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WO2021133777A1 - Inhibiteurs de monoamine oxydase en tant que modificateurs de la vulnérabilité des cellules bêta dans le diabète de type 1 - Google Patents

Inhibiteurs de monoamine oxydase en tant que modificateurs de la vulnérabilité des cellules bêta dans le diabète de type 1 Download PDF

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WO2021133777A1
WO2021133777A1 PCT/US2020/066560 US2020066560W WO2021133777A1 WO 2021133777 A1 WO2021133777 A1 WO 2021133777A1 US 2020066560 W US2020066560 W US 2020066560W WO 2021133777 A1 WO2021133777 A1 WO 2021133777A1
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cells
beta
diabetes
rnls
subject
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Stephan KISSLER
Peng YI
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Joslin Diabetes Center Inc
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Joslin Diabetes Center Inc
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Priority to CN202411358074.2A priority Critical patent/CN119524129A/zh
Priority to AU2020412477A priority patent/AU2020412477A1/en
Priority to CA3174673A priority patent/CA3174673A1/fr
Priority to EP20907245.3A priority patent/EP4081197A4/fr
Priority to CN202080097338.7A priority patent/CN115551491A/zh
Publication of WO2021133777A1 publication Critical patent/WO2021133777A1/fr
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
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    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
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    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
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    • A61P3/00—Drugs for disorders of the metabolism
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Definitions

  • This application relates to monoamine oxidase inhibitors (MAOIs) for treating diabetes, including autoimmune diabetes.
  • MAOIs monoamine oxidase inhibitors
  • Type 1 diabetes is caused by the immune-mediated killing of beta cells in the pancreas (1).
  • Several groups have developed effective differentiation protocols to generate insulin-producing beta-like cells from human embryonic or induced pluripotent stem cells (2). These advances have raised the prospect of replacing lost beta cells in T1D patients using autologous stem cell-derived beta cells, a strategy with the potential to provide an unlimited supply of cells while also circumventing issues of transplant rejection.
  • key hurdles persist. In the absence of immune suppression, recurrent autoimmunity rapidly destroys transplanted beta cells. Immune therapies that would induce tolerance to beta cells in T1D patients have not yet been successfully translated from animal models into human (1).
  • MAO inhibitors monoamine oxidase inhibitors
  • this application describes a method of lowering blood glucose or increasing insulin secretion in response to glucose in a subject comprising administering a monoamine oxidase inhibitor (MAOI), wherein the monoamine oxidase inhibitor binds renalase, binds flavin adenine dinucleotide (FAD), and/or produces an active agent that binds renalase or FAD.
  • MAOI monoamine oxidase inhibitor
  • FAD flavin adenine dinucleotide
  • the subject has autoimmune diabetes.
  • the subject has type 1 diabetes.
  • the subject has autoimmune diabetes induced by an immunotherapy.
  • the monoamine oxidase inhibitor is administered in combination with an additional treatment.
  • the additional treatment is insulin.
  • the insulin is a rapid acting, intermediate-acting, or long-acting insulin.
  • the additional treatment is a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thiazobdinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or sodium/glucose transporter 2 inhibitor.
  • the subject has a blood sugar level higher than 11.1 mmol/liter or 200 mg/dl.
  • a method of preventing the death of pancreatic beta cells or beta-like cells comprises administering a monoamine oxidase inhibitor (MAOI), wherein the monoamine oxidase inhibitor binds renalase, binds flavin adenine dinucleotide (FAD), and/or produces an active agent that binds renalase or FAD.
  • MAOI monoamine oxidase inhibitor
  • FAD flavin adenine dinucleotide
  • the beta cells are those of the subject. In some embodiments, the beta cells are not those of the subject. In some embodiments, the subject is being treated with an immunotherapy. In some embodiments, the immunotherapy is a checkpoint antibody.
  • the checkpoint antibody is an anti -PD- 1 antibody, anti- PD -LI antibody, or anti-CTLA-4 antibody.
  • the beta cells or beta-like cells are transplanted.
  • the beta cells or beta-like cells are transplanted into a patient with autoimmune diabetes.
  • the beta cells or beta-like cells are administered by transplant into the pancreas, liver, or fat pads via surgery, injection, or infusion.
  • a method of preventing the development of type 1 diabetes comprises screening a subject for risk factors for type 1 diabetes; determining if the subject has increased risk of developing type 1 diabetes; and administering a monoamine oxidase inhibitor if the subject has an increased risk of type 1 diabetes.
  • screening a subject for risk factors comprises obtaining data on a genetic risk score that is based on the known type 1 diabetes-associated gene variants, a family history of type 1 diabetes, the presence of one or more autoantibodies against beta cell antigens that are known to predict disease risk, and/or abnormal glucose tolerance.
  • the subject is a mammal. In some embodiments, the mammal is a human.
