Ion channels are key components of cell excitability and are associated with many diseases. In this article, the inventors have demonstrated that NALCN plays a vital role in epithelial stem cell-like cell shedding and non-malignant cell metastasis. NALCN is a non-selective monovalent cation channel and is the only member of different branches of voltage-gated sodium and calcium channels that regulate the resting membrane potential and excitability of neurons. NALCN is most abundantly expressed in the nervous system and conducts a sustained sodium leak current, which contributes to tonic neuronal excitability. The sequence of NALCN is known and can include sequences provided in ENSG00000102452 (ensemble), 259232 (NCBI Entrez gene), 19082 (HGNC), Q8IZF0 (UniProtKB / Swis-Prot) or 611549 (OMIM). In one embodiment, the sequence of NALCN includes SEQ ID NO.1. There are a variety of splice variants of NALCN, and the present invention extends to these variants. NALCN forms a polypeptide chain of 24 transmembrane helices (TMs) forming four homologous functional repeat units, also called α subunits, connected by an intracellular linker. Each functional repeat unit includes a voltage sensing domain, a pore helix, and an ion selectivity filter.

[0047] It has been shown that loss of NALCN function contributes to the shedding of epithelial stem-like cells in non-cancerous models. These cells mobilize to different tissues, such as the lung, liver, kidney, pancreas, and peritoneum, and form normal structures in these organs. Therefore, such modified cells and ion channel modulators that reduce ion flow through NALCN may be used in regenerative medicine, i.e., repairing or regenerating organs or tissues.

[0048] Thus, a first aspect of the present invention relates to a modified stem-like cell for use in organ regeneration or repair, wherein the modified stem-like cell exhibits reduced ion flux through the sodium leak channel NALCN (referred to herein as NALCN).

[0049] A second aspect of the present invention relates to an ion channel modulator for organ regeneration or repair, wherein the ion channel modulator reduces ion flow through the sodium leak channel NALCN.

[0050] The inventors have shown that by inhibiting ion flux through NALCN, either by mutation or knockout of NALCN, or by treatment with NALCN modulators, shedding of epithelial-derived stem-like cells is increased. These cells mobilize to different tissues and / or organs and form normal structures in these organs. Thus, the inventors have determined that inhibiting or reducing ion flux through NALCN can be used to repair or regenerate damaged or diseased organs and / or tissues.

[0051] Organ repair and regeneration

[0052] Those skilled in the art will appreciate that the phrases organ regeneration or repair as used herein are used to refer to the repair of damaged or defective organs or tissues. Repair may not be a complete repair, but rather an improvement. For example, the present invention may result in an improvement in the function of an organ or tissue compared to an untreated individual. For example, the present invention may improve the function of an organ or tissue by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to an untreated individual.

[0053] The present invention can be used to repair or regenerate one or more of the following organs or organ systems: thymus, adrenal glands, thyroid gland, intestines, lungs, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidneys and bladder, skin, hair follicles, pancreas, bones and cartilage. In embodiments, the present invention can be used to repair or regenerate one or more of the following organs or organ systems: intestines, lungs, liver, kidneys, bladder, pancreas. In embodiments, the present invention can be used to repair or regenerate kidneys, liver, pancreas, intestines, lungs or bladder. In embodiments, the present invention can be used to repair or regenerate kidneys. Therefore, the present invention can be used to treat chronic kidney disease, such as glomerulonephritis and / or renal failure. The present invention can also be used to treat lung diseases, such as COPD or liver fibrosis.

[0054] cell

[0055] In the present invention, modified stem cell-like cells are used for the repair or regeneration of organs or tissues. It will be readily appreciated by those skilled in the art that stem cells can be classified from other cells in a variety of ways. The properties of stem cells can be illustrated in vitro using, for example, clonogenic assays, in which the differentiation and self-renewal capabilities of individual cells are assessed. Stem cells can also be identified by a unique set of surface markers present on the surface of stem cells.

[0056] The term "stem cell-like cells" as used herein refers to cells that have common properties with stem cells, and the term includes stem cells. In particular, these cells are progenitor cells and highly plastic cells, i.e., have the ability to form various cell types. Stem cell-like cells can further differentiate into or have not yet differentiated into terminally differentiated cell types and / or somatic cells. The inventor's transcriptome profiles show that stem cell-like cells have transcriptome profiles very similar to stem cells, such as gastric stem cells, gastric isthmus cells, small intestinal stem cells, duodenal transit amplifying cells, and human circulating tumor cells that exhibit a high degree of plasticity to regenerate entire tumors. The epithelial stem-like cells of the present invention have been shown to be enriched in the following epithelial (e.g., Cdh1, Epcam, Krt8, Krt18, Krt80, Krt222) and stem cell / epithelial to mesenchymal transition / metastasis initiating cell / circulating tumor cell markers (e.g., Zeb2, Tgfb1, Tgfbr1, Cd36, Cd44, L1cam, Fn1, Lgals3, Hba-a1, Hba-a2, Hbb-bs, Hbb-bt). Using histology / antibody-based methods, enrichment of these markers can be observed at the transcriptional and / or protein levels. Therefore, these markers can be used to identify the stem-like cells of the present invention.

[0057] Thus, in an embodiment, the stem-like cells of the invention exhibit enrichment of one or more of the following markers: Cdh1, Epcam, Krt8, Krt18, Krt80, Krt222, Zeb2, Tgfb1, Tgfbr1, Cd36, Cd44, L1cam, Fn1, Lgals3, Hba-a1, Hba-a2, Hbb-bs, Hbb-bt, Cacybp, Ceacam1.

[0058] Stem cell-like cells can be epithelial in origin. As will be understood by those skilled in the art, "epithelial in origin" means that the cell is epithelial-derived, i.e., epithelial stem cell-like cells. The inventors have shown that epithelial stem cell-like cells exhibit enrichment of the following markers by immunofluorescence analysis: CDX2, CDH1, and KRT80. In addition, transcriptome data have shown enrichment of epithelial-specific markers Cdh1, Epcam, Cacybp, Ceacam1, Krt8, Krt18, Krt80, and Krt222. Using histology / antibody-based methods, enrichment of these markers can be observed at the transcriptional and / or protein levels. Therefore, these markers can be used to identify epithelial stem cell-like cells of the present invention.

[0059] Thus, in embodiments, the epithelial-derived stem-like cells exhibit enrichment for one or more of the following markers: CDX2, CDH1, KRT80, Epcam, Cacybp, Ceacam1, Cdh1, Krt8, Krt18, Krt80, and Krt222.

[0060] Herein, the inventors have shown that modified stem-like cells can be mobilized to different tissues and / or organs and form normal structures in these organs. In particular, it is shown herein that cells can be mobilized to the kidneys and subsequently begin to express markers of mature kidney cells, including Ly6a, Clic5, Calb1, Clcnkb, Lrp2.

[0061] In a related aspect of the invention, the stem cell-like cell may be a stem cell. In another aspect of the invention, the stem cell is an epithelial stem cell and / or an epithelial stem cell. The stem cell according to the invention is a cell that is capable of further differentiation into or has not yet differentiated into a terminally differentiated cell type and / or a somatic cell.

[0062] Stem cell can be any cell type selected from the following: pluripotent stem cell, multipotent stem cell, oligopotent stem cell or unipotent stem cell.It is obvious to those skilled in the art that although pluripotent stem cell directly derives from the totipotent cell from embryo, pluripotent stem cell can also be artificially derived from non-pluripotent stem cell, such as somatic cell.The pluripotent stem cell of this artificial origin is called as induced pluripotent stem cell.Stem cell of the present invention does not need to derive from human embryo.

[0063] Reduced ion flow through NALCN

[0064] We have shown herein that loss of NALCN function contributes to increased shedding of circulating non-tumor cells (ntCZC) and seeding of these cells in distant organs. Thus, by modulating the activity of NALCN, repair and / or regeneration of organs or tissues may be increased or promoted.

[0065] In the present invention, cells are modified so that the modified stem-like cells exhibit reduced ion flow through NALCN. Ion flow refers to the maximum number of ion molecules that can flow through NALCN in a given time unit. According to an embodiment of the present invention, reducing ion flow involves preventing or impairing the passage of ion molecules through NALCN so that fewer ion molecules flow through NALCN in the same given time unit. Since NALCN is a cell responsible for resting Na + NALCN is a permeable ion channel, so the activity of NALCN can be assessed using a variety of techniques. Activity can be assessed by whole-cell electrophysiology, fluorescence assays, membrane potential sensing dyes, and / or ion flux assays.

