Inhibiting ZHX2 to improve outcomes in all systemic diseases

Abstract

The present invention provides a novel approach to the treatment of various disease conditions that cause altered cytokine release or cytokine storm. These diseases include viral infections, immune diseases and other non-immune diseases. In particular, the present invention provides methods of targeting ZHX2, including methods of inhibiting, blocking or depleting ZHX2, to treat various disease conditions.

Description

[Technical field]

[0001] Government Subsidy Statement This invention was made with government support under several grants (1R01DK109713, 1R01DK111102, 1R01DK129522, 1R01DK128203) awarded by the National Institutes of Health. The Government has certain rights.

[0002] The general field of the present disclosure is novel approaches to the treatment of various disease states that result in altered cytokine release or cytokine storm, including viral and non-viral infections, immune and other non-immune diseases. [Background technology]

[0003] Zinc finger and homeobox (ZHX) family transcription factors (ZHX1, ZHX2, and ZHX3) are expressed in multiple organs in the body and regulate a variety of structurally and functionally important genes. See Mace et al., "The Zinc Fingers and Homeoboxes 2 protein ZHX2 and its interacting proteins regulate upstream pathways in podocyte diseases," (2020) Kidney Int 97(4):753-764; Kawata H et al., "The mouse zinc -fingers and homeoboxes (ZHX) family; ZHX2 forms a heterodimer with ZHX3," (2003) Gene. 323:133-140. In the liver, ZHX2 is the major transcriptional repressor of α-fetoprotein expression in adult mice. BALB / cJ mice, unlike the 25 other mouse strains tested, harbor a murine endogenous retrovirus in the first intron of the ZHX2 gene, resulting in a predominantly non-functional transcript, causing downregulation of ZHX2 and high alpha-fetoprotein protein levels in adult BALB / cJ mice. See Perincheri et al., "Hereditary persistence of alpha-fetoprotein and H19 expression in liver of BALB / cJ mice is due to a retrovirus insertion in the ZHX2 gene," (2005) Proc Natl Acad Sci USA 102:396-401. In kidney podocytes, ZHX2 is one of the most potent transcriptional repressors of WT1 and is very likely involved in the pathogenesis of FSGS (Mace et al. 2020). In the kidney, podocytes (specialized glomerular cells) express ZHX proteins primarily in the plasma membrane, whereas in tubular cells, as in many other organs, ZHX protein expression occurs primarily in the nucleus (Mace et al. 2020).In the setting of appropriate combinations and altered ZHX2 expression status, systemic cytokine release can induce translocation of ZHX proteins from normal (aminopeptidase A / APA, ephrinB1) or putative alternative plasma membrane anchors to the podocyte nucleus. Human and experimental MCD and most forms of FSGS are associated with reduced podocyte expression of the transcription factor ZHX2. See Mace et al., "ZHX2 and its interacting proteins regulate upstream pathways in podocyte diseases," (2020) Kidney Int. 97:753-764.

[0004] Viral infection triggers the production of cytokines as part of the innate and adaptive immune responses. We previously suspected that the widespread cytokine storm documented early in the pandemic may be involved in organ damage and developed a novel evidence-based model of cytokine-mediated end-organ damage. See Huang et al., "Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China" (2020) Lancet 395(10223):497-506; (online supplement). Of the three organs tested, the literature on cardiac involvement shows elevated cardiac troponin I levels (similar to acute myocardial infarction), myocarditis, myocardial necrosis, pericarditis, arrhythmias and heart failure. See Topo 2020; Inciardi et al., "Cardiac involvement in a patient with coronavirus disease 2019 (COVID-19)", (2020) JAMA Cardiol. 5:819-824. Evidence of liver injury includes elevated aminotransferase levels, hepatocellular injury, inflammation, and hepatic steatosis. See Herta et al., "COVID- 19 and the liver - Lessons learned," (2021) Liver Int. 41 Suppl 1: pp. 1-8. Renal manifestations are highly prevalent in hospitalized COVID-19 patients, with nearly 40% developing proteinuria and about one-third developing acute kidney injury (AKI). See Cheng et al., "Kidney disease is associated with in-hospital death of patients with COVID- 19," (2020) Kidney Int. 97:829-838; Hirsch et al., "Acute kidney injury in patients hospitalized with COVID-19," (2020) Kidney Int. 98:209-218.Renal biopsy studies of COVID-19 patients with severe proteinuria and / or impaired renal function have most commonly documented collapsing variants of focal and segmental glomerulosclerosis (FSGS) and acute kidney injury. See Kudose et al., "Kidney Biopsy Findings in Patients with COVID-19," (2020) J Am Soc Nephrol. 31:1959-1968; Nasr et al., "Kidney Biopsy Findings in Patients With COVID-19, Kidney Injury and Proteinuria," (2021) Am J Kidney Dis. 77:465-468 (2021). Early autopsy studies suspected viral particles, but renal biopsies of surviving patients did not confirm any viral particles. See also Bradley et al., "Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series," (2020) Lancet 396:320-332.

[0005] There are many other inflammatory and non-inflammatory diseases that affect cytokine release, including non-viral infections such as non-respiratory viral, bacterial, fungal or parasitic infections, immune-mediated diseases, cardiovascular pathologies, diabetes, metabolic syndrome, neurodegeneration and cancer, and ageing.

[0006] However, little is known about the potentially beneficial effects of ZHX2 inhibition on cytokine release and the treatment of various disease states. The present invention addresses these needs. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Mace et al., “The Zinc Fingers and Homeoboxes 2 protein ZHX2 and its interacting proteins regulate upstream pathways in podocyte diseases”, (2020) Kidney Int 97(4):753–764 [Non-Patent Document 2] Kawata H et al., "The mouse zinc -fingers and homeoboxes (ZHX) family; ZHX2 forms a heterodimer with ZHX3", (2003) Gene. 323:133-140 [Non-Patent Document 3] Perincheri et al., "Hereditary persistence of alpha-fetoprotein and H19 expression in liver of BALB / cJ mice is due to a retrovirus insertion in the ZHX2 gene", (2005) Proc Natl Acad Sci USA 102:396-401 [Non-Patent Document 4] Huang et al., "Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China" (2020) Lancet 395(10223):497-506; (online supplement) [Non-Patent Document 5] Inciardi et al., "Cardiac involvement in a patient with coronavirus disease 2019 (COVID-19)", (2020) JAMA Cardiol. 5:819-824 [Non-Patent Document 6] Herta et al., "COVID-19 and the liver - Lessons learned", (2021) Liver Int. 41 Suppl 1: pp. 1-8 [Non-Patent Document 7] Cheng et al., “Kidney disease is associated with in-hospital death of patients with COVID-19”, (2020) Kidney Int. 97:829–838 [Non-Patent Document 8] Hirsch et al., "Acute kidney injury in patients hospitalized with COVID-19," (2020) Kidney Int. 98:209-218 [Non-Patent Document 9] Kudose et al., “Kidney Biopsy Findings in Patients with COVID-19”, (2020) J Am Soc Nephrol. 31:1959~1968 [Non-Patent Document 10] Nasr et al., "Kidney Biopsy Findings in Patients With COVID-19, Kidney Injury and Proteinuria", (2021) Am J Kidney Dis. 77:465-468 (2021) [Non-Patent Document 11] Bradley et al., “Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series”, (2020) Lancet 396:320–332 Summary of the Invention [Problem to be solved by the invention]

[0008] Summary of the Invention The present invention provides a mechanism for targeting ZHX2. The present invention describes methods for inhibiting, blocking or depleting ZHX2 to treat various disease conditions.

[0009] We have already demonstrated how depletion of ZHX2 in a ZHX2 hypomorphic model impacts conditions associated with cytokine storm. In early studies, we found that ZHX2 flox / flox However, we subsequently used BALB / cJ mice, which are established models of the ZHX2 hypomorphic state, and BALB / c mice (ZHX2 + / +) to explain how ZHX2 expression influences cytokine storm-associated morbidity and mortality.See Mace et al. 2020; Perincheri et al., "Hereditary persistence of alpha-fetoprotein and H19 expression in liver of BALB / cJ mice is due to a retrovirus insertion in the Zhx2 gene." (2005) Proc Natl Acad Sci USA 102: 396-401; Perincheri et al., "Characterization of the ETnII-alpha endogenous retroviral element in the BALB / cJ Zhx2 (Afrl) allele", (2008) Mamm Genome 19: 26-31; Gargalovic et al., "Quantitative trait locus mapping and identification of Zhx2 as a novel regulator of plasma lipid metabolism", (2010) Circ Cardiovasc Genet. 3: 60-67; Creasy et al., "Zinc Fingers and Homeoboxes 2 (Zhx2) Regulates Sexually Dimorphic Cyp Gene Expression in the Adult Mouse Liver", (2016) Gene Expr. 17: 7-17; Jiang et al., "Zhx2 (zinc fingers and homeoboxes) regulates major urinary protein gene expression in the mouse liver", (2017) J Biol Chem 292: 6765-6774 (2017); Erbilgin et al., "Transcription Factor Zhx2 Deficiency Reduces Atherosclerosis and Promotes Macrophage Apoptosis in Mice", (2018) Arterioscler Thromb Vase Biol. 38: 2016-2027.

[0010] At low doses, the COVID-19 cocktail, unlike individual cytokines, induced glomerular injury and albuminuria in ZHX2 hypomorphic and ZHX2+ / + mice, mimicking COVID-19-associated proteinuria. At high doses, the COVID-19 cocktail, unlike individual cytokines, induced common clinical manifestations of SARS-CoV-2 disease in ZHX2+ / + mice, including acute cardiac injury, myocarditis, pericarditis, liver and kidney injury, and high mortality, while ZHX2 hypomorphic mice were relatively protected. Activation of signal transduction and activator of transcription 5 (STAT5), STAT6, and NFκB pathways in these organs was reduced and / or desynchronized in the ZHX2 hypomorphic state. Using genome sequencing and CRISPR-Cas9, an insertion upstream of ZHX2 was identified as the cause of the human ZHX2 hypomorphic state.

[0011] Therefore, given that glomerular expression of ZHX proteins, including ZHX2, is distinct (cell membrane associated with podocytes) from ZHX proteins in other organs and other parts of the kidney (mainly the nucleus), the studies described in this invention were performed to determine how the ZHX2 hypomorphic state affects other organs and other parts of the body in disease instances. [Means for solving the problem]

[0012] Thus, in embodiments of the invention, methods are provided for inhibiting, blocking or depleting ZHX2 to treat various disease conditions in patients.

[0013] In other embodiments of the invention, the method of inhibiting, blocking or depleting ZHX2 may further comprise asynchronous activation of STAT5 or STAT6.

[0014] In some embodiments, ZXH2 can be inhibited, neutralized or depleted by administering to the patient an agent comprising an adeno-associated virus (AAV) or lentivirus containing a short hairpin RNA (shRNA) against a ZXH2.

[0015] In some embodiments, the shRNA is commercially available and may be associated with or be part of any vector known in the art, including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes.

[0016] In other embodiments, the agent comprises a monoclonal antibody directed against ZXH2. In yet other embodiments, the agent comprises a monoclonal antibody directed against ZXH2. In yet other embodiments, the agent is a siRNA or antisense oligonucleotide targeted to ZXH2.

[0017] In yet other embodiments, the agent is a pharmacological agent that decreases expression of ZHX2.

[0018] In another embodiment, the agent is a pharmacological agent that reduces ZHX2 expression by directly or indirectly binding to or interacting with the ZHX2 gene or upstream or downstream of the ZHX2 gene.