  • the monoamine oxidase inhibitor is a propargylamine, hydrazine, propylamine, or oxazolidinone derivative.
  • the monoamine oxidase inhibitor is clorgyline, pargyline, rasagiline, selegiline, ladostigil, ASS234, isocarboxazid, toloxatone, or tranylcypromine.
  • Figure 1 shows that a genome-scale CRISPR/Cas9 screen identifies Rnls as a modifier of beta cell survival in the NOD mouse model.
  • Rnls gRNA (MGLibA_46009, 5’-CTACTCCTCTCGCTATGCTC-3’ (SEQ ID NO: 1)) as one of only 11 gRNAs detected at high frequency in mice with beta cell autoimmunity.
  • FIGS 2A-2L show that Rnls mutation protects NIT-1 and primary NOD beta cells against autoimmune destruction in vivo.
  • A Experimental approach used to test NIT-1 beta cell survival after transplantation and induction of autoimmunity. Control (NT) and Rnls mut NIT-1 cells (10 7 ) carrying a luciferase reporter were implanted on opposite flanks of NOD. sad mice. Autoimmunity was induced by injection of 10 7 splenocytes from diabetic (DM) NOD mice.
  • E Experimental approach used to test NIT-1 cell survival transplanted into diabetic NOD mice. Control (NT) and Rnls" m NIT-1 cells (10 7 ) were implanted on opposing flanks of overtly diabetic NOD mice.
  • F Representative images of graft luminescence at day 0, 5, 8, 10, 14 and 18 post-transplantation
  • G Proportion of remaining luminescence relative to day 0 (100%).
  • FIGS 3A-3H shows that Rnls deficiency diminishes immune recognition of beta cells in vitro.
  • A-D Representative flow cytometry data and summary data for MHC-I (A, B, MFI: mean fluorescent intensity) and MHC-II (C, D, expressed as % MHC-lE cells) expression in control and Rnls mut cells treated with thapsigargin (TG) or vehicle (DMSO). Data are representative of three independent experiments.
  • E and F BDC2.5-TCR transgenic CD4 + T cells were co-cultured with NIT-1 cells and irradiated splenocytes from NOD. scid mice.
  • IFN-g expression in CD4 + T cells was measured at 24 h by flow cytometry. Representative (E) and combined data (F) from technical triplicates are shown. Data are representative of 5 independent experiments.
  • FIGS 4A-4E show that Rnls deficiency confers ER stress resistance in vitro.
  • C-E Measurement of the unfolded protein response (UPR) in response to TG challenge.
  • ER stress pathway protein phosphorylation PERK, eIF2a and IRE la
  • expression ATF4, Txnip, NRF2
  • cleavage ATF6, Caspase3
  • D Xbpl splicing
  • Chop and Txnip mRNA levels E were measured in control (NT) and /rii/.v”’ ul NIT- 1 cells treated with or without 1 mM TG for 5 h.
  • Data represent mean ⁇ SEM, *# P ⁇ 0.05, **## P ⁇ 0.01,
  • Figures 5A-5H show that pargyline binds RNLS and protects beta cells against autoimmunity.
  • A Schematic representation of the predicted interaction of pargyline with the FAD co-factor within the RNLS active site.
  • B Human recombinant RNLS protein denaturation profile in the presence and absence of pargyline (PG) by a SYPRO orange protein-dye-based thermal shift assay.
  • D Experimental approach used to test pargyline for the protection of NIT-1 cells transplanted into diabetic NOD mice.
  • E Representative images of graft luminescence at days 0, 3, 5, 7,
  • FIGS. 6A-6B show autoimmune killing of NIT-1 cells in NOD mice can be visualized by bioluminescence imaging.
  • a and B Bioluminescence imaging of 10 7 NIT-1 cells transplanted subcutaneously into NOD.sc/r/ mice. Transplanted cells were engineered to carry a CMV-luciferase2 (Luc2) reporter. Some recipient mice were also injected intravenously with 10 7 splenocytes isolated from spontaneously diabetic (DM) NOD mice to cause beta cell killing. Images were taken at day 1 (A) and 15 (B) post-injection.
  • FIGS 7A and 7B show validation of Rnls mutation by CRISPR-Cas9 targeting.
  • A T7 endonuclease I assay. Genomic DNA from NIT-1 wild-type (WT) and Rnls mut cells was tested for CRISPR-Cas9 gene editing events. Cleavage at heteroduplex mismatch sites by T7 endonuclease I digestion was analyzed by agarose gel electrophoresis. DNA from Rnls" m cells segregated into multiple digested fragments, indicating efficient mutation of the targeted region in the Rnls gene.
  • B Genomic DNA from Rnls""" cells was sequenced to identify individual mutations (SEQ ID NOs: 17-23). The Rnls gRNA targeting site in the wildtype (SEQ ID NO: 16) is labelled with underlining. The mutations with the highest frequencies in Rnls mut cells are shown.