[0066] The ion channel modulators of the present invention regulate NALCN, thereby reducing the ion flow through NALCN. Ion channel modulators can be classified as ion channel blockers or ion channel openers. Ion channel blockers are generally antagonist compounds, which act to prevent the response usually provided by channel opening. In a preferred embodiment, the ion channel modulator is an ion channel blocker or a compound that antagonizes NALCN. As described above, ion flow refers to the maximum number of ion molecules that can flow through NALCN in a given time unit. According to an embodiment of the present invention, reducing ion flow involves preventing or impairing the passage of ion molecules through NALCN so that fewer ion molecules flow through NALCN in the same given time unit. Since NALCN is a cell responsible for resting Na + NALCN is a permeable ion channel, so the activity of NALCN can be assessed using a variety of techniques. Activity can be assessed by whole-cell electrophysiology, fluorescence assays, membrane potential sensing dyes, and / or ion flux assays.

[0067] Ion channel modulators

[0068] Ion channel modulators of the present invention may include small molecules, antibodies or fragments thereof, peptides, synthetic peptides, transcriptional regulators such as triplex-forming oligonucleotides, synthetic polyamides, zinc finger proteins, or post-transcriptional regulators such as RNAi, siRNA, miRNA.

[0069] Ion channel modulators can modulate the activity of NALCN by acting directly on the protein NALCN. For example, as a small molecule, antibody, peptide or synthetic peptide. Alternatively, or in addition, ion channel modulators can modulate the activity of NALCN by changing the expression level of the Nalcn gene or NALCN protein. For example, as a transcriptional or post-transcriptional regulator.

[0070] When an ion channel modulator acts on the NALCN protein, regulation can be achieved by binding to one or more α subunits of NALCN. This regulation can be achieved by the modulator disrupting the protein-protein interaction between the α subunit and the auxiliary subunit of NALCN, or disrupting the interaction between NALCN and NALCN-related proteins. Regulation can be achieved by the modulator interfering with or enhancing the movement of other elements of the voltage sensor or gating mechanism.

[0071] As mentioned above, ion channel regulators reduce the ion flow through NALCN. Ion channel regulators can reduce the ion flow through NALCN by directly targeting NALCN. Ion channel regulators can reduce the ion flow through NALCN by targeting one of the proteins associated with NALCN. NALCN protein forms a channelosome complex in the cell membrane. The channelosome includes various proteins associated with NALCN, including G protein coupled receptors, UNC-79, UNC-80, SLO2.1, FAM155A, FAM155B, NCA localization factor-1 and src family tyrosine kinase. Therefore, one of these proteins associated with NALCN can be targeted by ion channel regulators. Proteins associated with NALCN that can be targeted by ion channel regulators are selected from: GPCR (M3 muscarinic receptor M3R, TACR1, CaSR), UNC80, UNC79, FAM155A (NLF1A), Fam155B, SLO2.1, src family tyrosine kinase.

[0072] In embodiments, ion channel modulators may include, for example, gadolinium chloride, verapamil, high levels of exogenous Ca 2+ , 2-aminoethoxydiphenyl borate, nifedipine, nimodipine, flunarizine, esoxazolidone, N-benzhydryl quinuclidine compounds and / or L703606.

[0073] The NALCN protein comprises multiple domains, and therefore, ion channel modulators can target one or more of these domains to reduce ion flow through NALCN. Ion channel modulators can target one or more of the pore turret domain, voltage sensing domain, or linker domain of NALCN. The linker domain can be a linker domain that extends extracellularly or intracellularly. The mutation can be in one or more domains, and the one or more domains comprise any one of the amino acid sequences listed in SEQ ID NO. 2 to 23. The domains of NALCN and their sequences are listed in the following table:

[0074] Table 1

[0075]

[0076]

[0077]

[0078]

[0079] Ion channel modulators can also interact with specific residues in NALCN. For example, ion channel modulators can interact with one or more amino acid residues of NALCN, and the one or more amino acid residues are selected from, but not limited to: L588M, P573, R855, K1213, T71, P225, D1527, D416, C1348, R297, V1386, A1091, V1229, D134, T272, R43, A1157, V1036, M520, R1500, V320, V53, W1085, E1458, N1274, V1542, Y1300. , R1174, H1523, F332, Q549, L999, F540, A1421, R1384, H569, A1435, M55, R1495, C245, F110, V510, C970, E454, V273, R15 56. S174, S1068, V385, S384, A401, S902, R1495, A276, R1540, L517, R295, R382, H876, F300, R164, E257, R995, G1526, D29 1. V1239, E1552, N1475, M55, L1553, Y1349, E323, A1044, T1281, V1007, L253, L564, F1427, V949, Q279, T539, R159, K452 , R1127, V1490, G555, E62, L1461, L942, R166, P65, D952, I322, F154, K1163, L305, R152, W1085, R143, A1444, R989, R143, R1193, D1466, M520, V1285, S52, I51, E1518, E532, L1279, V1329, T57, A1378, S121, K498, R1094, V120, A88, A401, L1548, G1303, M150, D1277, E432, L1442, P1082, T1165, G1316, R1273, E128, E906, F1311, R1481, T204, T552, F389, D1527, P908,A1166, I577, G954, G1013, P65, E1016, N1070, S980, A1217, V1503, T1320, A223, A310, R1127 , D1504, D1277, E128, K1491, Q553, V511, F1250, S1374, D211, T1149, D1099, M1425, M1003, P4 67, R43, L222, V400, M1244, A424, F1410, G193, H39, W219, F1018, R1193, K1069, V50, R1498, K1230, S403, S1264, R995, Q238, I1433, P66, L428, D1171, A1107, S1033, I1017, K1259, M986. The inventors have demonstrated that mutations in each of these positions result in closure of the NALCN pore. Without wishing to be bound by theory, it is hypothesized that these amino acid residues may be involved in regulating the opening of the NALCN pore. Therefore, by targeting one or more of these residues, ion flux through NALCN may be reduced. ,

[0080] The reduction in ion flow through NALCN can be temporary (i.e., short-lived) or permanent. In a preferred embodiment, the reduction in ion flow is short-lived. In such an embodiment, cells can be treated with ion channel modulators to temporarily reduce the ion flow through NALCN. This short-lived reduction will allow stem-like cells to be transported to distal organs or tissues that need regeneration or repair. Once stem-like cells are integrated in these organs or tissues and form normal structures, normal ion flow through NALCN can be restored.

[0081] The ion channel modulators of the present invention may not completely block the ion flow through NALCN, but simply reduce the ion flow through NALCN. For example, compared to the ion flow through untreated cells / subjects, the ion channel modulator can reduce the ion flow through NALCN by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0082] Modification of stem-like cells

[0083] The present invention provides modified stem-like cells for organ repair or regeneration. The modified stem-like cells can:

[0084] i) comprises a mutation in Nalcn that reduces ion flux through NALCN; or

[0085] ii) with knockout of Nalcn;

[0086] iii) exhibit reduced expression of Nalcn; or

[0087] iv) have been treated with an ion channel modulator to reduce ion flux through NALCN.

[0088] As will be appreciated by those skilled in the art, the knockout of Nalcn means that the cell does not have the Nalcn gene or the Nalcn gene has been destroyed (e.g., because the gene has been removed or inactivated by genetic engineering). Methods for producing Nalcn knockout are well known to those skilled in the art, including, for example, random mutagenesis and selection, such as using radiation or chemical mutagenesis, methods based on homologous recombination, or new technologies such as CRISPR.

[0089] It is shown herein that the loss of Nalcn can result in upregulation of one or more genes selected from Mmp10, Mmp19, Mmp7, Mmp9, Fn1, Zeb1, Fstl1, Sparc, Sfrp4, Cdh6 and / or Timp3. Therefore, cells with a loss or mutation of Nalcn can be characterized by enrichment of one or more of Mmp10, Mmp19, Mmp7, Mmp9, Fn1, Zeb1, Fstl1, Sparc, Sfrp4, Cdh6 and / or Timp3.

[0090] In embodiments where the modified cell comprises a Nalcn mutation, the mutation reduces the ion flow through NALCN. Therefore, the mutation may be a loss-of-function mutation. This mutation may not completely block the ion flow through NALCN, but only reduce the ion flow through NALCN, and therefore may also be a reduction in functional mutations. For example, compared with the ion flow through unmutated Nalcn, the mutation may reduce the ion flow through NALCN by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. Any suitable method may be used to introduce mutations into Nalcn, including site-directed mutagenesis zinc finger nucleases, transcription activator-like effector nucleases (TALENs) or CRISPR technology. Mutations may be introduced into Nalcn in vitro, in vitro or in vivo. In the case of introducing mutations in vitro or in vitro, cells may be obtained from a subject before the step of introducing the mutation.