[0019] In any embodiment, the invention provides methods of treating various disease states by inhibiting, blocking or depleting ZHX2, including viral infections such as SARS-Cov-1, SARS-Cov-2, other coronaviruses, influenza, parainfluenza, respiratory syncytial virus, adenovirus, enterovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Middle East Respiratory Syndrome (MERS) and Ebola, non-viral infections such as non-respiratory viral infections, bacterial infections, fungal infections and parasitic infections, immune-mediated disorders, cardiovascular pathologies, diabetes, metabolic syndrome, organ transplantation, neurodegeneration and cancer, and aging, as well as inflammatory and non-inflammatory diseases that affect cytokine release. [Brief description of the drawings]

[0020] [Figure 1a] Development and characterization of the COVID-19 cytokine storm model. (Figure 1a) Schematic representation of COVID-19 induced cytokine storm in the context of human disease. [Figure 1b] Development and characterization of the COVID-19 cytokine storm model. (Fig. 1b) Composition of dose X of COVID cocktail A-D. [Figure 1c] Development and characterization of COVID-19 cytokine storm model. (Fig. 1c) Albuminuria after injection of different doses of cocktail D in BALB / cJ mice (n=4 mice per group). Half-fold (X / 2) is the threshold nephritogenic dose in BALB / cJ mice. [Figure 1d] Development and characterization of the COVID-19 cytokine storm model. (Fig. 1d) Albuminuria after injection of dose X of individual COVID cocktail components in BALB / cJ mice (n=4 mice per group). [Figure 1e] Development and characterization of the COVID-19 cytokine storm model. (Fig. 1e) Albuminuria after injection of dose X / 2 of COVID cocktail A-D in BALB / c mice (n=6 mice per group). [Figure 1f] Development and characterization of COVID-19 cytokine storm model. (Fig. 1f) Albuminuria after injection of dose X / 2 of COVID cocktail A-D in BALB / cJ mice (n = 6 mice per group). [Figure 1g] Development and characterization of the COVID-19 cytokine storm model. (Fig. 1g) Albuminuria after injection of BALB / c mice with dose X / 2 of intact cocktail C or with dose X / 2 of cocktail C lacking individual podocyte-targeting components (n=6 mice per group). P<0.05; **P<0.01; ***P<0.001. All significance values ​​are two-sided. [Figure 2a] Figure 2a shows reduced systemic damage induced by high dose of cocktail D (three-fold (3X)) in ZHX2 hypomorph BALB / cJ mice compared to ZHX2+ / +BALB / c mice. Dose 3X also has greater damage compared to low dose or dose 3X of individual components. (Figure 2a) Acute myocardial damage assessed by cardiac troponin I (cTPI3) levels (n=8 mice per group). [Figure 2b] Figure 2b shows reduced systemic damage induced by high dose of cocktail D (three-fold (3X)) in ZHX2 hypomorph BALB / cJ mice compared to ZHX2+ / +BALB / c mice. The 3X dose also has greater damage compared to the individual components at low dose or 3X dose. (Figure 2b) Acute liver damage assessed by alanine aminotransferase (ALT) activity levels (n=8 mice per group). [Figure 2c] Figure 2c shows reduced systemic damage induced by high dose of cocktail D (three-fold (3X)) in ZHX2 hypomorph BALB / cJ mice compared to ZHX2+ / +BALB / c mice. Dose 3X also has greater damage compared to lower doses or dose 3X of individual components. (Figure 2c) Acute kidney damage assessed by serum creatinine measured using mass spectrometry (n=8 mice per group). [Figure 2d]Figure 2 shows reduced systemic damage induced by a high dose of Cocktail D (3x) in ZHX2 hypomorph BALB / cJ mice compared to ZHX2+ / +BALB / c mice. The 3x dose also has greater damage compared to the individual components at the low dose or 3x dose. (Figure 2d) Histological characterization of acute cardiac damage using H&E stained sections from mice injected with Cocktail D 3x dose (n=3 mice per group). Cardiomylysis (red arrow), inflammation (black arrow), fibrosis (blue arrow), eosinophilia (green arrow), and pericarditis (orange arrow) were observed. [Figure 2e] Figure 2 shows reduced systemic damage induced by a high dose of Cocktail D (3x) in ZHX2 hypomorph BALB / cJ mice compared to ZHX2+ / +BALB / c mice. The 3x dose also has greater damage compared to the individual components at the low dose or 3x dose. (Figure 2e) Histological characterization of acute liver injury using H&E stained sections from mice injected with Cocktail D 3x dose (n=3 mice per group). Hepatocellular injury (red arrow), inflammation (black arrow), prominent Kupffer cells (green arrow), regenerative changes (yellow arrow), and pericentral venous injury (blue arrow) were observed. [Figure 2f] Figure 2f shows reduced systemic damage induced by a high dose of Cocktail D (three-fold (3X)) in ZHX2 hypomorph BALB / cJ mice compared to ZHX2+ / +BALB / c mice. The 3X dose also has greater damage compared to the individual components at the low dose or 3X dose. (Figure 2f) Histological evaluation of acute kidney injury using PAS-stained sections (rows 1, 2, 4) and kidney electron microscopy (row 3) from mice injected with the 3X dose of Cocktail D (n=3 mice per group). The bottom three rows show the proximal tubules and the top row shows the distal tubules. In the proximal tubules, there was vacuolization (red arrows), brush border destruction (green arrows), and tubular degeneration (black arrows). In the distal tubules, there was evidence of desquamation (blue arrows). Foam cells were also seen (white arrows). Electron microscopy scale bars BALB / c, 2.66 μm; BALB / cJ, 2 μm. [Figure 2g]Figure 2g shows reduced systemic damage induced by high dose of cocktail D (3x) in ZHX2 hypomorph BALB / cJ mice compared to ZHX2+ / +BALB / c mice. The 3x dose also has greater damage compared to the individual components at low dose or 3x dose. (Figure 2g) Table showing morphometric analysis and comparison of histological changes in the hearts of BALB / c and BALB / cJ mice. [Figure 2h] (FIG. 2h) Table showing morphometric analysis and comparison of histological changes in the liver of BALB / c and BALB / cJ mice. [Figure 2i] (FIG. 2i) Table showing morphometric analysis and comparison of histological changes in the kidneys of BALB / c and BALB / cJ mice. Light microscopy scale bar 20 μm. *P<0.05; **P<0.01; ***P<0.001, all values ​​are based on two-tailed tests. [Diagram 3] Figure 1 shows cytokine storm-induced mortality with and without depletion strategies on the impact of severe cytokine storm on systemic disease in ZHX2 hypomorphic BALB / cJ mice. The number of mice injected per group is shown. The mortality rate of 1 / 6 or 16.6% in the control IgG group in mice housed in metabolic cages reflects the mortality rate of groups without any antibody depletion strategy. All depleting antibodies or control IgG were injected intravenously 1 hour after model induction. Mortality is reduced after several depletion strategies. [Figure 4] Cytokine storm-induced mortality with and without depletion strategies on the impact of severe cytokine storm on systemic disease in ZHX2+ / +BALB / c mice. The number of mice injected per group is shown in panel a. The mortality rate of 5 / 6 or 83.3% in the control IgG group in mice housed in metabolic cages reflects the mortality rate of the group without any antibody depletion strategy. This is a figure to compare with the mortality rate of BALB / cJ mice in Figure 3. The remaining data in this table were obtained by housing the mice in regular cages, where the mortality rate was 2 / 6 or 33.3%. Under these circumstances, routine urine collection for albuminuria was not performed. [Figure 5a] Genomic basis of the ZHX2 hypomorphic state. Non-coding DNA insertions and deletions (InDels) affect ZHX2 expression in patients with glomerular diseases. (Figure 5a) Schematic representation of ZHX2 and nearby genes on chromosome 8. [Figure 5b] Genomic basis of the ZHX2 hypomorphic state. Non-coding DNA insertions and deletions (InDels) affect ZHX2 expression in patients with glomerular disease. (Fig. 5b) Mapping of shared insertions and deletions between MCD patients (n = 9 patients), FSGS patients (n = 19 patients) and COVID-19 associated FSGS collapsing variant (n = 8 patients). The most common shared insertions of 122, 533 and 694 could be mapped to or near the theoretical start of the rodent expressed gene Slc22a22, which is non-functional in humans. Another shared insertion of 122, 293 and 423 was located near the theoretical end of Slc22a22. These InDels were not found in control subjects (n = 33) or in the 1000 Genomes Project. Ex is exon. [Figure 5c] Genomic basis of the ZHX2 hypomorphic state. Non-coding DNA insertions and deletions (InDels) affect ZHX2 expression in patients with glomerular diseases. (Figure 5c) Tabular representation of the shared insertions mentioned above. [Figure 5d] Genomic basis of the ZHX2 hypomorphic state. Non-coding DNA insertions and deletions (InDels) affect ZHX2 expression in patients with glomerular diseases. (Fig. 5d) Schematic representation of CRISPR Cas9-assisted genome edited clones of a single-cell-derived cultured human podocyte cell line containing an 8 bp insertion common between patient and control subjects (CRISPR A) or 10 bp shared insertions of 122, 533, and 694 not present in control subjects and the 1000 Genomes Project (CRISPR B). [Figure 5e]Genomic basis of the ZHX2 hypomorphic state. Non-coding DNA insertions and deletions (InDels) affect ZHX2 expression in patients with glomerular disease. (Figure 5e) Relative ZHX2 mRNA expression in the above genomically modified clones (CRISPR A, 3 clones, pooled data; Test CRISPR B, 2 clones) was compared to a parental single-cell derived cultured human podocyte cell line (n=7 templates per clone). The dotted line represents expression in the parental cell line. [Figure 5f] Genomic basis of the ZHX2 hypomorphic state. Non-coding DNA insertions and deletions (InDels) affect ZHX2 expression in patients with glomerular diseases. (Fig. 5f) Western blot comparing ZHX2 expression in a control single-cell-derived parental cell line and one of two mutant clones with insertions at 122, 533, and 694. **P<0.01; ***P<0.001, all values ​​based on two-tailed tests. [Figure 6a] Figure 6 shows the difference in mechanism of COVID cocktail-induced systemic injury (heart, liver and kidney) between ZHX2 hypomorphic BALB / cJ and ZHX2+ / +BALB / c mice. Asynchronous activation of STAT5 and STAT6 signaling pathways in heart, liver and kidney by cocktail D was observed in BALB / cJ compared to BALB / c mice. Mice injected with 3x dose of cocktail D (n=3 mice per group, organs of each mouse evaluated individually) were examined at 15, 30 and 60 min after injection. Mice injected with saline did not activate STAT pathway signaling and data are not shown. (Figure 6a) Graphical comparison of pSTAT5 western blot densitometry expressed as a ratio to lamin B1 (nuclear extracts) and STAT5 (cytoplasmic extracts) in heart, liver and kidney of BALB / c (ZHX2 normal expression) and BALB / cJ (ZHX2 low expression) mice. [Figure 6b]Figure 6b shows the difference in mechanism of COVID cocktail-induced systemic injury (heart, liver and kidney) between ZHX2 hypomorphic BALB / cJ and ZHX2+ / +BALB / c mice. Asynchronous activation of STAT5 and STAT6 signaling pathways in heart, liver and kidney by cocktail D was observed in BALB / cJ compared to BALB / c mice. Mice injected with 3x dose of cocktail D (n=3 mice per group, organs of each mouse evaluated individually) were examined at 15, 30 and 60 min after injection. Mice injected with saline did not activate STAT pathway signaling and data are not shown. (Figure 6b) Graphical comparison of pSTAT6 western blot densitometry expressed as a ratio to lamin B1 (nuclear extracts) and STAT6 (cytoplasmic extracts) in heart, liver and kidney of BALB / c and BALB / cJ mice. *P<0.05; **P<0.01; ***P<0.001, all values ​​are based on two-tailed tests. [Figure 7a] Mechanisms of glomerular injury associated with COVID cytokine storm. (Fig. 7a) Western blot to assess activation of pSTAT6 signaling upon incubation of wild-type and ZHX2 hypomorph (CRISPR B) cultured human podocytes with the human counterpart of cocktail C (final concentration x / 100,000, n=3 plates per condition). [Figure 7b] Mechanism of glomerular injury associated with COVID cytokine storm. (Fig. 7b) Densitometry of western blot of cocktail C incubated in wild type and CRISPR B podocytes in panel c. *P<0.05; **P<0.01; ***P<0.001, all values ​​are based on two-tailed tests. [Figure 8a] Examples of in vivo signaling mechanisms activated by COVID cocktail injected into mice are shown. (Fig. 8a) Example of NFκB / p-p65 (liver, 30 min), pSTAT6 (kidney, 60 min) and pSTAT5 (heart, 15 min) activation by qualitative Western blot of whole organ protein extracts from mice injected with cocktail D3x or control saline (n=3 per group). [Figure 8b] We show examples of in vivo signaling mechanisms activated by COVID cocktails injected into mice. (Fig. 8b) Example of purity test of nuclear extracts by Western blot of nuclear and cytoplasmic fractions of equal protein loading from heart, kidney and liver using anti-Lamin B1 antibody. Traces of Lamin B1 in the cytoplasm may be related to protein synthesis before transport to the nucleus. [Figure 8c] Shown are examples of in vivo signaling mechanisms activated by COVID cocktails injected into mice. (Fig. 8c) Example assessment of GAPDH protein in cardiac cytoplasmic and nuclear fractions of equivalent protein loading from multiple mice. As expected, GAPDH is expressed in both fractions, with higher expression in the cytoplasmic extract. [Figure 8d] Examples of in vivo signaling mechanisms activated by COVID cocktails injected into mice are shown. (Fig. 8d) Example quantitative Western blots of BALB / c mouse heart nuclear protein extracts (20 μg protein per lane) in cocktail D (C'tail D) or control saline injection studies, evaluating pSTAT6 and Lamin B1 in separate blots developed on the same film. Abbreviations for mouse numbers are shown in each lane. [Figure 8e] Examples of in vivo signaling mechanisms activated by COVID cocktail injected into mice are shown. (Fig. 8e) Example quantitative Western blots of BALB / cJ mouse heart cytoplasmic protein extracts (20 μg protein per lane) in cocktail D (C'tail D) or control saline injection studies, evaluating pSTAT5 and STAT5 in separate blots developed on the same film. Abbreviations for mouse numbers are shown in each lane. [Figure 9a] (FIG. 9a) Plasma IL-4Rα levels assessed by ELISA in general COVID-19 patients, age-, sex-, and race-matched healthy controls, and COVID-19 patients with proteinuria. The number of patient samples assayed is shown below. [Figure 9b](FIG. 9b) Electron microscopy image of a BALB / cJ mouse glomerulus 1 day after injection of cocktail D dose X / 2, showing focal areas of foot process effacement (black arrow), endothelial vacuolization (green circle), and endothelial hypertrophy (blue circle). [Figure 9c] (FIG. 9c) Serum creatinine measured by mass spectrometry is not increased in the COVID cytokine cocktail dose X / 2 model (BALB / c and BALB / cJ mice; n=6 mice per group). Scale bar 0.5 μm. [Figure 10a] Further data on the cytokine storm model and characteristics of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10a) Plasma creatine kinase, a marker of skeletal muscle damage, in BALB / cJ mice (n=4 mice per group) 24 hours after injection of different doses of Cocktail D. [Figure 10b] Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10b) Serum cardiac troponin I level data from FIG. 2a replotted to show with higher resolution the smaller increase in levels in some single cytokine injection groups. [Figure 10c] Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10c) Serum ALT level data from FIG. 2b replotted to show with higher resolution the smaller increase in levels in several single cytokine injection groups. [Figure 10d] Further data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10d) Albuminuria at 18 hours in BALB / cJ mice injected with cocktail D 3x or a single cytokine dose 3x, corresponding to FIG. 2a-c. [Figure 10e]Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (Figure 10e) 18-h albuminuria of BALB / c mice injected with a single cytokine dose 3x, corresponding to Figures 2a-c. Considering their high mortality after cocktail D 3x, metabolic cage housing for timed urine collection is not feasible in BALB / c mice. [Figure 10f] Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (Fig. 10f) Fold difference in cardiac and skeletal muscle ZHX2 mRNA expression in BALB / cJ compared to BALB / c mice assessed by real-time PCR (n=6 templates per group). Low levels of ZHX2 mRNA expression in liver have been previously published and serve as a positive control for this phenomenon. A 3-fold difference was considered significant. [Figure 10g] Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10g) Real-time PCR comparison of expression of cytokine receptors, Ace2, Zhx1, Zhx3, and signaling pathway proteins STAT5, STAT6, and P-65 (NFκB) between BALB / cJ and BALB / c mouse hearts (n=6 templates per group). A 3-fold difference was considered significant. [Figure 10h] Additional data on the cytokine storm model and characteristics of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (Fig. 10h) Real-time PCR comparison of expression of cytokine receptors, Ace2, Zhx1, Zhx3, and signaling pathway proteins STAT5, STAT6, and P-65 (NFκB) between BALB / cJ and BALB / c mouse livers (n=6 templates per group). A 3-fold difference was considered significant. [Figure 10i]Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (Figure 10i) Real-time PCR comparison of expression of cytokine receptors, Ace2, Zhx1, Zhx3, and signaling pathway proteins STAT5, STAT6, and P-65 (NFκB) between BALB / cJ and BALB / c mouse skeletal muscles (n=6 templates per group). A 3-fold difference was considered significant. STAT5 expression was 3.06±0.40-fold higher in BALB / c mice. [Figure 10j] Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (Figure 10j) Real-time PCR comparison of the expression of cytokine receptor, Ace2, and signaling pathway proteins STAT5, STAT6, and P-65 (NFκB) between BALB / cJ and BALB / c mouse glomeruli (n=6 templates per group). A 3-fold difference was considered significant. The other IL-2R chains are not expressed in mouse glomeruli, and Zhx1 and Zhx3 have been previously published (Mace et al. 2020). [Figure 10k] Additional data on the cytokine storm model and characteristics of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10k) Electron micrograph of a kidney glomerulus from a BALB / cJ mouse 24 hours after injection of a triple dose of cocktail D. There was multifocal foot process effacement (red arrows), endothelial hypertrophy (green arrows), and remodeling of the glomerular basement membrane (GBM) (blue arrows). [Figure 10l] Additional data on the cytokine storm model and characteristics of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10l) Electron micrograph of a kidney glomerulus from a BALB / c mouse 24 hours after injection of a triple dose of cocktail D. There was extensive foot process effacement (red arrow), endothelial hypertrophy (green arrow), and remodeling of the glomerular basement membrane (GBM) (blue arrow). [Figure 10m]Further data on the cytokine storm model and characteristics of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10m) Hematoxylin and eosin stained skeletal muscle from a BALB / cJ mouse 24 hours after injection of a triple dose of cocktail D. Focal inflammation (black arrows) was noted in some sections. [Figure 10n] Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown (FIG. 10n). Induction of the cocktail D model in BALB / cJ mice (n=6 mice per group; dose x / 2) and albuminuria in combination with control IgG or antibody depletion 1 hour after model induction. [Figure 10o] Additional data on the cytokine storm model and characterization of the ZHX2 hypomorphic state in BALB / cJ mice are shown. (FIG. 10o) Albuminuria after receptor blockade using antibodies against IL-4Rα, TNFR1, and IL-10Rβ, or control IgG, after cocktail C induction in BALB / c mice (n=6 mice per group; ½ dose). Scale bars (k) 0.5 μm, (l) 0.5 μm, (m) 20 μm. *P<0.05; **P<0.01; ***P<0.001. [Figure 11a] (Figure 11a) Single and shared insertions and deletions (InDels) in the study population. [Figure 11b] (Figure 11b) InDels of FSGS patients expanded by disease subcategory. [Figure 11c] (Figure 11c) Shared InDels in patients with diabetic nephropathy (n=13). [Figure 12a] (Figure 12a) List of single InDels in the study population. [Figure 12b] (FIG. 12b) The Slc22a22 gene between Has2 and ZHX2 in rodents and its absence in large animals and humans, ordered by size and heart rate. [Figure 12c] (Figure 12c) Ponceau red stained membrane from the blot shown in Figure 5f. [Figure 13a](FIG. 13a) Confocal expression of cytokine receptors in BALB / c mouse glomeruli. White arrows indicate receptor expression in podocytes (P), endothelial cells (E), and mesangial cells (M). TNFR1 is expressed in podocytes and endothelial cells, so there is only partial colocalization with the podocyte protein nephrin (blue). Green is the nuclear stain. [Figure 13b] (FIG. 13b) Confocal expression of ACE-2 and cytokine receptors (red) in renal tubules of BALB / c mice. Most images show proximal tubules, whereas images of IL-10Rβ are of collecting ducts. [Figure 13c] (FIG. 13c) Characterization of the antibodies used in the depletion studies with recombinant proteins constituting the cytokine cocktail. Scale bars (a) 20 μm (b) 20 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention provides a mechanism for targeting ZHX2. The present invention describes a method for inhibiting, blocking or depleting ZHX2 to treat various disease conditions. The inventors believe that any of the disclosed methods for inhibiting, blocking or depleting ZHX2 may further include altering the activation of STAT5, STAT6 or NFκB.