  • FIG. 8 shows that Rnls mutation does not impair insulin secretion.
  • FIGS 9A-9B show that Rnls mutation does not prevent allo-rejection of NIT-1 beta cells.
  • Figures 10A-10B show that Rnls mut cells are not resistant to non-ER stress induced cell death.
  • FIGS 11 A-l ID show that Rnls knockout replicates the ER stress-resistant phenotype of Rnls mut cells.
  • Rnls knockout NIT-1 cell lines were generated by deleting either exons 2-4 or exon 5. Deletion efficiency was confirmed by qPCR of genomic DNA.
  • Rnls AEx2/4 cells showed -60% deletion of exons 2-4 genomic DNA qPCR (A) while Rnls AEx5 cells showed -87% deletion of exon 5 (B).
  • C and D Cell viability of Rnls deficient cells was measured 72 h after thapsigargin (TG, C) and tunicamycin (TC, D) treatment. Data show mean ⁇ SEM, *** P ⁇ 0.001, calculated by unpaired t-test (A and B) and ANOVA with Sidak’s multiple comparisons test (C and D).
  • FIGS 12A-12B show that Rnls overexpression sensitizes NIT-1 cells to ER stress-induced death and reverses the resistant phenotype of Rnls mut cells.
  • a and B NIT-1 cell viability was measured 24 h after treatment with thapsigargin (TG) at the indicated concentrations.
  • TG thapsigargin
  • Overexpression of Rnls in WT NIT-1 cells increased sensitivity to low dose- TG-induced killing (A).
  • n 4 technical replicates per group.
  • FIGS 13A-13C sho that Rnls overexpression increases sensitivity to autoimmune killing in vivo.
  • Control (WT) and Rnls overexpressing (Rnls 0E ) NIT-1 cells carrying a luciferase reporter were implanted on opposing flanks of NOD.sc/ ⁇ i mice. Some graft recipients were also injected intravenously with splenocytes from diabetic NOD mice (DM NOD splenocytes). Graft bioluminescence was imaged on days 0, 2, 3 and 7 (A). The relative luminescence of Rnls 0E and control grafts over time, normalized to day 0, is shown in (B). Data for all mice analyzed on day 3 is shown in (C).
  • FIGS 14A-14B show that Rnls overexpression restores the sensitivity of Rnls mut cells to autoimmune killing in vivo.
  • FIG. 15 shows that islet Rnls expression is elevated in the diabetes-prone NOD mouse strain.
  • Figures 16A-16H show that Rnls deficiency diminishes the UPR following ER stress using a variety of different markers.
  • Figure 17 shows Rnls deficiency confers resistance to oxidative stress.
  • Control (Ctrl) and Rnls mut NIT-1 cells were cultured overnight with or without hydrogen peroxide (H2O2) at the indicated concentrations.
  • Cell viability was assessed using the CellTiter-Glo luminescence Cell Viability Assay. Data show mean ⁇ SEM of triplicate cultures and are representative of three independent experiments. **** P0.0001.
  • FIG. 18 shows that pargyline treatment preserves insulin expression in NOD mice with long-duration diabetes.
  • Pancreases were isolated from control and pargyline- treated diabetic NOD mice described in Figure 5 that were euthanised at day 20 post-beta cell-transplantation.
  • Pancreatic sections were stained with anti -insulin (DAKO, #A0564), anti-CD3 (Bio-rad, #MCA500), and DNA dye Hoechst 33342 (Invitrogen, #H3570).
  • Goat anti -guinea pig Alexa Flour 488 and donkey anti -rat Alexa Flour 594 secondary antibodies were used to detect insulin and CD3 antibodies, respectively.
  • FIGS. 19A-19B show that pargyline treatment does not prevent beta cell destruction after allo-transplantation.
  • Wild-type NIT- 1 cells (10 7 ) carrying aluciferase reporter were implanted into C57BL/6 mice that were treated or not with oral pargyline via addition to the drinking water.
  • Graft bioluminescence was measured on days 1, 2, 3 and 4 after transplantation.
  • Figures 20A-20F show thermal shift assay results for human recombinant renalase in the presence of pargyline (A), rasagiline (B), selegiline (C), tranylcypromine (D), isocarboxazid (E), or toloxatone (F). All drugs were used at lOOmM, and caused a destabilization of the enzyme, as evidence by a decrease in the unfolding temperature (left- shift of the curves).
  • Figures 21A and 21B show in vivo effects of pargyline in diabetes models.
  • Pargyline treatment decreases diabetes onset after cyclophosphamide injection. Groups of 10- week-old male NOD mice were fed pargyline via the drinking water (5pg/ml). Diabetes was induced by intraperitoneal injection of cyclophosphamide (200mg/kg).
  • Pargyline treatment decreases diabetes onset after PD-1 blockade. Groups of 10-week-old female NOD mice were fed pargyline via the drinking water (10pg/ml). Diabetes was induced by intravenous injection of anti-PD-1 antibody (250pg/mouse).