[0091] As described above, the NALCN protein comprises multiple domains, and thus, the modified cells may comprise mutations in one or more of these domains to reduce ion flow through NALCN. The mutations may be in one or more of the pore turret domain, the voltage sensing domain, or the linker domain of NALCN. The linker domain may be a linker domain that extends extracellularly or intracellularly. The mutations may be in one or more domains comprising any one of the amino acid sequences listed in SEQ ID NO. 2 to 23. The domains of NALCN and their sequences are listed in Table 1.

[0092] The modified stem-like cells may comprise a mutation in Nalcn that reduces ion flow through NALCN. The one or more mutations within NALCN may be present at a position selected from, but not limited to, L588M, P573, R855, K1213, T71, P225, D1527, D416, C1348, R297, V1386, A1091, V1229, D134, T272, R43, A1157, V1036, M520, R1500, V320, V53, W1085, E1458, N1274, V1542, Y1300, R1174, H1523, F332, Q549, L999, F540, A1421, R1384, H569, A1435, M55, R1495, C245, F110, V510, C970, E454, V273, R1556, S174, S1068, V385, S3 84. A401, S902, R1495, A276, R1540, L517, R295, R382, H876, F300, R164, E257, R995, G1526, D291, V1239, E1552, N1475, M55, L1553, Y1349, E323, A1044, T1281, V1007, L253, L564, F1427, V949, Q279, T539, R159, K452, R 1127, V1490, G555, E62, L1461, L942, R166, P65, D952, I322, F154, K116 3. L305, R152, W1085, R143, A1444, R989, R143, R1193, D1466, M520, V128 5. S52, I51, E1518, E532, L1279, V1329, T57, A1378, S121, K498, R1094, V 120, A88, A401, L1548, G1303, M150, D1277, E432, L1442, P1082, T1165, G 1316, R1273, E128, E906, F1311, R1481, T204, T552, F389, D1527, P908, A 1166, I577, G954, G1013, P65, E1016, N1070, S980, A1217, V1503, T1320, A223, A310, R1127, D1504, D1277, E128, K1491, Q553, V511, F1250, S1374 , D211, T1149, D1099, M1425, M1003, P467, R43, L222, V400, M1244, A424,F1410, G193, H39, W219, F1018, R1193, K1069, V50, R1498, K1230, S403, S1264, R995, Q238, I1433, P66, L428, D1171, A1107, S1033, I1017, K1259, M986. It has been shown that mutations in each of these positions lead to closure of the NALCN pore, i.e., a decrease in the size of the NALCN pore, thereby reducing NALCN activity. It is hypothesized that these amino acid residues may be involved in regulating the opening of the NALCN pore, as mutations in one or more of these positions may lead to a decrease in the pore size, thereby reducing NALCN activity (i.e., a decrease in ion flux through NALCN).

[0093] In the embodiment where Nalcn expression is reduced, this can be achieved by any suitable method, such as RNAi, siRNA, zinc finger nuclease, transcription activator-like effector nuclease (TALENs) or CRISPR technology, and suitable methods for reducing expression using these methods are known to those skilled in the art. The reduction of expression can be temporary (i.e., transient) or permanent. In a preferred embodiment, the reduction of expression is temporary. In these embodiments, cells can be treated with RNAi or siRNA to temporarily reduce the expression of Nalcn. The expression of Nalcn can be modified using the above-mentioned ion channel modifiers. In order to reduce the expression of Nalcn, cells can be exposed to RNAi, siRNA, CRISPR and / or ion channel modulators or contacted with RNAi, siRNA, CRISPR and / or ion channel modulators, and the step of exposing or contacting cells can be performed in vitro, in vitro or in vivo. When cells are exposed or contacted in vitro or in vitro, cells can be obtained from a subject before the step of contacting or exposing the cells.

[0094] In embodiments where stem-like cells have been treated with an ion channel modulator to reduce ion flux through NALCN, the modulator may not completely block ion flux through NALCC, but may simply reduce ion flux through NALCAN. For example, the ion channel modulator may reduce ion flux through NALCN by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% compared to ion flux through untreated cells / subjects.

[0095] Pharmaceutical composition

[0096] In one aspect, the invention relates to a pharmaceutical composition comprising a modified stem-like cell, wherein the modified stem-like cell exhibits reduced ion flux through NALCN.

[0097] In one aspect, the invention relates to a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator reduces ion flux through NALCN.

[0098] Modified stem cell-like cells, ion channel modulators or pharmaceutical compositions can be administered by any suitable route, such as any parenteral or enteral route. For example, any suitable route includes but is not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, administration can be oral, sublingual, buccal, intravenous, intramuscular, subcutaneous, rectal or intranasal. Various oral dosage forms can be used, including solid forms such as tablets, capsules, liquids, granules and bulk powders. Tablets can be compressed, tablet grinding, enteric coated, sugar coated, film coated or multi-compressed, containing suitable binders, lubricants, diluents, disintegrants, colorants, flavoring agents, flow inducers and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.

[0099] The modified stem-like cells, ion channel modulators or pharmaceutical compositions may be in the form of one or more dosage units.

[0100] In embodiments, the pharmaceutical composition of the present invention can also include one or more other activating agents, pharmaceutically acceptable carriers, diluents or adjuvants.Pharmaceutically acceptable carriers, diluents or adjuvants can vary according to the dosage form used.For example, various oral dosage forms can be used, including solid forms such as tablets, capsules, liquids, particles and bulk powders.Tablets can be compressed, tablet grinding, enteric coating, sugar coating, film coating or multiple compressions, containing suitable adhesives, lubricants, diluents, disintegrants, coloring agents, flavoring agents, flow inducing agents and melting agents.Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstructed by non-effervescent particles, and effervescent preparations reconstructed by effervescent particles, containing suitable solvents, preservatives, emulsifiers, suspensions, diluents, sweeteners, melting agents, coloring agents and flavoring agents.Can be in the form of a liquid, such as a solution, emulsion or suspension. Liquid compositions, whether solutions, suspensions or other similar forms, may also include one or more of the following: sterile diluents such as water, saline solution (preferably physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic monoglycerides (monogylceride) or diglycerides (digylceride), polyethylene glycol, glycerol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; and agents for adjusting tonicity, such as sodium chloride or glucose. The modified stem-like cells, ion channel modulators or pharmaceutical compositions may be enclosed in ampoules, disposable syringes or multiple-dose vials made of glass, plastic or other materials.

[0101] The intravenous preparation of modified stem-like cells, ion channel modulators or pharmaceutical compositions can be in the form of sterile injectable aqueous or non-aqueous (such as oily) solutions or suspensions. Sterile injectable preparations can also be sterile injectable solutions or suspensions, dissolved in nontoxic parenteral acceptable diluents or solvents, such as 1,3-butanediol solutions. Acceptable vehicles and solvents that can be used are water, phosphate buffered solution, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils can be used as solvents or suspension media. For this reason, any mild fixed oil can be used, including synthetic monoglycerides (monoglyceride) or diglycerides (diglyceride). In addition, fatty acids such as oleic acid can be used for the preparation of intravenous preparations of the present invention.

[0102] The modified stem cell-like cells, ion channel modulators or pharmaceutical compositions can be prepared using methods well known in the pharmaceutical field. For example, a composition intended to be administered by injection can be prepared by combining with water to form a solution. A surfactant can be added to promote the formation of a uniform solution or suspension.

[0103] Methods of treatment / organ regeneration or repair

[0104] In one aspect, the present invention relates to a method for regenerating or repairing an organ in a subject, comprising administering to the subject a therapeutically effective amount of a modified stem-like cell or a pharmaceutical composition comprising a modified stem-like cell, wherein the modified stem cell exhibits a reduced ion flow through NALCN. The details of the modified stem-like cell are as described above.

[0105] In one aspect, the present invention relates to a method for regenerating or repairing an organ in a subject, comprising administering to the subject a therapeutically effective amount of an ion channel modulator or a pharmaceutical composition, the pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator reduces ion flow through NALCN. The details of the ion channel modulator are as described above.

[0106] The inventors have unexpectedly shown that by inhibiting ion flux through NALCN, either by mutation or knockout of NALCN, or by treatment with NALCN modulators, the shedding of epithelial-derived stem-like cells is increased. These cells mobilize to different tissues and / or organs and form normal structures in these organs. Thus, the inventors have determined that inhibiting ion flux through NALCN can be used to repair or regenerate damaged or diseased organs and / or tissues.

[0107] Those skilled in the art will appreciate that the phrases organ regeneration or repair as used herein are used to refer to the repair of damaged or defective organs or tissues. Repair may not be a complete repair, but rather an improvement. For example, the present invention may result in an improvement in the function of an organ or tissue compared to an untreated individual. For example, the present invention may improve the function of an organ or tissue by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to an untreated individual.