[0022] A method is disclosed in which ZHX2 can be inhibited, neutralized or depleted by administering a drug to a patient, the drug comprising an adeno-associated virus (AAV) or lentivirus containing a short hairpin RNA (shRNA) against one ZXH2.The inventors believe that shRNA can be purchased and can be attached to or part of any vector known in the art, including plasmids, viral vectors, bacteriophages, cosmids and artificial chromosomes.

[0023] Methods are disclosed in which ZHX2 can be inhibited, neutralized or depleted by administration of polyclonal or monoclonal antibodies directed against ZXH2.

[0024] Disclosed are methods in which ZHX2 can be inhibited, neutralized or depleted by administering siRNA or antisense oligonucleotides targeted to ZXH2.

[0025] Methods are disclosed in which ZHX2 can be inhibited, neutralized or depleted by administration of pharmacological agents that reduce ZHX2 expression.

[0026] Methods are disclosed in which ZHX2 can be inhibited, neutralized or depleted by pharmacological agents that directly or indirectly bind to or interact with the ZHX2 gene or upstream or downstream of the ZHX2 gene, or that effect reversible or irreversible changes at these sites.

[0027] It is also believed that ZHX2 can be indirectly silenced or "turned off" by pharmacological agents that inhibit or block proteins that interact with ZHX2 in the nucleus. Examples of such proteins include ZHX proteins, such as ZHX1 and ZHX3, nuclear factor YA, nuclear factor YB, nuclear factor YC, FoxC1 and ephrin B1 / B2, or any other protein known to interact with ZHX2.