  • Figures 22A-22C show in vivo effects of pargyline treatment (25pg/ml pargyline in the drinking water) in mouse models of type 1 diabetes.
  • Pargyline treatment improved survival when diabetes was induced by adoptive transfer (A) or when diabetes was induced by low doses of streptozotocin (STZ, B).
  • Pargyline also delayed the onset of diabetes following STZ treatment (C).
  • Table 1 provides a listing of certain sequences referenced herein. DESCRIPTION OF THE EMBODIMENTS
  • autoimmune disease refers to an attack by the subject’s immune system against cells that are part of the subject.
  • an autoimmune disease is an abnormal immune response to a normal body part.
  • the autoimmune attack is predominantly against the beta cells of the pancreas that normally secrete insulin in a glucose-dependent manner.
  • autoimmune diabetes relates to any diabetes induced an autoimmune attack, such as type 1 diabetes or diabetes induced by an immunotherapy.
  • Beta-like cell refers to any cell that secretes insulin in response to glucose.
  • a pancreatic beta cell is a “beta-like cell.”
  • Beta-like cells may be derived from cells that do not normally produce insulin in response to glucose.
  • a beta-like cell may be a stem cell that is induced to differentiate into a “beta-like cell” that produces insulin in a glucose-responsive manner (see FW Pagliuca et ak, Cell 159:428-439 (2014); E Kroon et ak, Nature Biotech 26(4):443-452 (2008); and A Rezania et ak, Nature Biotech 32(11): 1121-1133 (2014).
  • a “beta-like cell” may also be a pancreatic exocrine cell (see Q Zhou et ak, Nature 455:627-633 (2008)), pancreatic alpha cell (see Li et al, Cell 168:86-100 (2017), or gut cell (see Ariyachet C et ak, Cell Stem Cell 18(3):410-21 (2016)) that is induced to produce insulin in response to glucose.
  • pancreatic exocrine cell see Q Zhou et ak, Nature 455:627-633 (2008)
  • pancreatic alpha cell see Li et al, Cell 168:86-100 (2017)
  • gut cell see Ariyachet C et ak, Cell Stem Cell 18(3):410-21 (2016)
  • beta-like cells also includes cells that become glucose responsive insulin secretors after transplantation into a subject.
  • treatment covers any administration or application of a therapeutic for disease in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, or preventing reoccurrence of one or more symptoms of the disease.
  • treatment of diabetes type 1 subjects may comprise alleviating hyperglycemia as compared to a time point prior to administration or reducing the subject’s need for exogenous insulin administration.
  • This application relates to lowering blood glucose or increasing insulin secretion in response to glucose in a subject comprising administering a monoamine oxidase inhibitor (MAOI) wherein the monoamine oxidase inhibitor binds renalase (RNLS), binds flavin adenine dinucleotide (FAD), and/or produces an active agent that binds RNLS or FAD.
  • MAOI monoamine oxidase inhibitor
  • Flavin adenine dinucleotide (FAD) binding sites are conserved across flavoprotein oxidases, such as RNLS and MAO (See Gaweska and Fitzpatrick Biomol Concepts. 2(5): 365-377 (2011)). These flavoprotein oxidases may have structural diversity in other regions, while retaining homology in the FAD binding site.
  • FAD is comprised of an adenine nucleotide (adenosine monophosphate) and a flavin mononucleotide bridged together through their phosphate groups.
  • Human RNLS comprises a flavin adenine dinucleotide (FAD) binding site and uses FAD as a co-factor for catalysis.
  • MAO including MAO A and MAO B isoforms
  • FAD flavin adenine dinucleotide
  • the MAOI binds renalase (RNLS).
  • MAOIs that bind RNLS can be determined using standard binding assays, such as radioligand binding, functional assays, or thermal shift assays. This application describes a variety of MAOIs that can bind FAD and can also bind to RNLS.
  • a thermal shift assay can be used to determine binding of an MAOI to RNLS, such as data shown in Figures 20A-20F.
  • the MAOI binds RNLS via binding to the FAD bound to RNLS. In some embodiments, binding of the MAOI to RNLS blocks or inhibits function of RNLS.
  • the MAOI binds FAD. In some embodiments, the MAOI binds to the flavin of FAD. In some embodiments, the MAOI binds to the N5 atom of flavin.
  • the MAOI produces an active species that can bind RNLS. In some embodiments, the MAOI produces an active species that can bind FAD. In some embodiments, the MAOI is modified by RNLS to an active form, which can bind to FAD or RNLS. In some embodiments, the MAOI or its active species forms a covalent adduct with FAD or RNLS. [0054] In some embodiments, the MAOI binds reversibly to FAD or RNLS. In some embodiments, the MAOI binds irreversibly to FAD or RNLS.