[0108] The present invention can be used to repair or regenerate one or more of the following organs or organ systems: thymus, adrenal glands, thyroid gland, intestines, lungs, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidneys and bladder, skin, hair follicles, pancreas, bones and cartilage. In embodiments, the present invention can be used to repair or regenerate one or more of the following organs or organ systems: intestines, lungs, liver, kidneys, bladder, pancreas. In embodiments, the present invention can be used to repair or regenerate kidneys, liver, pancreas, intestines, lungs or bladder. In embodiments, the present invention can be used to repair or regenerate kidneys. Therefore, the present invention can be used to treat chronic kidney disease, such as glomerulonephritis and / or renal failure. The present invention can also be used to treat pulmonary diseases or liver fibrosis diseases, such as COPD.

[0109] In an embodiment, the modified cells, ion channel modulators or pharmaceutical compositions are used to treat or prevent organ and / or tissue loss and / or damage. Organ and / or tissue loss and / or damage may be caused by disease, injury and / or congenital abnormalities.

[0110] In embodiments, the modified cells are used to treat organ and / or tissue loss and / or damage in one or more of the following organs or organ systems: thymus, adrenal glands, thyroid gland, intestines, lungs, heart, liver, blood vessels, germ cells, nervous system, eye tissue, hair cells, kidneys and bladder, skin, hair follicles, pancreas, bones and cartilage. In embodiments, the present invention can be used to treat organ and / or tissue loss and / or damage in one or more of the following organs or organ systems: intestines, lungs, liver, kidneys, bladder, pancreas. In embodiments, the present invention can be used to treat organ and / or tissue loss and / or damage in kidneys, liver, pancreas, intestines, lungs or bladder. In embodiments, the present invention is used to treat organ and / or tissue loss and / or damage in kidneys. Therefore, the present invention can be used to treat chronic kidney disease, such as glomerulonephritis and / or renal failure. The present invention can also be used to treat pulmonary diseases or liver fibrosis diseases, such as COPD.

[0111] When the modified stem-like cells or ion channel modulators are used to prevent a disease, the modified stem-like cells or ion channel modulators can be administered prophylactically. When the modified stem-like cells or ion channel modulators are used to treat a disease, disease progression monitoring can be used to identify subjects who need further therapy.

[0112] The modified stem-like cells, ion channel modulators or pharmaceutical compositions can be used in combination with further treatments. Further treatments can include other organ repair or regenerative therapies, such as tissue engineering and / or cell therapy. Alternatively or in addition, further treatments can include drug therapy to improve the function of an organ or tissue.

[0113] As used herein, the term "in combination with" is meant to encompass the administration of the modified stem-like cells, ion channel modulators, or pharmaceutical compositions simultaneously, separately, or sequentially with the administration of the further treatment.

[0114] The present invention provides a method for repairing or regenerating an organ in a subject, comprising administering to the subject a therapeutically effective amount of modified stem-like cells.

[0115] In some embodiments, the stem-like cells are autologous. In these embodiments, the stem-like cells can be obtained from a subject to whom the modified stem-like cells are to be administered. Thus, in embodiments, the method comprises:

[0116] i) Obtaining stem-like cells from the subject to be treated.

[0117] In some embodiments, the stem-like cells are allogeneic. In these embodiments, the stem-like cells can be obtained from a suitable subject or donor person. Therefore, in embodiments, the method comprises:

[0118] i) obtaining stem-like cells from a suitable subject or donor.

[0119] Those skilled in the art will understand how to obtain such cells from a subject, for example, by obtaining such cells from a blood sample or tissue sample obtained from a subject. Methods for selecting stem-like cells from such samples include, for example, size exclusion and enrichment methods (magnetic bead sorting or FAC) using defined antibody enrichment methods for cell surface markers of a specific population.

[0120] Then, the stem-like cells can be modified ex vivo so that the stem-like cells exhibit reduced ion flux through NALCN. Therefore, the present invention further comprises:

[0121] i) Modifying a stem-like cell such that the stem-like cell exhibits reduced ion flux through NALCN.

[0122] Methods of modifying stem-like cells to reduce ion flux through NALCN are discussed above. Stem-like cells can be modified by:

[0123] a) contacting the stem-like cells with an ion channel modulator, wherein the ion channel modulator inhibits ion flux through NALCN; and / or

[0124] b) introducing a Nalcn mutation and / or a Nalcn deletion and / or reducing the expression of Nalcn in stem-like cells, wherein the mutation and / or the deletion and / or the reduced expression results in a reduced ion flux through NALCN.

[0125] The method may also include amplifying modified stem-like cells that have been modified to reduce ion flow through NALCN. Those skilled in the art will understand how to amplify these cells. As described herein, the term "amplification" of stem-like cells or any other cell type or "amplification" of stem-like cells or any other cell type describes an increase in the number of cells due to cell division. Culture media that allow stem-like cells to amplify are known to those skilled in the art, and include but are not limited to IPS-Brew, PS-Brew XF, E8, StemFlex, mTeSR1, PluriSTEM, StemMACS, TeSRTM2, Corning NutriStem hPSC XF culture media, Essential 8 culture media (ThermoFisher Scientific), StemFit Basic02 (Ajinomoto Co.Inc), just to name a few examples to illustrate. Temperature may affect whether the conditions are suitable for the amplification of stem-like cells. Therefore, the temperature of the culture medium may be about 30°C to 50°C, about 35°C to 40°C, about 36°C to 38°C or about 37°C. Oxygen levels may affect whether the conditions are suitable for the amplification of stem-like cells. Thus, the oxygen percentage of the culture medium can be varied over a range from normoxic (20% O2) along a gradient of hypoxic conditions (0.5% O2).

[0126] For the cultivation of stem cell-like cells, culture surfaces such as culture plates or culture containers can be coated with extracellular matrix (ECM) to provide support for the attachment and growth of cells. ECM is an extracellular surface matrix, mainly composed of proteins such as collagen, elastin and laminin. ECM is widely used in the cultivation of mammalian cells and is known to those skilled in the art. Non-limiting examples of ECM that can be used to coat solid supports or culture surfaces (such as culture plates) include Matrigel. TM , laminin, polylysine, a combination of polyornithine (PO) / fibronectin (FN) / laminin (lam), fibronectin, etc.

[0127] In an embodiment, the expansion may include culturing the modified stem-like cells in a culture medium. The culture medium includes a mixture of nutrients required for the growth of a certain type of cell. The culture medium is typically prepared by adding supplements to a basal culture medium. Supplements refer to additional ingredients that are not present in the basal culture medium but may be required for cell culture, including but not limited to proteins, lipids, amino acids, vitamins, hormones, cytokines, and growth factors. The culture medium may contain specific growth factors, including, for example, one or more of insulin, growth factors such as EGF, bFGF, N2, B27, wnt conditioned medium, r-spondin, and TGFβ inhibitors. When more than one culture medium is used, the culture medium may be replaced at any time point during the culture process by methods well known to those skilled in the art.

[0128] A therapeutically effective amount of the modified stem-like cells may then be administered to the subject.

[0129] The method of organ regeneration or repair in a subject may include different steps, depending on the stage of organ damage or the disease causing the organ damage. For example, where a subject is determined to have an early but high-risk disease, the subject may initially be treated with a modulator targeting NALCN. The modulator targeting NALCN will allow the mobilization of circulating stem-like cells, preferably epithelial stem-like cells.

[0130] Wherein, in subjects treated with NALCN modulators, close monitoring of disease progression can be used to monitor the subject to determine whether continued treatment is needed. If the disease progresses further or further progresses to a severe stage, further treatment may be required, including treatment with modified stem-like cells. Treatment with modified stem-like cells may include the above method. In an embodiment, treatment with modified stem-like cells may include the following steps:

[0131] i) obtaining stem-like cells from said subject, a suitable subject or a donor,

[0132] ii) modifying a stem-like cell such that the stem-like cell exhibits reduced ion flux through NALCN:

[0133] a. contacting the stem-like cells with an ion channel modulator, wherein the ion channel modulator inhibits ion flow through NALCN; and / or

[0134] b. Introducing a Nalcn mutation and / or a Nalcn deletion and / or reducing the expression of Nalcn in stem-like cells, wherein the mutation and / or the deletion and / or the reduced expression results in a reduced ion flux through NALCN.

[0135] iii) introducing the modified cells into said subject.

[0136] When a subject is determined to have a severe disease, e.g., where significant organ damage has occurred, the subject can be treated with a modulator targeting NALCN in combination with modified stem-like cells that have been prepared according to the methods described herein.

[0137] The present invention also relates to the use of the modified stem cell-like cells or ion channel regulators in preparing drugs for organ regeneration or repair.