[0028] In any embodiment, the invention provides methods of treating various disease states by inhibiting, blocking or depleting ZHX2, including viral infections such as SARS-Cov-1, SARS-Cov-2, other coronaviruses, influenza, parainfluenza, respiratory syncytial virus, adenovirus, enterovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Middle East Respiratory Syndrome (MERS) and Ebola, non-viral infections such as non-respiratory viral infections, bacterial infections, fungal infections and parasitic infections, immune-mediated disorders, cardiovascular pathologies, diabetes, metabolic syndrome, organ transplantation, neurodegeneration and cancer, and aging, as well as inflammatory and non-inflammatory diseases that affect cytokine release.

[0029] Throughout this disclosure, various quantities, e.g., amounts, sizes, dimensions, ratios, etc., are presented in range format. It should be understood that the description of quantities in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, the description of a range shall be considered to have specifically disclosed all possible subranges and all individual numerical values ​​within that range, unless the context clearly indicates otherwise. For example, description of a range such as 1-6 shall be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual values ​​within that range, e.g., 1.1, 2, 2.3, 4.62, 5, 5.9, etc. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges and are encompassed within the scope of the disclosure, subject to any specifically excluded limitations within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly indicates otherwise.

[0030] The terminology used herein is merely for the purpose of describing certain embodiments and is not intended to limit any embodiment. As used herein, the singular forms "a", "an" and "the" are intended to include the plural unless the context clearly indicates otherwise. It will be further understood that the terms "includes", "comprises", "including" and / or "comprising" as used herein specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. It will further be noted that items included in a list in the format "at least one of A, B, and C" can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B, and C). Similarly, an item listed in the format "at least one of A, B, or C" can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B and C).

[0031] Unless otherwise specified or clear from the context, as used herein, the term "about" in reference to a numerical value or numerical range is understood to mean, for the stated numerical value and values ​​+ / - 10% thereof, or for values ​​recited as ranges, 10% below the recited lower limit and 10% above the recited upper limit.

[0032] In any embodiment disclosed herein, the term "treating" or "to treat" includes inhibiting, slowing, halting, or reversing the progression or severity of an existing condition or disorder.

[0033] In any embodiment disclosed herein, the term "patient" refers to a human.

[0034] ZHX2 inhibitors The present invention contemplates that ZHX2 can be neutralized or inhibited by several different non-limiting methods. For example, as described herein, ZHX2 can be neutralized or inhibited by administration of a therapeutically effective amount of an agent comprising an adeno-associated virus (AAV) or lentivirus containing a short hairpin RNA (shRNA) against ZHX2 (sh-ZHX2). In some embodiments, sh-ZHX2 is commercially available and can be bound to or part of any vector known in the art, including plasmids, viral vectors, bacteriophages, cosmids, and artificial chromosomes. Alternatively, as described herein, ZHX2 can be neutralized or inhibited by administration of a therapeutically effective amount of an agent comprising an antibody, bivalent antibody, or monoclonal antibody against ZHX2. Additionally, as described herein, ZHX2 can be neutralized or inhibited by administration of a therapeutically effective amount of an agent comprising an siRNA or antisense oligonucleotide targeting ZHX2. Also contemplated herein, ZHX2 can be neutralized or inhibited by administration of a therapeutically effective amount of an agent, which can be a pharmacological agent that can include an antagonist or an antagonist that binds to a ZHX2 binding protein or DNA sequence to prevent binding of ZHX2. Also contemplated herein, the pharmacological agent can directly or indirectly bind or interact with the ZHX2 gene, or upstream or downstream of the ZHX2 gene. The ZHX2 inhibitor or composition thereof can be administered once a day, more than once a day, or once a week. The ZHX2 inhibitor or composition containing it can be administered to a subject by any conventional means, including oral, intramuscular, intraperitoneal, or intravenous administration. When injected, it can be injected into a single site per administration, or into multiple sites per administration.

[0035] ZHX2 antibodies and related inhibitors More specifically, the ZHX2 inhibitor is a polyclonal or monoclonal antibody directed against ZHX2. Examples of suitable antibodies directed against ZHX2 are disclosed herein and are known to those skilled in the art. The ZHX2 antibody may also include an antibody fragment or bivalent antibody or a fragment thereof that inhibits ZHX2. As described herein, the ZHX2 inhibitor may be part of a pharmaceutical composition, which may include either an antibody against ZHX2 or a fragment thereof.

[0036] The anti-ZHX2 antibody described herein can be produced or obtained by any means known in the art, including commercially available.It is also contemplated that the antibody can specifically react with ZHX2 or a specific ZHX2 polypeptide can also be used as an antagonist.Anti-ZHX2 herein can be an antibody or fragment thereof that binds to ZHX2 or a cytokine, or a bivalent antibody that binds to ZHX2 and another suitable target.

[0037] As used herein, the term "antibody" refers to an immunoglobulin (Ig), whether natural or partially or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain that is, or is homologous to, an antigen-binding domain. The term further includes "antigen-binding fragments" and other interchangeable terms that refer to similar binding fragments, such as those described below.

[0038] Native antibodies and native immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is typically linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced disulfide bridges within the chain. Each heavy chain contains a variable domain ("V") at one end. H " or "VH") followed by several constant domains ("C HEach light chain has at one end a variable domain ("V" or "CH"). L " or "VL") at one end, and a constant domain ("C L " or "CL"). The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0039] The ZHX2 described herein may be a "synthetic polypeptide" derived from a "synthetic polynucleotide" derived from a "synthetic gene", meaning that the corresponding polynucleotide sequence or a portion thereof, or the amino acid sequence or a portion thereof, is derived from a designed or newly synthesized or modified sequence compared to the equivalent natural sequence. Synthetic polynucleotides (antibodies or antigen-binding fragments) or synthetic genes can be prepared by methods known in the art, including but not limited to chemical synthesis of nucleic acid or amino acid sequences. Synthetic genes typically differ from natural genes at the amino acid level or polynucleotide level (or both) and are typically placed in the context of synthetic expression control sequences. Synthetic gene polynucleotide sequences do not necessarily code for proteins with different amino acids compared to natural genes. For example, these sequences can also include synthetic polynucleotide sequences that incorporate different codons but code for the same amino acids (i.e., the nucleotide changes represent silent mutations at the amino acid level).

[0040] With respect to anti-ZHX2 antibodies, the term "antigen" refers to ZHX2 or any fragment of its protein molecule.

[0041] The terms "antigen-binding portion of an antibody," "antigen-binding fragment," "antigen-binding domain," "antibody fragment," or "functional fragment of an antibody" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to ZHX2.

[0042] The ZHX2 antibody is also intended to include a "diabody," which refers to a small antibody fragment having two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. See, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993).

[0043] It is contemplated that ZHX2 may also include "chimeric" forms of non-human (e.g., murine) antibodies, including chimeric antibodies that contain minimal sequences derived from non-human Ig. In most cases, chimeric antibodies are murine antibodies into which at least a portion of the immunoglobulin constant region (Fc), usually of a human immunoglobulin, has been inserted in place of the mouse Fc. See, e.g., Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0044] It is contemplated that ZHX2 antibodies may also include "monoclonal antibodies," which refers to antibodies obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that may include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be produced by hybridoma methods, recombinant DNA methods, or isolated from phage antibodies.

[0045] As used herein, "immunoreactive" refers to a binding agent, antibody or fragment thereof that is specific for a sequence of amino acid residues on ZHX2 ("binding site" or "epitope"), but is non-toxic at levels formulated for administration to human use, even if cross-reactive with other peptides / proteins. The term "binding" refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions, including interactions such as salt bridges and water bridges, as well as other conventional binding means. The term "preferentially binds" means that the binding agent binds to the binding site with higher affinity than it binds to an unrelated amino acid sequence.

[0046] As used herein, the term "affinity" refers to the equilibrium constant of reversible binding of two agents, expressed as Kd. The affinity of a binding protein to a ligand, e.g., the affinity of an antibody to an epitope, can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM). As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA), or any other technique familiar to those of skill in the art. Avidity can be determined by methods such as Scatchard analysis, or any other technique familiar to those of skill in the art.

[0047] "Epitope" refers to that portion of an antigen or other macromolecule capable of forming a binding interaction with the variable region binding pocket of an antibody.

[0048] The term "specific" refers to the situation where the antibody does not show any significant binding to molecules other than the antigen containing the epitope recognized by the antibody. The term is also applicable, for example, when the antigen-binding domain is specific for a particular epitope carried by some antigens, in which case the antibody can bind to various antigens carrying the epitope. The term "preferentially binds" or "specifically binds" means that the antibody binds to the epitope with higher affinity than it binds to an unrelated amino acid sequence, and is non-toxic at the levels formulated for administration to human use, even if there is cross-reactivity with other polypeptides containing the epitope.

[0049] The term "binding" refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding interactions under physiological conditions, and includes interactions such as salt bridges and water bridges as well as any other conventional binding means.

[0050] As discussed herein, ZHX2 inhibitors can be produced by gene expression technology. The term "RNA interference" or "RNAi" refers to the silencing or reduction of gene expression by siRNA. It is a process of sequence-specific post-transcriptional gene silencing in animals and plants, initiated by siRNA that is homologous in the duplex region to the sequence of the silenced gene. The gene may be endogenous or exogenous to the organism, and may be present integrated in a chromosome or present in a transfection vector that is not integrated in the genome. The expression of the gene is completely or partially inhibited. RNAi may also be considered to inhibit the function of the target RNA. The function of the target RNA may be complete or partial.

[0051] The term "siRNA" refers to small interfering RNA. In some embodiments, siRNA comprises a duplex or double-stranded region of about 18-25 nucleotides in length. Often, siRNA contains about 2-4 unpaired nucleotides at the 3' end of each strand. At least one strand of the duplex or double-stranded region of the siRNA is substantially homologous or substantially complementary to a target RNA molecule. The strand complementary to the target RNA molecule is the "antisense strand" and the strand homologous to the target RNA molecule is the "sense strand" and is also complementary to the siRNA antisense strand. siRNA may also contain additional sequences. Non-limiting examples of such sequences include linking sequences, or loops, and stems, as well as other folded structures. siRNA appears to function as an important intermediary in triggering RNA interference in invertebrates and vertebrates, and in triggering sequence-specific RNA degradation during post-transcriptional gene silencing in plants.

[0052] It is also contemplated that ZHX2 can be silenced or "turned off" using CRISPR technology, as disclosed in the Examples herein.

[0053] It is also believed that ZHX2 may be silenced or "turned off" by pharmacological agents that directly or indirectly bind to or interact with the ZHX2 gene, or upstream or downstream of the ZHX2 gene, or cause reversible or irreversible changes at these sites.

[0054] It is also believed that ZHX2 can be indirectly silenced or "turned off" by pharmacological agents that inhibit or block proteins that interact with ZHX2 in the nucleus. Examples of such proteins include other ZHX proteins, such as ZHX1 and ZHX3, nuclear factor YA, nuclear factor YB, nuclear factor YC, FoxC1 and ephrin B1 / B2, or any other protein known to interact with ZHX2.