  • the MAOI binds covalently to FAD or RNLS. In some embodiments, the MAOI binds non-covalently to FAD or RNLS.
  • the monoamine oxidase inhibitor is a propargylamine, hydrazine, propylamine, or oxazolidinone derivative.
  • the monoamine oxidase inhibitor is clorgyline, pargyline, rasagiline, selegiline, ladostigil, ASS234, isocarboxazid, toloxatone, or tranylcypromine.
  • a method of treating diabetes mellitus comprising administering a MAOI is encompassed. This method may be for treating diabetes, including autoimmune diabetes. In some embodiments, the subject has type 1 diabetes or autoimmune diabetes induced by an immunotherapy.
  • a method of lowering blood glucose levels comprising administering a MAOI is also encompassed. This method may be for treating subjects with diabetes, including autoimmune diabetes. In some embodiments, the subject has type 1 diabetes or autoimmune diabetes induced by an immunotherapy.
  • a method increasing insulin secretion in response to glucose comprising administering a MAOI is also encompassed.
  • This method may be for treating subjects with diabetes, including autoimmune diabetes.
  • the subject has type 1 diabetes or autoimmune diabetes induced by an immunotherapy.
  • a method of preventing or slowing the death of pancreatic beta cells or beta like cells comprising administering a MAOI is also encompassed.
  • This method may be for treating subjects with diabetes, including autoimmune diabetes.
  • the subject has type 1 diabetes or autoimmune diabetes induced by an immunotherapy.
  • Glucose levels in the blood are normally tightly regulated to maintain an appropriate source of energy for cells of the body.
  • Insulin and glucagon are principal hormones that regulate blood glucose levels.
  • insulin is released from beta cells of the pancreas.
  • Insulin regulates the metabolism of carbohydrates and fats by promoting uptake of glucose from the blood into fat and skeletal muscle. Insulin also promotes fat storage and inhibits the release of glucose by the liver. Regulation of insulin levels is a primary means for the body to regulate glucose in the blood.
  • glucose levels in the blood are decreased, insulin is no longer released and instead glucagon is released from the alpha cells of the pancreas. Glucagon causes the liver to convert stored glycogen into glucose and to release this glucose into the bloodstream. Thus, insulin and glucagon work in concert to regulate blood glucose levels.
  • treatment of diabetes mellitus is to administer a MAOI to a subject to lower blood glucose.
  • Hyperglycemia refers to an increased level of glucose in the blood. Hyperglycemia can be associated with high levels of sugar in the urine, frequent urination, and increased thirst. Diabetes mellitus refers to a medical state of hyperglycemia.
  • ADA American Diabetes Association
  • FPG fasting plasma glucose
  • the ADA states that a diagnosis of diabetes mellitus may be made in a number of ways.
  • a diagnosis of diabetes mellitus can be made in a subject displaying an HbAlc level of >6.5%, an FPG levels of >126 mg/dL, a 2-hour plasma glucose of >200 mg/dL during an OGTT, or a random plasma glucose level >200 mg/dL in a subject with classic symptoms of hyperglycemia.
  • the subject has a blood sugar level higher than 11.1 mmol/liter or 200 mg/dl.
  • Type 1 diabetes mellitus can be broken into Type 1 and Type 2.
  • Type 1 diabetes mellitus (previously known as insulin-dependent diabetes or juvenile diabetes) is an autoimmune disease characterized by destruction of the insulin-producing beta cells of the pancreas.
  • Classic symptoms of Type 1 diabetes mellitus are frequent urination, increased thirst, increased hunger, and weight loss.
  • Subjects with Type 1 diabetes mellitus are dependent on administration of insulin for survival.
  • the subject treated has diabetes mellitus based on diagnosis criteria of the American Diabetes Association.
  • the subject with diabetes mellitus has an HbAlc level of >6.5%.
  • the subject with diabetes mellitus has an FPG levels of >126 mg/dL.
  • the subject with diabetes mellitus has a 2-hour plasma glucose of >200 mg/dL during an OGTT.
  • the subject with diabetes mellitus has a random plasma glucose level >200 mg/dL or 11.1 mmol/L.
  • the subject with diabetes mellitus has a random plasma glucose level >200 mg/dL or 11.1 mmol/L with classic symptoms of hyperglycemia.
  • the subject treated is a mammal.
  • the mammal is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent.
  • the subject is a human subject.
  • the subject has autoimmune diabetes. In some embodiments, the subject treated has Type 1 diabetes mellitus.
  • the subject treated has a relative decrease in insulin levels. In some embodiments, the subject treated has decreased beta cell mass. In some embodiments, the decrease in beta cell mass in a subject is due to an autoimmune disease.
  • immunotherapies Treatment of patients with therapeutics targeted to increase the body’s immune response to cancers, termed immunotherapies, has also been associated with the development of autoimmune diabetes (See Alrifai T et al., Case Reports in Oncological Medicine 2019: Article ID 8781347).