[0138] The subject to be treated can be any subject in need of repair or regeneration of an organ or tissue. In an embodiment, the subject is a mammal, preferably a human.

[0139] As used herein, the terms "treat," "treatment," or "treating" refer to the administration of a compound to a subject for preventive and / or therapeutic purposes. As explained herein, the modified cells of the present invention can be used to repair or regenerate an organ or tissue. Thus, in one embodiment, the modified cells can be used to prevent a reduction or further reduction in organ or tissue function and are administered prophylactically. Thus, the term "prevention" refers to the prevention of organ degeneration, and in particular, the modified cells can be used to prevent organ degeneration (or further organ degeneration). For example, the modified cells can be used to treat subjects who are determined to have a high risk of organ damage or degeneration or further damage or degeneration. The term "prevention" may refer to a reduced risk of organ degeneration or damage. The term "prevention or preventive treatment" refers to the treatment of a subject who has not yet shown symptoms of a disease or condition, but is susceptible to or at risk of a particular disease or condition, so that the treatment reduces the likelihood of the patient developing the disease or condition. The term "therapeutic treatment" refers to the administration of treatment to a subject who already has a disease or condition.

[0140] As used herein, the terms "effective amount" and "therapeutically effective amount" refer to an amount of an active therapeutic agent sufficient to produce the desired therapeutic response without excessive adverse side effects (such as toxicity, irritation or allergic reactions). The specific "effective amount" will vary depending on factors such as the specific disease being treated, the patient's physical condition, the type of subject being treated, the duration of treatment, the nature of concomitant treatment (if any), the specific formulation used, and the structure of the compound or its derivatives.

[0141] Other methods

[0142] The present invention also provides a method for increasing the level of solid tissue cell shedding of stem-like cells, the method comprising introducing Nalcn mutation and / or deletion of Nalcn and / or reducing the expression of Nalcn in the stem-like cells, wherein the mutation and / or deletion and / or reduced expression leads to reduced ion flow through NALCN.

[0143] Mutation and deletion of Nalcn and reduction of Nalcn expression are described above.

[0144] In one aspect, the invention also relates to a method of increasing the level of solid tissue cell shedding of stem-like cells, the method comprising contacting the cells with an ion channel modulator, wherein the ion channel modulator reduces ion flux through NALCN.

[0145] The inventors have shown that loss of NALCN function significantly increases the level of circulating stem-like cells. Therefore, by reducing ion flux through NALCN, the level of circulating stem-like cells can be increased.

[0146] In embodiments, the cells are contacted in vitro, ex vivo, or in vivo.

[0147] Ion channel modulators are as described above.

[0148] The solid tissue may be epithelial tissue.

[0149] The present invention also provides a method for preparing modified stem-like cells, the method comprising:

[0150] i) contacting the stem-like cells with an ion channel modulator, wherein the ion channel modulator inhibits ion flux through NALCN; and / or

[0151] ii) introducing a Nalcn mutation and / or a Nalcn deletion and / or reducing the expression of Nalcn in stem-like cells, wherein the mutation and / or the deletion and / or the reduced expression results in a reduced ion flux through NALCN.

[0152] Ion channel modulators and stem-like cells are as described above.

[0153] The method may also include expanding the modified stem-like cells that have been modified to reduce ion flow through NALCN. Those skilled in the art will understand how to expand these cells. Expansion of cells, including appropriate growth medium and additional factors, is as described above.

[0154] In embodiments, the method may include depleting terminally differentiated cells from the ex vivo culture. Suitable methods for depleting terminally differentiated cells include, for example, antibody enrichment methods.

[0155] In embodiments, the stem-like cells to be modified can be obtained from a subject to be treated with the modified stem-like cells. Methods for obtaining such cells are described above.

[0156] Reagent test kit

[0157] The present invention also provides a kit comprising a modified stem-like cell and optionally instructions for use, wherein the modified stem-like cell exhibits reduced ion flux through NALCN.

[0158] The kit may also include various other components. In an embodiment, the kit includes a NALCN modulator, which may be any NALCN modulator described herein. In an embodiment, the kit further includes a component for culturing the modified stem-like cells. The cell culture component may be any culture medium, buffer, excipient, container, etc. described herein.

[0159] The invention also provides kits comprising a device for obtaining a cell sample from a subject and a NALCN modulator and / or a component for modifying cells to reduce ion flux through NALCN, as described herein.

[0160] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those skilled in the art. Although the above disclosure provides a general description of the subject matter contained within the scope of the present disclosure, including the preparation and use of the methods of the present disclosure and the best mode thereof, the following examples are provided to further enable those skilled in the art to practice the present disclosure. However, it will be understood by those skilled in the art that the details of these embodiments should not be construed as limitations on the present invention, and the scope of the present invention should be understood from the claims attached to the present disclosure and their equivalents. In view of the present disclosure, various other aspects and embodiments of the present disclosure will be apparent to those skilled in the art.

[0161] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and patent publications.

[0162] As used herein, "and / or" should be considered as a specific disclosure of each of the two specified features or components, including or excluding the other. For example, "A and / or B" should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually listed herein. Unless the context indicates otherwise, the descriptions and definitions of the above features are not limited to any specific aspect or embodiment of the invention, and apply equally to all aspects and embodiments described.

[0163] As used herein, the term "comprising" or "comprises" means including the specified components but does not exclude the presence of other components. The term "consisting essentially of" or "consists essentially of" means including the specified components but excluding other components, except for materials present as impurities, materials that are inevitably present due to the process used to provide the components, etc.

[0164] The term "consisting of" or "consists of" means that the specified components are included but other components are excluded.

[0165] Depending on the context, where appropriate, the use of the terms "comprise" or "including" may also be considered to include the meaning of "consists essentially of" or "consisting essentially of", and may also be understood to include the meaning of "consists of" or "consisting of".

[0166] The optional features described herein may be used alone or in combination with each other where appropriate, particularly in the combinations listed in the appended claims. As listed herein, the optional features of each aspect or exemplary embodiment of the invention are also applicable to all other aspects or exemplary embodiments of the invention where appropriate. In other words, the skilled person reading this specification should consider that the optional features of each aspect or exemplary embodiment of the invention are interchangeable and combinable between different aspects and exemplary embodiments.

[0167] The present invention is further illustrated in the following non-limiting examples.

[0168] method

[0169] Culture of gastric stem cells

[0170] Gastric glands were isolated by perfusing mice with 30 mM EDTA / PBS, gastrectomizing, and scraping the pyloric mucosa into 10 mM EDTA / PBS at 4°C. The dissociated, filtered and resuspended cells were placed in Matrigel (354230, BD Bioscience) and culture medium: Advanced DMEM / F12 406 (31330038, ThermoFisher), B27 (12587010, ThermoFisher), N2 (A1370701, ThermoFisher), N-407 acetylcysteine ​​(A9165, Sigma-Aldrich) and 10 nM gastrin (G9145, Sigma-Adrich), containing growth factors (50 ng / ml EGF [Peprotech], 1 mg / ml R-spondin1 [120-38, PeproTech.], 100 ng / ml Noggin [250-38, Peprotech], 100 ng / ml FGF10 [100-26, Preprotech] and Wnt3A conditioned medium). Gastric spheroids were digested and dissociated into single cells by dispase (D4818, Sigma-Aldrich). Accutase, Life Technologies) was used for passage. Gadolinium (439770, Sigma-Aldrich) was diluted in culture medium and overlaid on Matrigel-embedded cells. CreERT2 / LacZ ;Rosa26 ZSG ;Nalcn Flx / Flx Isolated gastric glands from mice were treated with 0.25 μM 4-hydroxytamoxifen in vitro to induce the loss of Nalcn. ZsGreen+ gastric organoids were harvested by FAC 72 hours after induction and subjected to RNA isolation and transcriptome analysis.

[0171] Generation of the NalcnFlx allele

[0172] Mice were derived from targeted ES cells (UCDAVIS KOMP Repository Knockout Mouse Project clone EPD0383_5_C01). ES cells were screened using the KOMP PCR strategy of Nalcntm1a(KOMP)Wstsi. ES cells were implanted into recipient C57 / Bl6 mice according to an IACUC-SJ approved protocol. Standard polymerase chain reaction and primers were used to detect wild-type Nalcn and Nalcn FlxAlleles (UCDAVIS KOMP Repository Knockout Mouse Project clone EPD0383_5_C01): 5'-ATTGTCCGTGAGATTGCTCATCACC-3' (SEQ ID NO. 24) and 5'-GCACCAGCTATATGTCCCTCTCACG-3' (SEQ ID NO. 25) for Nalcn wild type or Flx It is 5"-GGAAAATGACCACTTCCTAGCAGAAGC-3" (SEQ ID NO. 26).