[0055] Common methods COVID cytokine cocktail and related animal studies All animal studies performed were approved by the IACUC of Rush University or the University of Alabama at Birmingham. All animals were treated humanely according to protocols. Methods for isolation of mouse glomeruli by Dynabeads, isolation of rat glomeruli by sieving, tissue preservation for tissue sections, 18-hour urine collection in unfed metabolic cages, evaluation of albuminuria and proteinuria, real-time PCR, confocal imaging, electron microscopy and sample processing, histology for light microscopy, western blotting and co-immunoprecipitation have been previously described and are known. The following were assayed using commercially available kits using serum samples; mouse ALT (BioVision K752-100), mouse cardiac troponin I type 3 (Novus Biologicals NBP3-00456), mouse creatine kinase (Abeam ab155901) and human IL-4Rα ELISA (Abeam ab46022). The following antibodies were purchased for Western blot: anti-pSTAT6 (Cell Signaling Technology, Inc. Danvers MA, USA; cat # 56554, 1:500 dilution); anti-STAT6 (Cell Signaling Technology, Inc. Cat # 5397, 1:500 dilution). Antibodies against ZHX1, ZHX2, and ZHX3 have been previously described. Mace et al. 2020; Liu et al., "ZHX proteins regulate podocyte gene expression during the development of nephrotic syndrome," (2006) J. Biol. Chem. 281:39681-39692; Clement et al., "Early changes in gene expression that influence the course of primary glomerular disease," (2007) Kidney Int. 72:337-347.

[0056] All cytokines, soluble receptors, and antibodies were injected intravenously into rodents and are listed below:

[0057] [Table 1]

[0058] Antibodies used in the depletion studies were characterized by Western blot using the corresponding recombinant proteins. Each dose of the cytokine cocktail was dissolved in a final volume of 100 μL of sterile 0.9% saline. BALB / cJ (Jackson Labs) and BALB / c (Envigo) mice were purchased at 8 weeks of age and allowed to acclimate for 2 weeks before baseline 18-h urine and tail blood sample collection. Additional baseline urine collections were performed on BALB / cJ mice. Most in vivo studies were performed between 10 and 15 weeks of age. The nephritogenic dose spectrum of the cytokine cocktail was determined for BALB / cJ, BALB / c, IL4r, and IL5R. - / - (Jackson Labs), ZHX2 flox / flox , NPHS2 cre / cre The threshold nephritogenic dose (BALB / cJ, BALB / c, I14r - / - on BALB / cJ background, X / 2;ZHX2 flox / flox , NPHS2 cre / cre During mouse cytokine studies using 100 μL of 0.9% saline was administered intraperitoneally immediately after intravenous administration of the cytokine cocktail to maintain intravascular hydration. In the medium and high cocktail models, two additional intraperitoneal injections of 100 μL of 0.9% saline were administered at 6 and 23 hours. In cytokine depletion studies, different groups of mice were administered 50 μg of control IgG or the respective antibody or antibody combination intravenously 1 hour after administration of the mouse cytokine cocktail.

[0059] Mass spectrometric assay of plasma creatinine Serum creatinine was measured by LC / MS / MS using an Agilent 1290 Infinity II LC system coupled with a 2x50mm, 2μm Tosoh Bioscience TSK-GEL amide-80 LC column connected to an Agilent 6495 triple quadrupole mass spectrometer. The oven temperature was fixed at 40°C. The mobile phase consisted of 10mM ammonium acetate in LCMS grade water (35%) and LCMS grade acetonitrile (ACN; 65%). Synthetic creatinine (20μg / ml-0.16μg / ml range; Sigma) and isotopically labeled creatinine (D3-creatinine, 10μg / ml; Sigma) were used as standards and internal standards, respectively. 10ul of sample or standard was then combined with 5ul of internal standard and 235ul of 100% ACN, vortexed, and centrifuged at 15000rpm for 15min at 4°C. The supernatant was transferred to a new tube containing 200 μL of 10 mM ammonium acetate and 65% acetonitrile in LCMS grade water, vortexed, and centrifuged at 15,000 rpm for 15 min at 4° C. before measurement. All samples were measured in duplicate.

[0060] Sources of human genomic DNA and human kidney biopsies Genomic DNA samples from 36 patients with nephrotic syndrome, 33 control subjects, and 16 patients with diabetic nephropathy were obtained from the following sources: (a) Immortalized monocytes from plasma of patients with nephrotic syndrome at the University of Alabama at Birmingham, obtained via IRB-approved protocol X080813001 for collecting DNA, blood, and urine samples; (b) IRB-approved studies at the Instituto Nacional De Cardiologia, Mexico City, including archived kidney biopsies from patients with glomerular disease or pre-transplant kidney biopsies from healthy living related kidney donors (CONACYT 34751M, CONACYT 11-05, DPAGA-UNAM IN-201902); (c) IRB-exempt archived kidney biopsies from Hospital Nacional Alberto Sabogal Essalud, Lima, Peru; (d) Duke Molecular Physiology Institute, Duke University ... (e) the Coriell Cell Repositories, which store DNA from the 1000 Genomes Project and the HAPMAP Project. For analytical comparison between cases and controls, the 1000 Genomes Project Phase 3 Ensambl v84 was included as a single additional control.

[0061] Agilent Custom Capture and High-Throughput Illumina Sequencing A custom capture sequencing panel was created to isolate the genomic interval between HAS2 and ZHX2 on chromosome 8. Target intervals were uploaded to the SureDesign website for Agilent SureSelect capture probe design and synthesis (Agilent Technologies, Santa Clara CA). Genomic DNA library preparation and interval capture were performed using the QXT SureSelect kit according to the manufacturer's instructions (Agilent Technologies). The resulting DNA libraries were quantified by QPCR (Kapa Biosystems, Wilmington MA) and sequenced by paired-end 100 bp sequencing on an Illumina HiSeq 2500 or NextSeq 500 according to standard protocols. Approximately 15 million sequences were obtained per reaction. Generation of FASTQ files was performed using the bcl2fastq converter from Illumina (Illumina, Inc., San Diego CA). Paired Illumina sequences were compared to the hg38 database (GRCh38.pl3 Primary Assembly) using CLC Genomics software (version 12, Qiagen, Venlo, The Netherlands). Insertions and deletions ≥ 3 bp in size and a minimum of 20 sequence reads were selected for analysis. Fisher test comparisons of insertions and deletions in study and control subjects were exported in Excel format, after which all insertions and deletions present in the controls were excluded, both software-assisted and manually. Only insertions and deletions that were subsequently confirmed using IGV browser software (Broad Institute, Boston MA) were included. Establishment of homozygosity required the presence of InDels in > 85% of sequences, followed by confirmation by IGV. Minor discrepancies (1–2 base pair position differences) at the sites of insertions or deletions were occasionally observed between the two software and were resolved by Sanger sequencing during the design of the CRISPR Cas9 study.All genome numbering is based on hg38 and CLC genomics software.

[0062] A hypothetical projection of the location of Slc22a22 in the human genome. The BLAST-based margins used the two flanking parts of the mouse gene that matched the human genome. The BLAST- and size-based projections extended the BLAST-based margins to the size of both ends of the mouse gene.

[0063] Genome editing of cultured human podocytes using CRISPR / Cas9 The basic methodology for CRISPR Cas9 has been previously published, see Cong et al., "Multiplex Genome Engineering using CRISPR / Cas Systems," (2013) Science 339:819-823. Single-cell derived cell clones were generated from an established early passage immortalized human podocyte cell line51 and used for genome editing studies. The oligonucleotides and primers used are listed in Table 4.

[0064] [Table 2] TIFF2024543325000004.tif178161

[0065] CRISPR B Generation of sgRNA plasmids: To introduce a 10 bp insertion (CACACACACA), sgRNAs recognizing specific sites (Chr8-122, 533, 694-122, 533, 695) 45 bp downstream of the insertion site were designed using the Benchling website (https: / / benchling.com). Oligos G0016 and G0017 were phosphorylated and annealed using T4 polynucleotide kinase (NEB), digested with BbsI, and ligated into the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (kindly provided by Feng Zhang, Addgene plasmid #42230) using T7 DNA ligase (New England Biolabs). The ligation products were treated with PlasmidSafe exonuclease (Epicentre) to prevent unwanted recombination products and then transformed into One Shot TOP10 cells (Invitrogen). Ten colonies were picked and plasmids were isolated using the QIAprep Spin Miniprep kit (QIAgen). Plasmid DNA was sequenced using primer KI145 (see Table 4).

[0066] Donor Plasmid Creation: The human genomic sequence of patient E58-13 containing the insert of interest was amplified using the KAPA HiFi HotStart PCR Kit (Kapa Biosystems), specific patient genomic DNA, and primers KI195 and KI196, and cloned into the pBlueScript II KS+ vector between the BamHI and HindIII restriction sites. Plasmid DNA was sequenced using KI207 to confirm the presence of the insert. A single mutation was created in the PAM sequence using the Quikchange Mutagenesis Kit (Agilent Technologies) and primers K1215 and K1216 to prevent cleavage of the donor template plasmid, and the change was confirmed by sequencing. The plasmid was then linearly amplified using primers K1219 and K1220, and the PCR product was digested with DpnI to remove any remaining circular template plasmid. An antibiotic selection cassette (puromycin resistance and truncated thymidine kinase) flanked by ITR sequences was amplified by PCR from the PB-MV1 Puro-TK plasmid (Transposagen) using primers K1217 and K1218 and ligated into the linearized plasmid (see above) at the TTAA region 78 bp upstream of the insertion using Gibson assembly Master Mix (NEB). NEB® 5-alpha competent E. coli cells were transformed with 2 μl of the assembly reaction product. Plasmid DNA was isolated from 10 colonies and sequenced using primer K1217 to confirm correct assembly. Genome editing using sgRNA and donor plasmid: To in vitro replicate the InDels identified in kidney disease patients, cultured human podocytes derived from single cells were transfected by electroporation (Biorad Gene Pulser Xcell™ electroporation system, 0.2 cm cuvette, square wave mode, 150V, 10 ms pulse) with CRISPR / Cas9 vectors containing specific sgRNAs and donor plasmids containing donor sequences and antibiotic selection cassettes. After 15 days of incubation with 1 μg / ml puromycin dihydrochloride (Gibco) to remove non-transfected cells, 10 μg of an excision-only piggyBac transposase expression vector (Transposagene) was transfected to remove the antibiotic selection cassette without scarring. Four days after transfection, cells were incubated with 2.5 μM ganciclovir (Sigma) to remove cells with residual truncated thymidine kinase activity. Single cells were picked, clones were established, genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAgen), and the target region was PCR amplified using Platinum HiFi DNA polymerase (Invitrogen) and primers KI189 and KI188. PCR products were gel purified using the QIAquick Gel Extraction Kit (QIAgen), cloned into pCR2.1 vector using the TA Cloning™ kit (Invitrogen), and inserts were sequenced using the M13 forward sequencing primer. Sequences were aligned by BLAST with the native podocyte genome sequence and the donor template sequence.

[0067] CRISPR A The overall method was the same as for CRISPR B, except for the primers and oligonucleotides used, as well as the following site-specific details. An 8 bp insert (TGGATGGA) was introduced into Chr8-122, 304, 094-122, 304, 095) and sgRNA was designed to recognize a specific site 73 bp upstream of the insertion site. To generate the donor plasmid, patient-specific genomic DNA (patient SF3) was cloned between the SpeI and BamHI sites of the pBlueScript II KS+ vector. During Gibson assembly, an antibiotic resistance cassette was ligated with the linearized plasmid at the TTAA region 51 bp upstream of the insertion.