  • immune checkpoint antibodies have been reported to cause immune-mediated damage of islet cells leading to induction of autoimmune diabetes similar to type 1 diabetes.
  • the subject has autoimmune diabetes induced by an immunotherapy.
  • the immunotherapy is a checkpoint antibody.
  • the checkpoint antibody is an anti-PD-1 antibody, anti-PD -LI antibody, or anti-CTLA-4 antibody.
  • the method comprises lowering blood glucose levels in the diabetic subject to below about 200 mg/dL, 150 mg/dL, 100 mg/dL, or about 125 mg/dL.
  • treatment of diabetes is lowering blood glucose in the subject after administering a MAOI. In some embodiments, treatment of diabetes is increasing insulin levels in the subject after administering a MAOI. In some embodiments, treatment of diabetes is increasing insulin secretion in the subject after administering a MAOI.
  • administering a MAOI causes a decrease in blood glucose levels such that levels are less than 200 mg/dL.
  • the MAOI is administered in combination with an additional treatment.
  • the additional treatment is insulin.
  • the insulin is a rapid-acting, intermediate-acting, or long-acting insulin.
  • the additional treatment is a glucagon-like peptide analog or agonist, dipeptidyl peptidase-4 inhibitor, amylin analog, biguanide, thiazobdinedione, sulfonylurea, meglitinide, alpha-glucosidase inhibitor, or sodium/glucose transporter 2 inhibitor.
  • This application also encompasses methods of preventing the death of pancreatic beta cells or beta-like cells comprising administering a monoamine oxidase inhibitor.
  • Exemplary beta cells or beta-like cells include those that are transplanted in a subject as a means to treat diabetes. Currently, transplanted beta or beta-like cells are prone to cell death due to autoimmune atack on the transplanted cells.
  • the beta cells or beta-like cells are transplanted. In some embodiments, the beta cells or beta-like cells are transplanted into a subject with autoimmune diabetes. In some embodiments, the beta cells or beta-like cells are administered by transplant into the pancreas, liver, or fat pads via surgery, injection, or infusion.
  • the beta or beta-like cells are those of a subject who is being treated with an immunotherapy.
  • the immunotherapy is a checkpoint antibody.
  • the checkpoint antibody is an anti-PD-1 antibody, anti-PD -LI antibody, or anti-CTLA-4 antibody.
  • Beta cells of pancreas are the cells that normally can secrete insulin. These beta cells of the pancreas are located in pancreatic islets, also known as the islets of Langerhans.
  • a transplanted beta or beta-like cell refers to a cell that is placed in an individual, wherein the cell is from a different individual or is from the same individual but from a different original source in the body than the pancreatic islets.
  • the beta or beta-like cell has been reintroduced into the same or different individual from which it was isolated.
  • the beta or beta-like cell are those of the subject. When introduced into the same subject from which it was isolated it is an autologous beta or beta-like cell. When introduced into a different subject from which it was isolated it is a heterologous beta or beta-like cell.
  • the beta-like cell is a cell that does not normally produce insulin in response to glucose, but is induced or designed to have a phenotype of a beta-like cell, i.e., induced or designed to produce insulin in response to glucose.
  • Beta-like cells include “designer beta cells,” which have been described as using synthetic pathways to produce insulin ( see M Xie et ak, Science 354(6317): 1296-1301 (2016)). [0086] A variety of beta-like cells have been described. a) Stem cells
  • any stem cell capable of differentiating into a beta-like cell may be a beta-like cell according to the invention.
  • the beta-like cell may be differentiated from a hematopoietic stem cell, bone marrow stromal stem cell, or mesenchymal stem cell.
  • Beta-like cells capable of secreting insulin in response to glucose can be generated from pluripotent stem cells (PSCs) (see FW Pagliuca et ak, Cell 159:428-439 (2014)) or embryonic stem cells (ESCs) (see E Kroon et ak, Nature Biotech 26(4):443-452 (2008) and A Rezania et ak, Nature Biotech 32(11): 1121-1133 (2014)).
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • the stem cell may be an embryonic stem cell.
  • the embryonic stem cell is taken from a blastocyst.
  • the embryonic stem cell may be derived from an embryo fertilized in vitro and donated.
  • the embryonic stem cell undergoes directed differentiation.
  • the stem cell may be an adult stem cell.
  • An adult stem cells may also be referred to as a “somatic” stem cell.
  • the adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ.
  • the stem cell is an induced pluripotent stem cell (iPSC).
  • iPSC induced pluripotent stem cell
  • the stem cells may be from bone marrow, adipose tissue, or blood. In some embodiments, the cells may be from umbilical cord blood.
  • stem cells undergo directed differentiation into beta like cells.
  • the directed differentiation is based upon treatment of stem cells with modulators.
  • the directed differentiation is based on culture conditions.
  • beta-like cells are generated from human PSCs (hPSCs) in vitro.