[0173] Nalcn RNA expression was quantified by quantitative reverse transcriptase PCR and Biorad CFX96Touch real-time PCR detection system with the following primers: Nalcn sense 5"-GCCCTCAGCCCCCAAAC-3" (SEQ ID NO.27) (spanning exon 43 / 44), Nalcn antisense 5"-GGAAGCTGTGTCTGGCATGG-3" (SEQ ID NO.28) (exon 44), Gapdh sense 5"-AGGTCGGTGTGAACGGATTTG-3" (SEQ ID NO.29) and Gapdh antisense 5"-TGTAGACCATGTAGTTGAGGTCA-3" (SEQ ID NO.30).

[0174] Cycle ZsGreen + Cell harvest and injection

[0175] At necropsy, peripheral blood (500 μl-1 ml) of mice was collected into 10 μl of 0.5 M EDTA, diluted in PBS, and evaluated for ZSgreen expression (525 / 50 nm [FITC] vs 614 / 50 nm [PI]) by MACSQuant analyzer (Miltenyi Biotech Inc.). Cells for single-cell RNA sequencing and tail vein injection were sorted using a BD FACSAria II cell sorter (BD Biosciences) under 525 / 50 nm (FITC) and 614 / 50 nm (PI) excitation. Peripheral blood from non-tamoxifen-induced mice was used as a negative control to set gating parameters. 25,000 ZSgreen cells were added to the 4% 1% ZSgreen cell line. +Cells were sorted and injected into recipient NOD SCIDγ mice (Charles River) and allowed to age. For serial dilution assessment of tCZC metastatic initiation, tCZC were isolated from tumor-bearing donors by FACS based on ZSGreen expression and placed in culture. Culture medium: Advanced DMEM / F12 (31330038, ThermoFisher), 2 mM L-glutamine (25030024, ThermoFisher), B27 (12587010, ThermoFisher), N2 (A1370701, ThermoFisher), containing growth factors (50 ng / ml EGF [Petrotech], 100 ng / ml bFGF [10-18c, Peprotech], and 1% FBS (10500064, ThermoFisher). Cells were grown at 37°C in 5% CO2. Recipient NOD SCIDγ mice (Charles River) were injected via the tail vein with 10, 100, 1000, or 10,000 tCZC cells and allowed to age. Full necropsy and tissue harvest were performed as described above.

[0176] Induction of renal injury (acute model) and mobilization of ZSG+ntCZCs in vivo

[0177] Adult Villin1-CreERT2; Rosa26-ZSGreen; Nalcn+ / +; Villin1-CreERT2; Rosa26-ZSGreen; NalcnFlx / +; Villin1-CreERT2; Rosa26-ZSGreen; NalcnFlx / Flx male and female mice were given a total of 8 mg of tamoxifen over two days to induce recombination of the conditional allele in the intestinal epithelium. The animals were aged for 2 months and then received a single intraperitoneal injection of folic acid (250 mg / kg) to induce renal injury. The animals were monitored daily for weight loss for 7 days. At the endpoint, the kidneys were collected, fixed with 1% paraformaldehyde for 24 hours, then fixed with 10% sucrose solution, and finally embedded in OCT for tissue sectioning and histological assessment of fibrosis and the presence of ZSG+ cells in the kidney.

[0178] Histology

[0179] Hematoxylin and eosin staining was performed using standard procedures (7221, 7111, Thermo 511 Fisher Scientific). Immunohistochemistry was performed using standard procedures and primary antibodies: Ki67 (IHC-00375, Bethyl, 1:1000), ZSGreen (632474, Clontech, 1:2000), Pancytokeratin (AE1 / AE3) (901-011-091620, BioCare Medical, 1:100), CK5 (ab52635, Abcam, 1:100), Vimentin (5741S, Cell Signalling Technology, 1:200), Cleaved Caspase 3 (9664, Cell Signaling Technology, 1:200), CD31 (77699, Cell Signaling Technology, 1:100), α-smooth muscle actin (ab5694, Abcam 1:500), CD45 (ab25386, Abcam, 5 μg / ml). Secondary antibodies were anti-rabbit poly-HRP-IgG (included in the kit) or rabbit anti-rat (A110-322A, Bethyl Laboratories, 1:250). Digital images of whole tissue sections were captured using a Leica Aperio AT2 digital scanner (40×, resolution 0.25 μM / pixel), viewed using a Leica Aperio Image Scope v12.3.2.8013, and quantified by HALO (Indica Labs) image analysis.

[0180] For immunofluorescence detection, tissue sections were incubated with primary antibodies: rhodamine-conjugated DBA (RL-1032, Vector Laboratories, 1:100), rhodamine-conjugated UEAI (RL-1062, Vector Laboratories, 1:100), ZSGreen (TA180002, Origene, 1:1000), CD31 (102520, BioLegend, 1:100), CK7 (ab181598, Abcam, 1:200), CK20 (ab97511, Abcam, 1:200), E-cadherin (147308, BioLegend, 1:100), PDGFRb (136005, BioLegend, 1:100), N-cadherin (13116, Cell Signaling, 1:100). Technology, 1:100), Icam1 (ab179707, Abcam, 1:100), Cdx2 (ab76541, Abcam, 1:100), Ttf1 (ab76013, Abcam, 1:100), Krt80 (1683 5-1-AP, ProteinTech, 1:100), Hba-a1 (ab92492, Abcam, 1:100), Lgals3 (ab209344, Abcam, 1:200), CD45 (ab10558, Abcam, 1:200). Secondary antibodies include Alexa 488, 594, and 647 (A-11055, A-21207, A-31571, ThermoFisher, 1:500). Sections were counterstained (DAPI 4083; Cell Signaling, 1:10,000), and images were captured at 40× magnification using a Zeiss ImagerM2 and Apotome microscope or a Zeiss Axioscan.Z1 (Zeiss) and processed using ZEN2.3 (Zeiss) software.

[0181] Using Advanced Cell Diagnostics(ACD) 2.5LS Kit RED (ACD, 322150) and RNAscope 2.5LS Mm Nalcn (ACD, 415168) were used to detect Nalcn RNA expression in FFPE sections. Probe hybridization and signal amplification were performed according to the manufacturer's instructions. Fast Red detection of mouse Nalcn was performed on Bond Rx using the Bond Polymer RefineRed Detection Kit (Leica Biosystems, DS9390) according to the manufacturer's protocol. Whole tissue sections were imaged on Aperio AT2 (Leica Biosystems) and immunohistochemical analysis was performed using HALO (Indica Labs) imaging analysis software, and β-galactosidase staining was performed exactly as described.

[0182] Histological examination and classification of primary and metastatic tumors were performed by expert pathologists (Peter Vogel and Dr. Betania Mahler-Araujo) who were blinded to the genotype and clinical history of the mice. + The number of cell clusters or metastases was counted.

[0183] Whole tissue imaging

[0184] Kidneys were bled and perfused with PBS at 37°C and 80 rpm, fixed with 4% PFA, washed with PBS and immersed in reagent 1a [150 g ultrapure water, 20 g Triton X-100 (10254583, Fisher Scientific), 10 g 100% N,N,N",N"-tetrakis(2-hydroxypropyl)ethylenediamine solution (122262, Sigma), 20 g urea (140750010, ACROS organics), 1 ml 5M NaCl] (containing 10 μM DAPI (4083; Cell Signaling Technology). The solution was changed every 2 days until the tissue was clean. The clean tissue was washed and immersed in 50% PBS / 50% reagent 2 [15 g ultrapure water, 50 g sucrose (220900010, ACROS organics), 25 g urea (140750010, ACROS Organics) and 10g 2,2,2-nitrilotriethanol (90279, Sigma)] for 6 hours (room temperature, gently shaking), and then immersed in 100% reagent 2 (10ml) for 1 day (room temperature). The tissue was mounted on a TCS SP5 confocal laser scanning microscope (Leica) and scanned for endogenous expression of Dapi and ZSGreen with a 10x objective. Images were processed using Imaris x64v9.3.0 software (Oxford Instruments).

[0185] Serial two-photon tomography (STPT) imaging was performed on a TissueCyte 1000 instrument (TissueVision, MA USA), and a series of mosaic 2D images were taken of the tissue, which was then physically sectioned using a vibratome and subjected to a subsequent round of imaging. This continued in an automated fashion, generating 15 μm STPT sections that could be mounted on standard microscope slides and imaged by Axioscan fluorescence scanning (Zeiss) for section identification and realignment. Fiducial agarose marker beads labeled with GFP were distributed throughout the embedding medium to help realign the sample for subsequent use.