[0068] Examination of STAT5, STAT6 and NFκB pathways in animal models BALB / c and BALB / cJ mice (n=3 mice per group) were injected with normal saline or 3x the dose of Cocktail D and euthanized at 15, 30, and 60 min. Mice were perfused with protease inhibitors (Thermo Fisher Scientific, Catalog No. A32953) and phosphatase inhibitors (Thermo Fisher Scientific, Catalog No. A32957) by left ventricular injection before euthanasia. For qualitative studies, total protein was extracted from liver, heart, and kidney using RIPA buffer (Thermo Fisher Scientific, Catalog No. 89900) in the presence of protease and phosphatase inhibitors to confirm activation of STAT5 (STAT5 and pSTAT5), STAT6 (STAT6 and pSTAT6), and NFκB (p65 and phospho-p65) pathways. For quantitative studies, nuclear and cytoplasmic fractions were extracted separately from these organs of each mouse using a nuclear extraction kit (Nous Biological, Centennial CO, USA, Cat# NBP-2-29447). Western blots of the nuclear expressed protein Lamin B1 were performed on both fractions to confirm its predominant expression in the nuclear fraction. Western blots of GAPDH were performed to confirm its presence in both fractions. For relative quantification by Western blot, pSTAT proteins were expressed as a ratio to Lamin B1 in the nuclear fraction and to the corresponding STAT proteins in the cytoplasmic fraction. Both ratio components were always scanned from the same non-saturated film image, and densitometry was performed by manual detection of close cropped bands, background subtraction, band identification, and adjusted total lane volume calculation using Bio-rad Image Lab 6.1 software.Antibodies against the following proteins were purchased: STAT5 (D2O69, 1:500), p-STAT5 (D47E7, 1:1000), STAT6 (D3H4, 1:500), P-STAT6 (D8S9Y, 1:1000), NF-κB p-65 (D14E12, 1:1000), P-NF-κB Pp-65 (S536, 1:1000), GAPDH 14C10, 1:20,000, all from Cell Signaling Technology, Inc, Danvers MA, USA; lamin B1 (abl6048, 1:5,000, Abeam Cambridge UK); donkey anti-rabbit IgG HRP (1:20,000, Jackson Laboratories).

[0069] In vitro STAT6 signaling assay Wild-type (precursors of CRISPR-modified podocytes) and CRISPR-B podocytes were cultured at 33°C in RPMI 1640 medium containing heat-inactivated 10% fetal bovine serum, 1% insulin-transferrin-selenium (ITS-G, Thermo Fisher Scientific-cat. no. 41400045), and 1% penicillin-streptomycin (Thermo Fisher Scientific, cat. no. 15140122). Cells were subcultured and seeded at 50,000 cells / dish in 10 cm culture dishes for 3 days at 37°C. The culture medium was then replaced with RPMI 1640 containing heat-inactivated 0.2% FBS and 1% penicillin-streptomycin. After 24 hours, cells were treated with cocktail C or cold cocktail (X / 100,000) for 10, 20 and 30 min. Proteins were isolated using RIPA buffer (Thermo Fisher Scientific, Catalog No.: 89900) containing protease inhibitors (Thermo Fisher Scientific, Catalog No.: A32953) and phosphatase inhibitors (Thermo Fisher Scientific, Catalog No.: A32957). (10 ml of RIPA buffer contained one tablet each of protease inhibitor and phosphatase inhibitor.) Protein concentration was assessed using the Bradford protein assay.

[0070] Human plasma from COVID-19 and control patients for IL-4Rα assay 100 μL aliquots of human plasma were obtained from the following sources: (a) de-identified IRB-approved hospitalized COVID patient samples from the Rush University COVID-19 Registry and Biorepository, (b) de-identified IRB-approved hospitalized COVID patient samples from the Rush University COVID-19 Registry and Biorepository selected for the presence of proteinuria, and (c) de-identified plasma samples purchased from Zenbio (Durham NC, USA) matched to group a for age, sex, and race.

[0071] statistical analysis All graph values ​​are mean + SEM. For differences in proteinuria, albuminuria, or gene expression involving two groups, unpaired Student's t-tests were used in Microsoft Excel 2013. All significance is two-sided unless otherwise stated.

[0072] See further experimental examples below. EXAMPLES

[0073] The following examples are provided for the purpose of illustrating various embodiments of the present invention and are not intended to limit the disclosure in any manner. The examples, together with the methods described herein, are representative of currently preferred embodiments and are provided by way of illustration only and are not intended to limit the scope of the present invention. Modifications therein and other uses encompassed within the spirit of the disclosure as defined by the claims will be apparent to those skilled in the art.

[0074] [Example 1] Development of a novel COVID-19 cytokine storm cocktail Figure 1a-g shows the development and characterization of the COVID-19 cytokine storm model. (Figure 1a) Schematic of COVID-19 induced cytokine storm in the context of human disease. (Figure 1b) Composition of dose X of COVID cocktail A-D. (Figure 1c) Albuminuria after injection of different doses of cocktail D in BALB / cJ mice (n=4 mice per group). X / 2 is the threshold nephritogenic dose in BALB / cJ mice. (Figure 1d) Albuminuria after injection of dose X of individual COVID cocktail components in BALB / cJ mice (n=4 mice per group). (Figure 1e) Albuminuria after injection of dose X / 2 of COVID cocktail A-D in BALB / c mice (n=6 mice per group). (Figure 1f) Albuminuria after injection of dose X / 2 of COVID cocktail A-D in BALB / cJ mice (n=6 mice per group). (FIG. 1g) Albuminuria after injection of BALB / c mice with dose X / 2 of intact cocktail C or with dose X / 2 of cocktail C lacking individual podocyte-targeting components (n=6 mice per group). P<0.05; ** P < 0.01; *** P<0.001. All significance values ​​are two-sided.

[0075] Figure 9a-c: (Fig. 9a) Plasma IL-4Rα levels assessed by ELISA in general COVID-19 patients, age-, sex-, and race-matched healthy controls, and COVID-19 patients with proteinuria. Number of patient samples assayed is indicated below. (Fig. 9b) Electron microscopy image of a BALB / cJ mouse glomerulus 1 day after injection of cocktail D dose X / 2. Areas of focal foot process effacement (black arrow), endothelial vacuolization (green circle), and endothelial hypertrophy (blue circle) were observed. (Fig. 9c) Serum creatinine measured by mass spectrometry is not increased in the COVID cytokine cocktail dose X / 2 model (BALB / c and BALB / cJ mice; n=6 mice per group). Scale bar 0.5 μm. * P < 0.05; ** P<0.001.

[0076] COVID cocktails A-D were developed stepwise to model COVID-19 patients admitted to the intensive care unit (Fig. 1b). The first five cytokines (Fig. 1b) are common to all cocktails. Circulating IL-4Rα levels are also elevated in COVID patients with proteinuria (Fig. 9a). The COVID-19 receptor, ACE2, was included in the COVID-19 cocktail because plasma sACE2 levels were significantly higher in COVID-19 patients in the intensive care unit and in elderly and metabolic syndrome patients who are more susceptible to severe COVID-19 disease. High plasma IL-13 and IL-4 levels in COVID-19 patients suggest acute activation of allergic pathways in this disease. Cocktail B was created by removing sIL-4Rα from cocktail A and adding IL-4 and IL-13, while cocktail C was obtained by adding IL-4 to cocktail A. Cocktail D was obtained by adding IL-13 to cocktail C.

[0077] Dose-response studies showed that 1 / 2-fold was the threshold nephritogenic dose in BALB / cJ mice (Fig. 1c), which also induced histological changes (Fig. 9b). Individually, none of these cytokines used at the same doses as combination X caused albuminuria (Fig. 1d). Lower baseline albuminuria has been reported in BALB / cJ mice versus BALB / c mice. See Mace et al. 2020.

[0078] [Example 2] Systemic manifestations of synergistic multi-cytokine injury induced by COVID-19 cocktail Injection of a high dose (3-fold) of cocktail D induced albuminuria and elevated serum cardiac troponin type I3 ​​(cTPI3; myocardial injury, Fig. 2a), serum alanine aminotransferase (ALT; acute liver injury, Fig. 2b), serum creatinine (acute kidney injury, AKI; Fig. 2c), and plasma creatine kinase (CK, skeletal muscle injury; Fig. 10a).

[0079] Figure 2a-h shows the ZHX2+ / + Figure 2 shows reduced systemic damage induced by a high dose of Cocktail D (3x) in ZHX2 hypomorph BALB / cJ mice compared to BALB / c mice. The 3x dose also has greater damage compared to the individual components at the low dose or 3x dose. (Figure 2a) Acute myocardial damage assessed by cardiac troponin I (cTPI3) levels (n=8 mice per group). (Figure 2b) Acute liver damage assessed by alanine aminotransferase (ALT) activity levels (n=8 mice per group). (Figure 2c) Acute kidney damage assessed by serum creatinine measured using mass spectrometry (n=8 mice per group). (Figure 2d) Histological characterization of acute cardiac damage using H&E stained sections from mice injected with Cocktail D 3x dose (n=3 mice per group). Cardiolysis (red arrow), inflammation (black arrow), fibrosis (blue arrow), eosinophilia (green arrow), and pericarditis (orange arrow) were observed. (Fig. 2e) Histological features of acute liver injury using H&E stained sections from mice injected with 3x Cocktail D (n=3 mice per group). Hepatocellular injury (red arrow), inflammation (black arrow), prominent Kupffer cells (green arrow), regenerative changes (yellow arrow), and pericentral vein injury (blue arrow) were observed. (Fig. 2f) Histological evaluation of acute kidney injury using PAS stained sections (columns 1, 2, 4) and kidney electron microscopy (column 3) from mice injected with 3x Cocktail D (n=3 mice per group). The bottom three rows show proximal tubules, and the top row shows distal tubules. Vacuolization (red arrow), brush border destruction (green arrow), and tubular degeneration (black arrow) were observed in the proximal tubules. In the distal tubules, there was evidence of desquamation (blue arrows). Foam cells were also seen (white arrows). Electron microscopy scale bars BALB / c, 2.66 μm; BALB / cJ, 2 μm. (Figure 2g) Table showing morphometric analysis and comparison of histological changes in the hearts of BALB / c and BALB / cJ mice. (Figure 2h) Table showing morphometric analysis and comparison of histological changes in the livers of BALB / c and BALB / cJ mice. (Figure 2i) Table showing morphometric analysis and comparison of histological changes in the kidneys of BALB / c and BALB / cJ mice. Light microscopy scale bar 20 μm. *P < 0.05; ** P < 0.01; *** P<0.001, all values ​​are based on two-tailed tests.