  • beta-like cells are generated from hPSCs using directed differentiation.
  • beta-like cells are generated from hPSCs using a multi-step protocol.
  • beta-like cells are generated from hPSCs using sequential modulation of multiple signaling pathways.
  • beta-like cells are generated from hPSCs using a three-dimensional cell culture system.
  • beta-like cells are generated from human ESCs (hESCs) in vitro. In some embodiments, beta-like cells are generated from hESCs using directed differentiation. In some embodiments, beta-like cells are generated from hPSCs using a multi-step protocol. In some embodiments, beta-like cells are generated from hESCs using sequential modulation of multiple signaling pathways. In some embodiments, beta-like cells are generated from hESCs using a planar cell culture and air-liquid interface at different stages of differentiation. b) Non-stem cells
  • beta-like cells are produced from non-stem cells. In some embodiments, beta-like cells are produced from differentiated non-beta cells. In some embodiments, beta-like cells are produced from reprogramming or transdifferentiation of differentiated non-beta cells.
  • the beta-like cell is a reprogrammed non-beta cell. In some embodiments, the beta-like cell is a transdifferentiated non-beta cell.
  • any type of cell could be induced into a beta-like cell based on principles of reprogramming and transdifferentiation.
  • the invention is not limited by the original phenotype of the beta-like cell.
  • Pancreatic exocrine cells can be reprogrammed into beta-like cells that secrete insulin (see Q Zhou et ak, Nature 455:627-633 (2008)).
  • a pancreatic exocrine cell is reprogrammed into a beta-like cell.
  • the pancreatic exocrine cell is differentiated into a beta-like cell based on re-expression of transcription factors.
  • these transcription factors are NgnJ Pdxl, mdMafa.
  • Pancreatic alpha cells can be transdifferentiated into beta-like cells.
  • the anti-malarial drug inhibits the master regulatory transcription factor Arx (Aristaless related homeobox) and enhances gamma-amino butyric acid (GABA) receptor signaling, leading to impaired pancreatic alpha cell identity and transdifferentiation of alpha cells into a beta-like cell phenotype (see Li et al, Cell 168:86-100 (2017) and Ben-OthmanN et al, Cell 168(l-2):73-85 (2017)).
  • Arx master regulatory transcription factor
  • GABA gamma-amino butyric acid
  • the beta-like cell is a transdifferentiated cell.
  • an alpha cell is transdifferentiated into a beta-like cell.
  • the transdifferentiation into a beta-like cell is due to inhibition of Arx.
  • the transdifferentiation into a beta-like cell is due to enhancement of GABA receptor signaling.
  • Stomach tissue can be reprogrammed into beta-like cells (see Ariyachet C et al, Cell Stem Cell 18(3):410-21 (2016)).
  • a gut or stomach cell is reprogrammed into a beta-like cell.
  • the reprogramming is based on expression of beta cell reprogramming factors.
  • cells of the antral stomach are reprogrammed into beta-like cells.
  • these cells of the antral stomach are antral endocrine cells.
  • reprogrammed antral endocrine cells can be assembled into a mini-organ of beta-like cells.
  • Certain individuals can be predicted to have a high risk for developing type 1 diabetes based on one or more risk factors, such as family history.
  • a method of preventing the development of type 1 diabetes comprises screening a subject for risk factors for type 1 diabetes; determining if the subject has increased risk of developing type 1 diabetes; and administering a MAOI if the subject has an increased risk of type 1 diabetes.
  • screening a subject for risk factors comprises obtaining data on a genetic risk score that is based on the known type 1 diabetes-associated gene variants, a family history of type 1 diabetes, the presence of one or more autoantibodies against beta cell antigens that are known to predict disease risk, and/or abnormal glucose tolerance.
  • type 1 diabetes-associated gene variant Many individuals with type 1 diabetes have a genetic susceptibility because their genome comprises one or more type 1 diabetes-associated gene variant. The presence of one or more of these variants leads to an increased risk of type 1 diabetes. As these type 1 diabetes-associated gene variants can be inherited, a subject with a positive family history of type 1 diabetes may have an increased risk of developing the disease.
  • type 1 diabetes-associated gene variants have been described (See, for example, Watkins RA et ak, Transl Res. 164(2): 110-21 (2014)).
  • the one or more type 1 diabetes-associated gene variant are comprised in one or more HLA gene.
  • the one or more type 1 diabetes-associated gene variant are HLA polymorphisms conferring greater risk for type 1 diabetes.
  • the one or more type 1 diabetes-associated gene variant are comprised in one or more non-HLA gene.
  • a family history of type 1 diabetes is determined by patient history or a questionnaire. In some embodiments, a family history of type 1 diabetes is based on one or more sibling, parent, or grandparent having type 1 diabetes.
  • autoantibody levels against beta cell antigens are measured to determine increased risk of developing type 1 diabetes.