[0186] Single-cell RNA sequencing

[0187] The animals were perfused with PBS and then with 100 U / ml collagenase type IV in HBSS with a Ca content of 3 mM CaCl. 2+ and Mg 2+(Life Technologies) medium. Whole organs were dissected, dissociated and placed in 2 ml of appropriate dissociation buffer: lungs and stomach were dissociated with 200 U / ml collagenase type IV (Sigma) and 100 μg / ml DNAseI (Roche) in HBSS with 3 mM CaCl2. 2+ and Mg 2+ (Life Technologies) medium; liver was dissociated with collagenase type I (100 U / ml), dispase (2.4 U / ml), and DNAseI (100 μg / ml) in HBSS with 3 mM CaCl2 2+ and Mg 2+ (Life Technologies) medium; kidneys were dissociated with papain (20 U / ml) and DNAseI (100 mg / ml) in DMEM high glucose, 2 mM L-glutamine (Life Technologies), 1x Pen-605Strep, and 10% fetal bovine serum; uteri and epididymis were dissociated with collagenase type I (100 U / ml) and DNAseI (100 mg / ml) in HBSS with a Ca content of 3 mM CaCl2 2+ and Mg 2+ (Life Technologies) medium. The cell suspension was filtered, washed with HBSS without calcium and magnesium, and centrifuged at 300 x g for 5 min at 4°C.

[0188] Prior to sequencing, single cell suspensions of solid tissues were multiplexed and labeled with CellHashing conjugates: anti-mouse hashtags (0301-0315, BioLegend). All nucleated cells and ZSG isolated from peripheral blood were +Cells were not multiplexed but were placed into the 10x Genomics pipeline. Single cell RNA-seq libraries were prepared using the Chromium Single Cell 3′ Library & Gel Bead Kit v3, Chromium Chip B Kit, and Chromium Single Cell 3′ Kit v3 User Guide (Manual Section CG000183A; 10X Genomics). The cell suspension was loaded onto the Chromium instrument, expecting to collect the gel bead emulsion containing single cells. Subsequently, the RNA of the barcoded cells of each sample was reverse transcribed in a C1000Touch Thermal cycler (Bio-Rad), and all subsequent steps to generate single cell libraries were performed according to the manufacturer's protocol without any modifications (12 cycles for cDNA amplification for most samples and 16 cycles for samples with very low cell concentrations). The quality and quantity of cDNA were measured with an Agilent TapeStation 4200 (High Sensitivity 5000ScreenTape), and 25% of the material was then used for the preparation of gene expression libraries. The library size was estimated using an Agilent TapeStation 4200 (High Sensitivity D1000 Screen Tape) and a Qubit 4.0 Fluorometer (ThermoFisher Qubit TM Library quality was confirmed using the dsDNA HS Assay Kit to assess dsDNA quantity. Each sample was normalized and pooled at equimolar concentrations. To confirm concentration, the pool was subjected to qPCR on a QuantStudio6Flex using the KAPA Library Quantification Kit before sequencing. The pool was sequenced on an Illumina NovaSeq6000 sequencer with the following parameters: 28 bp, read 1; 8 bp, i7 index; 91 bp, read 2.

[0189] Raw RNA reads were processed with cellranger using mm10 from 10X as a reference genome to create a filtered gene expression matrix. Cell barcodes detected by cellranger were used as input to CITESeq for hashtagged sequence data (solid organs), generating a count matrix with cell barcodes and hashtag oligonucleotide sequences for each cell. The HTODemux function from Seurat was then used to identify clusters and classify cells based on their barcodes, including negative cells and doublet cells. Quality control metrics were generated using Scater, and single cell objects were then converted to Seurat objects, merged, and the analysis was run using the standard Seurat pipeline.

[0190] Single cell sequencing profiles of 500 cells from Illumina 10X of human CTCs (GSE75367; GSE74639; GSE60407; GSE67980; GSE114704; GSE144494) and human PBMC raw counts were merged in python (v3.7.3) using the pandas library. Only common genes between datasets were analyzed. Seurat objects were created from PBMCs and CTCs. After this step, the data were analyzed using the standard Seurat pipeline.

[0191] To directly compare human CTCs and mouse tCZCs, 15,328 orthologs were identified and profiles processed by the standard Seurat workflow, which includes per-cell normalization of expression counts for each gene. Enrichment for hemoglobin gene expression was performed in UCell, and enrichment scores were generated using the Mann-Whitney U statistic.

[0192] Example 1: NALCN and circulating non-cancer cells

[0193] The inventors have shown that loss of Nalcn in cells is associated with shedding of circulating tumor cells (CTCs) into the peripheral blood and metastasis; however, it has proven challenging to separate this process from the complex cascade of tumorigenesis. Flx / Flx Deletion of Nalcn in gastric stem cells rapidly upregulated genes associated with invasion (such as Mmp7, Mmp9, Mmp10 and Mmp19) and genes associated with gastric epithelial-mesenchymal transition (EMT) (such as Zeb1, Fstl1, Sparc, Sfrp4, Cdh6 and Timp3) (Table 2), indicating that NALCN may regulate cell shedding from solid tissues independently of transformation. To test this, the inventors in Prom1 cells that lack oncogenic alleles and have never developed tumors CreERT2 / LacZ ;Rosa26 ZSG ;Nalcn + / + (P1 R Nalcn + / + n=87)、P1 R Nalcn + / Flx (n=50) and P1 R NalcnF lx / Flx Nucleated ZSG autofluorescent circulating cells (CZCs) were identified in the peripheral blood of mice (n=37). Notably, the absence of Nalcn increased the number of CZCs in these mice to levels similar to those observed in tumor-bearing animals ( Figure 1 a). Similarly, blocking NALCN with gadolinium chloride (GdCl3) also significantly increased Prom1 CreERT2 / LacZ;Rosa26 ZSG ;Nalcn + / + (P1RNalcn + / + CZCs in mice (n=10) Figure 1 a). Single-cell RNA sequencing (SCS) profiles of CZCs isolated from non-tumor-bearing (ntCZCs) mice co-clustered with CZCs from tumor-bearing animals (tCZC; Figure 1 b). The vast majority of tCZCs and ntCZCs SCSs do not cluster with the SCS profiles of primary IACs, GACs, or normal tissues, but cluster with the SCS profiles of metastases ( Figure 1 b). The SCS profiles of both tCZCs and ntCZCs matched that of huCTCs and expressed genes associated with epithelial cells, stem cells, and progenitor cells similar to human CTCs (Table 3); although tCZCs were relatively more enriched in metastasis- and invasion-related gene sets ( Figure 2 a; Table 4). Co-immunofluorescence of blood smears confirmed that both ntCZCs and tCZCs shared markers of huCTCs, including Hba-a1, Krt80, and Lgals3 ( Figure 1 c). In addition, transcriptome analysis showed enrichment of various epithelial cell markers, stem cell markers, and progenitor cell markers (Table 3).

[0194] Table 2: Genes upregulated in Nalcn-deficient gastric stem cells

[0195]

[0196]

[0197] Table 3: Upregulated genes associated with CZCs

[0198] Gene Mean log fold change Adjusted p-value L1cam 0.895770782 4.76E-08 Krt80 0.816936447 8.12E-158 Krt18 0.274491431 1.10E-261 Krt222 0.379569529 0 Krt8 0.436565392 1.52E-251 Cdh1 1.161112119 8.78E-130 Epcam 0.400290103 1.93E-167 Cacybp 0.100023733 0.025964511 Ceacam1 0.95889773 1.79E-220 Zeb2 0.528985849 1.09E-93 Tgfb1 0.522272019 7.91E-82 Tgfbr1 0.905995223 2.37E-211 CD36 1.092748198 1.73E-280 Cd44 0.923327024 6.82E-08 L1cam 0.895770782 4.76E-08 Fn1 2.511520987 6.64E-128 Lgals3 1.131734887 1.83E-219 Hba-a1 4.014739639 2.53E-275 Hba-a2 3.96942072 0 Hbb-bs 3.976006038 7.47E-131 Hbb-bt 3.930386043 9.22E-211

[0199] Table 4: Enrichment of tCZCs / ntCZCs in metastasis- and invasion-related gene sets

[0200]

[0201]

[0202]

[0203] To understand the fate of ntCZCs, the inventors investigated the fate of ntCZCs in aged V1 R and Pdx1 R Nalcn + / + 、Nalcn + / Flx and / or Nalcn Flx / FlxFinding ZSG in the lungs and kidneys of mice + Notably, in Nalcn-deficient animals, ZSG was readily detected in these organs. + Cell clusters, but in Nalcn + / + mice, or were absent or detected at significantly lower levels; suggesting that ntCZCs traffic to and engraft in distal organs ( Figure 1 df; Figure 2 b, c). To test this more directly, the inventors isolated R Nalcn Flx / Flx Individual aliquots of 25,000 ntCZCs from mice were injected into the tail veins of six immunocompromised mice. After an average of 100 days, all recipient mice were clinically well, but their lungs, livers, kidneys, and peritoneum contained large amounts of ZSG. + / Cdh1 + / Icam1 + Donor cell clusters with frequencies similar to those of metastatic tumors formed by tail vein injection of tCZCs ( Figure 1 fh; Figure 2 c). Transported ntCZCs form apparently normal structures in target organs, the most extreme examples being the glomerulus and tubules ( Figure 1 fi). Thus, NALCN regulates cell shedding from solid tissues independently of cancer.