[0080] Figure 10a-n shows the characteristics of the ZHX2 hypomorphic state in BALB / cJ mice. (Figure 10a) Plasma creatine kinase, a marker of skeletal muscle damage, in BALB / cJ mice (n=4 mice per group) 24 hours after injection with different doses of cocktail D. (Figure 10b) Serum cardiac troponin I level data taken from Figure 2a replotted to show with higher resolution the smaller increase in levels in some single cytokine injection groups. (Figure 10c) Serum ALT level data taken from Figure 2b replotted to show with higher resolution the smaller increase in levels in some single cytokine injection groups. (Figure 10d) 18 hour albuminuria of BALB / cJ mice injected with cocktail D 3x or a single cytokine dose 3x, corresponding to Figure 2a-c. (Figure 10e) 18 hour albuminuria of BALB / c mice injected with a single cytokine dose 3x, corresponding to Figure 2a-c. Considering their high mortality after cocktail D 3x, metabolic cage housing for timed urine collection is not feasible for BALB / c mice. (Fig. 10f) Fold difference in cardiac and skeletal muscle ZHX2 mRNA expression in BALB / cJ compared to BALB / c mice assessed by real-time PCR (n=6 templates per group). Low levels of ZHX2 mRNA expression in liver have been previously published and serve as a positive control for this phenomenon. A 3-fold difference was considered significant. (Fig. 10g) Real-time PCR comparison of expression of cytokine receptors, Ace2, Zhx1, Zhx3, and signaling pathway proteins STAT5, STAT6, and P-65 (NFκB) between BALB / cJ and BALB / c mouse hearts (n=6 templates per group). A 3-fold difference was considered significant. (FIG. 10h) Real-time PCR comparison of expression of cytokine receptors, Ace2, Zhx1, Zhx3, and signaling pathway proteins STAT5, STAT6, and P-65 (NFκB) between BALB / cJ and BALB / c mouse livers (n=6 templates per group). A 3-fold difference was considered significant.(Fig. 10i) Real-time PCR comparison of the expression of cytokine receptors, Ace2, Zhx1, Zhx3, and signaling pathway proteins STAT5, STAT6, and P-65 (NFκB) between BALB / cJ and BALB / c mouse skeletal muscles (n=6 templates per group). A 3-fold difference was considered significant. STAT5 expression was 3.06±0.40-fold higher in BALB / c mice. (Fig. 10j) Real-time PCR comparison of the expression of cytokine receptors, Ace2, and signaling pathway proteins STAT5, STAT6, and P-65 (NFκB) between BALB / cJ and BALB / c mouse glomeruli (n=6 templates per group). A 3-fold difference was considered significant. No other IL-2R chains are expressed in mouse glomeruli, and Zhx1 and Zhx3 have been previously published. (Fig. 10k) Electron micrograph of kidney glomerulus from a BALB / cJ mouse 24 hours after injection of a triple dose of Cocktail D. There was multifocal foot process effacement (red arrow), endothelial hypertrophy (green arrow), and remodeling of the glomerular basement membrane (GBM) (blue arrow). (Fig. 10l) Electron micrograph of kidney glomerulus from a BALB / c mouse 24 hours after injection of a triple dose of Cocktail D. There was extensive foot process effacement (red arrow), endothelial hypertrophy (green arrow), and remodeling of the glomerular basement membrane (GBM) (blue arrow). (Fig. 10m) Hematoxylin and eosin stained skeletal muscle from a BALB / cJ mouse 24 hours after injection of a triple dose of Cocktail D. Focal inflammation (black arrow) was noted in some sections. (FIG. 10n) Induction of the cocktail D model in BALB / cJ mice (n=6 mice per group; ½ dose) followed by albuminuria in combination with control IgG or antibody depletion 1 hour after model induction. (o) Albuminuria after cocktail C induction in BALB / c mice (n=6 mice per group; ½ dose) followed by receptor blockade using antibodies against IL-4Rα, TNFR1, IL-10Rβ, or control IgG. Scale bars (k) 0.5 μm, (l) 0.5 μm, (m) 20 μm. * P < 0.05; ** P < 0.01; *** P<0.001.

[0081] cTPI3, ALT and albuminuria were also increased at the 3x dose for several individual cytokines, albeit at significantly lower levels than the cocktail (Fig. 2a, b; Fig. 10b-e). Timed urine collection in metabolic cages for albuminuria assessment was not performed given the high mortality in BALB / c mice injected with the 3x dose of cocktail D (see below). The 3x dose of cocktail D induced significantly more severe cardiac, hepatic and acute kidney injury in BALB / c mice compared to BALB / cJ mice, suggesting that the latter were protected by the ZHX2 hypomorphic state. Although the ZHX2 hypomorphic state of the liver, kidney and glomeruli of BALB / cJ mice has already been described (Mace et al., 2020; Perincheri et al., 2005; Perincheri et al., 2008), we found similar changes in the heart and skeletal muscle (Fig. 10f). mRNA expression of cytokine receptors, ACE2, other ZHX proteins and select signaling pathway proteins in heart, liver, skeletal muscle and glomeruli was similar between BALB / cJ and BALB / c mice (Fig. 10g, h, I, j; exceptions are higher Ace2 in BALB / cJ glomeruli and higher STAT5 in BALB / c skeletal muscle). Cardiac histology (Fig. 2d) revealed myocardial lysis, focal fibrosis and eosinophilia, inflammation (myocarditis) and pericarditis. Liver histology (Fig. 2e) showed parenchymal hepatocellular injury, prominent Kupffer cells, frequent degenerative and regenerative changes and mild inflammation. Histological evaluation of the kidney tubulo-interstitial compartment (Fig. 2f) revealed evidence of proximal tubule injury in the form of frequent vacuolization, luminal dilation, brush border destruction and detachment of tubular epithelial cells. Epithelial cell desquamation, foam cell and vacuole formation were also observed in the distal tubules. Morphometric differences in these organs were observed between BALB / c and BALB / cJ mice 24 h after triple injection of cocktail D (Fig. 2g, h, i). However, there was no evidence of severe or widespread inflammation.

[0082] [Example 3] Effect of ZHX2 expression on mortality following induction of a severe cytokine storm model (3x cocktail D dose).

[0083] Figures 3 and 4 show ZHX2 hypomorphic BALB / cJ mice (Figure 3) and ZHX2 + / + Mortality rates of BALB / c mice (Figure 4) are shown. The number of mice injected per group is shown in the denominator. The control IgG group in each figure represents mortality rates in each strain without any therapeutic cytokine depletion. Under comparable housing conditions, including metabolic cages, mortality rates in this group were much higher in BALB / c mice (5 / 6 mice or 83.3%; Figure 4) compared to BALB / c mice (1 / 6 mice or 16.6%; Figure 3). The remaining data in Figure 4 were generated by housing the mice in regular cages, where mortality rates were lower at 2 / 6 or 33.3%. All depleting antibodies or control IgG were injected intravenously 1 hour after model induction.

[0084] [Example 4] Genomic basis of the ZHX2 hypomorphic state and cloning of the ZHX2 hypomorphic state using CRISPR Cas9 Large-scale whole-exome sequencing studies did not identify any disease-causing forms of ZXH2 (MCD, FSGS, COVID-19-associated FSGS collapse type), so control subjects were sequenced from the start of HAS2 (the immediate upstream gene) (Figure 5a) to the end of ZHX2. The 1000 Genomes database was used as an additional control.

[0085] Figure 5a-f: Genomic basis of the ZHX2 hypomorphic state. Non-coding DNA insertions and deletions (InDels) affect ZHX2 expression in patients with glomerular disease. (Figure 5a) Schematic representation of ZHX2 and nearby genes on chromosome 8. (Figure 5b) Mapping of shared insertions and deletions between MCD patients (n = 9 patients), FSGS patients (n = 19 patients) and COVID-19-associated FSGS collapsed type (n = 8 patients). The most common shared insertions of 122, 533 and 694 could be mapped to or near the theoretical start of the rodent expressed gene Slc22a22, which is non-functional in humans. Another shared insertion of 122, 293 and 423 was located near the theoretical end of Slc22a22. These InDels were not observed in control subjects (n = 33) or in the 1000 Genomes Project. Ex is exon. (Fig. 5c) Tabular representation of the shared insertions described above. (Fig. 5d) Schematic of CRISPR Cas9-assisted genome edited clones of single-cell derived cultured human podocyte cell lines containing either an 8 bp insertion common between patient and control subjects (CRISPR A) or a 10 bp shared insertion at 122, 533, 694 (CRISPR B) not present in control subjects and the 1000 Genomes Project. (Fig. 5e) Relative ZHX2 mRNA expression in the above genome modified clones (CRISPR A, 3 clones, pooled data; test CRISPR B, 2 clones) was compared to the parental single-cell derived cultured human podocyte cell line (n=7 templates per clone). Dotted lines represent expression in the parental cell line. (Fig. 5f) Western blot comparing ZHX2 expression in the control single-cell derived parental cell line and one of the two mutant clones with insertions at 122, 533, 694. ** P < 0.01; *** P<0.001, all values ​​are based on two-tailed tests.

[0086] Figures 11a-c: (Figure 11a) Single and shared insertions and deletions (InDels) in the study population. (Figure 11b) InDels in FSGS patients expanded by disease subcategory. (Figure 11c) Shared InDels in diabetic nephropathy patients (n=13).

[0087] Figures 12a-c: (Figure 12a) List of single InDels in the study population. (Figure 12b) Slc22a22 gene between Has2 and ZHX2 in rodents and its absence in large animals and humans ordered by size and heart rate. (Figure 12c) Ponceau red stained membrane from the blot shown in Figure 5f.

[0088] Multiple insertions and deletions (InDels) of 3 bp or greater that were identified only in the patient population using CLC Genomics software were confirmed using IGV as a second screening method, and only InDels present by both methods were included. Six of nine MCD patients, 10 of 19 FSGS patients, and all eight COVID-19 CG patients had InDels. Three insertions and one deletion were shared by two or more patients. Insertions at 122, 533, and 694 were present only in patients with primary MCD, primary FSGS, or Hodgkin's lymphoma FSGS acral lesions.

[0089] Cross-species analysis of the genome showed that the gene Slc22a22 exists between HAS and ZHX2 in mice and rats, but is non-functional in higher species, including humans. Fine mapping and analysis of the remnants of mouse Slc22a22 (prostaglandin transporter) in the human genome showed that shared insertions at 122, 533, and 694 exist at or near the origin of this gene site. None of the above shared insertions were found in patients with diabetic nephropathy, the most common glomerular disease associated with chronic kidney disease in the Western world (Figure 11c).

[0090] To further explore the relative resistance of ZHX2 hypomorphic states to non-glomerular manifestations of the COVID-19 cytokine storm and previous literature on reduced podocyte ZHX2 expression in human MCD and FSGS, the insertions at chromosome 8 122, 533, and 694 were replicated in a single cell-derived cultured human podocyte cell line using CRISPR-Cas9 technology (CRISPR B study, Fig. 5d). For comparison, another insertion found in patients and controls (control CRISPR A) was also replicated. ZHX2 mRNA expression was unchanged in all CRISPR A cell line clones (pooled data) and was significantly downregulated in both clones generated for CRISPR B (Fig. 5e). Reduced ZHX2 protein expression was confirmed by western blot in CRISPR B lines compared to the parental cell lines (Fig. 5f, Fig. 12c).

[0091] [Example 5] ZHX2 + / + Asynchronous activation of cell signaling pathways in ZHX2 hypomorphic mice compared with normal mice.

[0092] ZHX2 hypomorphic BALB / cJ mice were found to have a high expression of ZHX2 after induction of a severe cytokine storm model. + / +As mortality was much lower compared to BALB / c mice, depletion of TNF-α with a combination of IL-2 or IL-4 or IL-13 eliminated mortality or reduced morbidity in BALB / c mice, and we examined the signaling pathways downstream of these receptors. Qualitative studies from whole organ protein extracts of heart, liver and kidney confirmed phosphorylation of NFκB pathway components p-p65 (downstream of TNFα receptor), pSTAT5 (downstream of IL-2 receptor) and pSTAT6 (downstream of IL-4 and IL-13 receptor complex) at 15, 30 and 60 min in BALB / c and BALB / cJ mice injected with Cocktail D, but not in saline-injected controls (example in Figure 8a). Nuclear and cytoplasmic proteins were then extracted from each organ and qualitatively tested for predominant expression of the nuclear protein Lamin B1 in the nuclear extracts (example in Figure 8b) and GAPDH in both fractions (example in Figure 8c). Since IL-2, IL-4 and IL-13 receptors are expressed in the same cells as ZHX2, whereas TNFα receptors are mostly expressed in blood vessels, Western blot and densitometric quantification of nuclear and cytoplasmic pSTAT5 and pSTAT6 proteins were compared with lamin B1, STAT5 and STAT6 in BALB / c and BALB / cJ mice (Fig. 6a, b, examples of Western blots in Fig. 8d and 8e). The saline group did not activate the signaling pathway and is not shown in Fig. 6. Higher and / or earlier expression of nuclear pSTAT5 was observed in all three organs in BALB / cJ compared to BALB / c mice, although in most scenarios relative pSTAT5 expression in the cytoplasmic fraction was similar or lower (Fig. 6a). Although overall relative cytoplasmic pSTAT6 production was lower in BALB / cJ mice, relative nuclear pSTAT6 expression was similar and in some cases higher in BALB / cJ compared to BALB / c mice (Fig. 6b).These data suggest that pSTAT5 and pSTAT6 translocate into the nucleus more rapidly and earlier in ZHX2-deficient BALB / cJ mice compared with BALB / c mice, leading to asynchronous activation of target genes in BALB / cJ mice and preventing the more severe damage and higher mortality in BALB / c mice. This means that the nuclear action of cell signaling pathways in BALB / cJ mice occurs very early in the disease process (asynchronous activation) and therefore other disease pathways in these conditions are not yet ready to synergize as they would be if activated at the right time in BALB / c mice.