[0204] discuss

[0205] The inventors' data show that NALCN is involved in the regulation of stem cell-like epithelial cell shedding. Within 72 hours after the loss of Nalcn from normal gastric stem cells, the inventors observed upregulation of genes associated with EMT and invasion (Table 2). The inventors also observed that the use of agents to block NALCN resulted in an increase in the level of stem cell-like cell shedding, which was comparable to the level of non-tumor-bearing mice with non-functional NALCN. The movement of epithelial cells to different tissues such as the lungs, liver, kidneys, and peritoneum and the formation of normal structures in target organs demonstrate the effectiveness of such modified cells in repairing or regenerating organs or tissues.

[0206] Embodiment 2:

[0207] The inventors conducted two sets of experiments to characterize the potential of non-tumor circulating ZS green-labeled cells (ntCZCs) to traffic to the kidney and engraft, potentially enabling kidney repair.

[0208] First, from Prom-1; Nalcn Flx / Flx Donor ntCZCs were isolated from mice and injected into immunocompromised mice ( Figure 1gi). We isolated ZSG from the kidneys of these mice. + We identified ntCZC cells from 12 samples of the human ntCZC gene and analyzed them by single-cell RNA sequencing (scRNAseq) analysis. We compared these transcriptomes with those of circulating ntCZCs and kidney cells taken from immunocompromised mice ( Figure 3 a). The transcriptome of transported ntCZCs implanted in the kidney is distinct from both ntCZC donor cells and renal cells. Transported ntCZCs express markers of mature renal cells, including markers of tubules and glomeruli ( Figure 3 be).

[0209] To test whether this process operates in a whole animal (non-donor) system of renal injury, we utilized an established model of acute kidney injury in which high-dose folate crystallizes in the kidney, leading to inflammation and fibrosis. + / + (n=2), Villin-1; Nalcn + / Flx (n=3) and Villin-1; Nalcn Flx / Flx These experiments were performed in mice (n=2) Figure 4 a). In this model, Cre recombination occurs in the intestinal epithelium and is reported using the lineage reporter gene ZSG. Recombination was induced in the intestinal epithelium 60 days before renal injury. Five days before folate exposure, we confirmed shedding and circulation of ntCZCs in the peripheral blood of all animals ( Figure 4 b) As expected, folic acid administration resulted in rapid weight loss: Villin-1; Nalcn + / + Mice reached the endpoint within three days, while Villin-1; Nalcn + / Flx and Villin-1; Nalcn Flx / Flx Mice reached the endpoint within six days ( Figure 4 c). Histological evaluation of the animals' kidneys at endpoint confirmed the presence of inflammation and fibrosis (PDGFRb staining), as well as the presence of ntCZCs trafficking to sites of renal injury ( Figure 4 dg). We were able to obtain + / Flx and Villin-1; NalcnF lx / Flx ZSG+ cells were further isolated from mice ( Figure 4 h). These data suggest that ntCZCs can migrate to and incorporate into the kidney to adopt the transcriptome of mature target tissues and contribute to renal function, thereby repairing kidney injury.

Claims

1. A modified stem-like cell for use in organ regeneration or repair, wherein the modified stem-like cell exhibits reduced ion flow through a sodium leak channel (NALCN).

2. An ion channel modulator for use in organ regeneration or repair, wherein the ion channel modulator reduces ion flow through a sodium leak channel (NALCN).

3. The ion channel modulator used according to claim 2, wherein the ion channel modulator targets NALCN and / or one of the proteins associated with NALCN, wherein the protein associated with NALCN is selected from: GPCR (M3 muscarinic receptor M3R, TACR1, CaSR), UNC80, UNC79, FAM155A (NLF1A), Fam155B, SLO2.

1.

4. The ion channel modulator for use according to claim 2 or 3, wherein the ion channel modulator targets the pore turret domain, voltage sensing domain or linker domain of NALCN.

5. A modified stem cell-like cell or ion channel modulator for use according to any one of the preceding claims, wherein the organ to be regenerated is selected from the group consisting of: thymus, adrenal glands, thyroid gland, intestines, lungs, heart, liver, blood vessels, reproductive cells, nervous system, eye tissue, hair cells, kidneys and bladder, skin, hair follicles, pancreas, bones and cartilage.

6. The modified stem-like cell or ion channel modulator for use according to claim 5, for treating chronic kidney diseases such as glomerulonephritis and / or renal failure, lung diseases such as COPD, or liver fibrosis.

7. The modified stem-like cell or ion channel modulator for use according to any one of the preceding claims, wherein the reduced ion flux through NALCN is transient.

8. A pharmaceutical composition comprising the modified stem-like cells of claim 1 or 7 or the ion channel modulator of any one of claims 2 to 4 or 7.

9. A method for repairing or regenerating an organ of a subject, comprising administering to the subject a therapeutically effective amount of the modified stem-like cells of claim 1 or 7, the ion channel modulator of claim 2 to 4 or 7, or the composition of claim 8.

10. The method of claim 9, wherein the method further comprises obtaining stem-like cells from the subject to be treated.

11. The method of claim 10, wherein the method further comprises modifying the stem-like cells such that the stem-like cells exhibit reduced ion flux through NALCN.

12. The method of claim 11, wherein the stem-like cells are modified by: contacting the stem-like cells with an ion channel modulator, wherein the ion channel modulator inhibits ion flux through NALCN; and / or Introducing a Nalcn mutation and / or a Nalcn deletion and / or reducing the expression of Nalcn in stem-like cells, wherein the mutation and / or deletion and / or reduced expression results in reduced ion flux through NALCN.

13. The modified stem-like cells for use according to claim 1 or 7, the pharmaceutical composition according to claim 8 or the method according to claims 9 to 12, wherein the modified stem-like cells are epithelial stem cells.

14. The modified stem-like cell for use according to claim 1 or 7, the pharmaceutical composition according to claim 8 or the method according to claims 9 to 12, wherein the modified stem-like cell: i) comprises a mutation in Nalcn that reduces ion flux through NALCN; or ii) with knockout of Nalcn; iii) exhibit reduced expression of Nalcn; or iv) have been treated with an ion channel modulator to reduce ion flux through NALCN.

15. The modified stem-like cell for use according to claim 14, wherein the reduced ion flux through NALCN is transient.

16. A method for preparing a modified stem-like cell, the method comprising: i) contacting the stem-like cells with an ion channel modulator, wherein the ion channel modulator inhibits ion flux through NALCN; or ii) introducing a Nalcn mutation and / or a deletion of Nalcn and / or reducing the expression of Nalcn in stem-like cells, wherein the mutation and / or the deletion and / or the reduced expression results in a reduced ion flux through NALCN. The method of claim 16 , wherein the stem-like cells are epithelial stem cells.

18. The method of claim 16 or 17, wherein the contacting is performed in vitro or ex vivo.

19. The method of any one of claims 16 to 18, wherein the method further comprises expanding the modified stem-like cells.

20. A method for increasing the level of solid tissue cell shedding of stem-like cells, comprising introducing a Nalcn mutation and / or deleting Nalcn and / or reducing the expression of Nalcn in stem-like cells, wherein the mutation and / or deleting and / or reduced expression results in a reduced ion flow through NALCN.

21. A method for increasing the level of solid tissue cell shedding of stem-like cells, comprising contacting the cells with an ion channel modulator, wherein the ion channel modulator reduces ion flux through NALCN.

22. The method of claim 21, wherein the cells are contacted in vitro, ex vivo or in vivo.

23. The method of claim 20 or 21, wherein the solid tissue is epithelial tissue.

24. A kit comprising modified stem-like cells and optionally instructions for use, wherein the modified stem-like cells exhibit reduced ion flux through NALCN.

25. The kit of claim 24, wherein the kit further comprises a NALCN modulator.

26. A kit comprising a device for obtaining a cell sample from a subject and components for modifying the cells to reduce ion flux through NALCN.

27. The kit of any one of claims 24 or 25, further comprising components for culturing the modified stem-like cells.

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