[0093] [Example 6] STAT6 pathway and ZHX-mediated mechanisms in cytokine cocktail-induced glomerular injury Similar to cytosolic pSTAT6 studies in the liver and kidney of BALB / cJ mice (Fig. 6b), cocktail C-induced pSTAT6 phosphorylation was significantly reduced at 30 min in CRISPR B ZHX2 hypomorphic podocytes compared to wild-type controls (Fig. 7a, b).

[0094] (Figure 7a) Western blot to assess activation of pSTAT6 signaling upon incubation of wild-type and ZHX2 hypomorph (CRISPR B) cultured human podocytes with the human counterpart of cocktail C (1 / 100,000x final concentration, n=3 plates per condition). (Figure 7b) Densitometry of western blot of wild-type and CRISPR B podocytes incubated with cocktail C in panel a. * P < 0.05; ** P < 0.01; *** P<0.001, all values ​​are based on two-tailed tests.

[0095] Figure 8a-e show examples of in vivo signaling mechanisms activated by COVID cocktail injected into mice. (Figure 8a) Example of NFκB / p-p65 (liver, 30 min), pSTAT6 (kidney, 60 min) and pSTAT5 (heart, 15 min) activation by qualitative Western blot of whole organ protein extracts from mice (n=3 per group) injected with cocktail D3x or control saline. (Figure 8b) Example of purity test of nuclear extracts by Western blot of nuclear and cytoplasmic fractions of equal protein loading from heart, kidney and liver using anti-Lamin B1 antibody. Tracing of Lamin B1 in the cytoplasm may relate to protein synthesis before being transported to the nucleus. (Figure 8c) Example of evaluation of GAPDH protein in cardiac cytoplasmic and nuclear fractions of equal protein loading from multiple mice. As expected, GAPDH is expressed in both fractions and is more highly expressed in the cytoplasmic extracts. (Figure 8d) Example of a quantitative Western blot of BALB / c mouse heart nuclear protein extracts (20 μg protein per lane) in cocktail D (C'tail D) or control saline injection studies, where pSTAT6 and Lamin B1 were assessed in separate blots developed on the same film. Mouse number abbreviations are shown in each lane. (Figure 8e) Example of a quantitative Western blot of BALB / cJ mouse heart cytoplasmic protein extracts (20 μg protein per lane) in cocktail D (C'tail D) or control saline injection studies, where pSTAT5 and STAT5 were assessed in separate blots developed on the same film. Mouse number abbreviations are shown in each lane.

[0096] Consideration The present inventors have discovered the following.

[0097] 1) ZHX2 hypomorphic BALB / cJ mice were exposed to a cytokine cocktail and subsequently + / + Compared to mice, rats exhibit less severe heart, liver and kidney damage.

[0098] 2) The genomic basis of the human hypomorphic state shows that this deletion at 122, 533, 694 induces the ZHX2 hypomorphic state using CRISPR Cas9-induced modification of a human podocyte cell line. See Figure 5.

[0099] 3) Cytokine storm-related disease development requires synergy between cytokines and synchronization between different pathways. Cytokine depletion after cocktail induction reduces synergy. In liver, heart and kidney cells, active signaling components of the STAT5 and STAT6 pathways (pSTAT5 and pSTAT6) enter the nucleus early during the cytokine storm in the ZHX2 hypomorphic state, out of sync with other molecular events. See Figure 6, Figure 8. This occurs despite a global reduction in activation of the STAT6 pathway in the ZHX2 hypomorphic state. This asynchronous and differential early activation of target genes results in less severe disease development in the ZHX2 hypomorphic state.

[0100] 4) A global reduction in activation of the STAT6 pathway is also observed in cultured human podocytes mutated to replicate with the 122, 533, 694 deletion (CRISPR B). See Figure 7a, b.

[0101] 5) ZHX2 hypomorph reduces mortality in cytokine storm. + / + When comparing the high-dose cytokine storm model in mice (Figure 4) and ZHX2 hypomorphic mice (Figure 3), the mortality rate of ZHX2 hypomorphic mice under the same conditions was significantly higher than that of ZHX2 + / + It was found that the IgG antibody titer was lower (1 / 6 or 16.6%, FIG. 3 , control IgG group) compared with mice (5 / 6 or 83.3%; FIG. 4 , control IgG group).

[0102] As will be appreciated from the description herein, a wide variety of aspects and embodiments are contemplated by the present disclosure, examples of which include, but are not limited to, the aspects and embodiments listed below.

[0103] A method is disclosed in which ZHX2 can be inhibited, neutralized or depleted by administering a drug to a patient, the drug comprising an adeno-associated virus (AAV) or lentivirus containing a short hairpin RNA (shRNA) against one ZXH2.The inventors believe that shRNA can be purchased and can be attached to or part of any vector known in the art, including plasmids, viral vectors, bacteriophages, cosmids and artificial chromosomes.

[0104] Methods are disclosed in which ZHX2 can be inhibited, neutralized or depleted by administration of polyclonal or monoclonal antibodies directed against ZXH2.

[0105] Methods are disclosed in which ZHX2 can be inhibited, neutralized or depleted by administration of siRNA or antisense oligonucleotides targeted to ZXH2.

[0106] Methods are disclosed in which ZHX2 can be inhibited, neutralized or depleted by administration of pharmacological agents that reduce ZHX2 expression.

[0107] Methods are disclosed in which ZHX2 can be inhibited, neutralized or depleted by pharmacological agents that directly or indirectly bind to or interact with the ZHX2 gene, or upstream or downstream of the ZHX2 gene, or that effect reversible or irreversible changes at these sites.

[0108] It is also believed that ZHX2 can be indirectly silenced or "turned off" by pharmacological agents that inhibit or block proteins that interact with ZHX2 in the nucleus. Examples of such proteins include other ZHX proteins, such as ZHX1 and ZHX3, nuclear factor YA, nuclear factor YB, nuclear factor YC, FoxC1 and ephrin B1 / B2, or any other protein known to interact with ZHX2.

[0109] In any embodiment, the invention provides methods of treating various disease states by inhibiting, blocking or depleting ZHX2, including viral infections such as SARS-Cov-1, SARS-Cov-2, other coronaviruses, influenza, parainfluenza, respiratory syncytial virus, adenovirus, enterovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Middle East Respiratory Syndrome (MERS) and Ebola, non-viral infections such as non-respiratory viral infections, bacterial infections, fungal infections and parasitic infections, immune-mediated disorders, cardiovascular pathologies, diabetes, metabolic syndrome, organ transplantation, neurodegeneration and cancer, and aging, as well as inflammatory and non-inflammatory diseases that affect cytokine release.

[0110] Although embodiments of the present disclosure are described herein, it should be understood by those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in implementing the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method for treating a cardiac, liver, kidney, or multi-organ disease in a patient in need of such treatment, comprising administering to the patient an inhibitor of ZHX2, wherein administration of the inhibitor of ZHX2 results in depletion of ZHX2 in the patient.

2. 2. The method of claim 1, wherein the cardiac, hepatic, renal, or multi-organ disease being treated is selected from the group consisting of viral infections such as SARS-CoV-1, SARS-CoV-2, other coronaviruses, influenza, parainfluenza, respiratory syncytial virus, adenovirus, enterovirus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Middle East Respiratory Syndrome (MERS), and Ebola; non-viral infections such as non-respiratory viral, bacterial, fungal, and parasitic infections; immune-mediated diseases; cardiovascular pathologies; diabetes; metabolic syndrome; organ transplantation; neurodegeneration; cancer; and aging, inflammatory and non-inflammatory diseases affecting cytokine release.

3. The method of claim 1, wherein the inhibitor of ZHX2 is selected from the group consisting of an adeno-associated virus (AAV) or lentivirus containing short hairpin RNA (shRNA), an antibody or antibody fragment against ZHX2, an siRNA or other antisense oligonucleotide targeting ZHX2, and a drug comprising an antagonist that binds to a ZHX2-mediated receptor.

4. The method of claim 3 , wherein the shRNA is linked to or is part of a vector.

5. 5. The method of claim 4, wherein the vector is selected from the group consisting of a plasmid, a viral vector, a bacteriophage, a cosmid, and an artificial chromosome.

6. The method described in claim 3, wherein the antibody or antibody fragment against ZHX2 is one or more antibodies selected from the group consisting of polyclonal antibodies, monoclonal antibodies, and bivalent antibodies.

7. The method of claim 3, wherein the inhibitor of ZHX2 is one or more DNA fragments encoding a modified ZHX2 gene.

8. The method of claim 7, wherein one or more DNA fragments encoding the ZHX2 gene are modified by CRISPR.

9. 3. The method of claim 2, wherein the cardiac, hepatic, renal, or multi-organ disease being treated is a disease that causes increased release of cytokines.

10. 3. The method of claim 2, wherein the respiratory viral infection is the result of infection with SARS-CoV-2.

11. 10. The method of claim 1, wherein administration of the ZHX2 inhibitor results in a ZHX2 hypomorphic state in the treated patient.

12. 10. The method of claim 1, further comprising altering activation of STAT5, STAT6 or NFκB protein.

13. A method for inhibiting, neutralizing or depleting ZHX2 in a patient in need thereof, comprising administering a pharmacological agent, wherein the pharmacological agent binds to or interacts with the ZHX2 gene, and binding of the pharmacological agent results in a reversible or irreversible change in the ZHX2 gene.

14. 14. The method of claim 13, wherein the pharmacological agent directly binds to or interacts with the ZHX2 gene.

15. 14. The method of claim 13, wherein the pharmacological agent indirectly binds to or interacts with the ZHX2 gene either upstream or downstream of the ZHX2 gene.

16. A method for reducing or silencing ZHX2 gene expression in a patient in need thereof, comprising administering a pharmacological agent that inhibits or blocks a protein that interacts with ZHX2 in the nucleus of a cell.

17. The method of claim 16, wherein the proteins that interact with ZHX2 in the nucleus are selected from one or more of ZHX1, ZHX3, nuclear factor YA, nuclear factor YB, nuclear factor YC, FoxC1 and ephrin B1 / B2, or other proteins known to interact with ZHX2.

18. 17. The method of claim 16, wherein a pharmacological agent that inhibits or blocks proteins that interact with ZHX2 in the nucleus of a cell causes a reversible or irreversible change in ZHX2 gene expression.

19. 14. The method of claim 13, wherein administration of the pharmacological agent results in a ZHX2 hypomorphic state in the treated patient.

20. 17. The method of claim 16, wherein administration of the pharmacological agent results in a ZHX2 hypomorphic state in the treated patient.

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