  • a wide variety of autoantibodies against beta cell antigens have described in the literature (See. for example, Watkins 2014).
  • Autoantibody panels are commercially available to identify individuals at risk of developing type 1 diabetes. Inclusion of certain antibodies, such as anti-ZnT8, in autoantibody levels can predict individuals at risk of developing type 1 diabetes.
  • the presence of one or more autoantibodies is used to determine an increased risk of developing type 1 diabetes.
  • the number of autoantibodies or the titer of a specific autoantibody is used to determine an increased risk of developing type 1 diabetes.
  • an abnormal glucose tolerance is used to determine an increased risk of developing type 1 diabetes.
  • a subject with increased risk of developing type 1 diabetes shows abnormal glucose tolerance results without presently meeting criteria for type 1 diabetes.
  • a subject is determined to have an increased risk of developing type 1 diabetes based on the presence of more than one risk factor. For example, a subject with a positive family history for type 1 diabetes may be determined to also have an abnormal glucose tolerance. Multiple risk factors for type 1 diabetes can assessed to determine a subject’s risk of developing type 1 diabetes. In some embodiments, a subject’s risk of developing type 1 diabetes is determined using an algorithm based on multiple risk factors (See. for example, Watkins 2014).
  • a subject having an increased risk of type 1 diabetes is administered a MAOI.
  • administration of a MAOI prevents the development of type 1 diabetes in a subject with increased risk.
  • administration of a MAOI slows the time period until development of type 1 diabetes in a subject with increased risk.
  • Example 1 CRISPR screen for beta cell protective mutations identifies the T1D GWAS candidate gene Rnls
  • NIT-1 beta cell line originally derived from a nonobese diabetic (NOD) mouse insulinoma (5). These cells are suitable for autologous transplantation into NOD mice, the most extensively studied animal model for type 1 diabetes (6). Of importance, NIT-1 cells transplanted into diabetic NOD mice are rapidly destroyed by autoimmunity (Figs. 6A-6B). NIT-1 cells were transduced with the mouse lentiviral GeCKO A CRISPR library that comprises ⁇ 60,000 gRNAs targeting a total of approximately 19,050 genes (7).
  • Rnls the candidate gene for a region in the human genome associated both with the overall risk of T1D (8) and with the age of diabetes onset (9) by GWAS. Based on its prior association with human autoimmune diabetes, Rnls was prioritized for validation.
  • a Rnls mutant NIT- 1 cell line (Rnls mM ) was generated using the Rnls gRNA identified in the screen (Figs. 7A-7B). NIT-1 cells were also engineered to carry a luciferase reporter for longitudinal non-invasive imaging of beta cells after transplantation (Figs. 6A-6B). A validation experiments was performed using an approach similar to the original genome-wide screen. As illustrated in Fig. 2A, Rnls mut cells and control NIT-1 cells transduced with a non-targeting (NT) gRNA were co-transplanted on opposing flanks of NOD. sad mice. Transplant recipients were then injected with splenocytes from diabetic NOD mice.
  • NT non-targeting
  • Pancreatic islets were isolated from immuno-deficient NODxc/ri mice that are devoid of autoimmune infiltrates in the pancreas. Dispersed islet cells were transduced with lentivirus encoding rat insulin promoter-driven Cas9 endonuclease and either the Rnls -targeting (Rnls"'" 1 ) gRNA or a non-targeting (NT) control gRNA (Fig. 21). Gene edited and control islets cells were transplanted each under one kidney capsule of the same NOD. sad mice.
  • graft recipients were injected with splenocytes from diabetic NOD mice to induce autoimmune beta cell killing.
  • islet grafts were also followed in NOD. sad recipients that did not receive splenocytes from diabetic mice.
  • autoimmunity decreased the size and insulin expression in control grafts (Figs. 2J-2L).
  • Rnls mut islets survived autoimmunity and maintained insulin expression.
  • the unfolded protein response (UPR) that is triggered by ER stress has been implicated in beta cell apoptosis in both T1D and type 2 diabetes (13-15).
  • ER stress was proposed to contribute not only directly but also indirectly to beta cell death in T1D owing its ability to increase the presentation of auto- and neoantigens, for example by affecting post-translational modifications (12, 16 18).
  • the Rnls mutation could affect the cellular response to ER stress and thereby diminish the stimulation of diabetogenic CD8+ T cells.
  • NIT-1 cells were challenged with the ER stressor thapsigargin (TG).
  • Control cells were highly sensitive to TG treatment, with concentrations greater than 50 nM killing a majority of cells. Remarkably, Rnls mutant cells withstood even a 20-fold greater concentration of TG (Fig. 4A). Similar results were seen with the alternative ER stressor tunicamycin (TC) (Fig. 4B). Of note, Rnls mut NIT-1 cells remained sensitive to mitomycin C and streptozotocin that cause ER stress-independent cell death (Figs. 10A-10B). These data indicate that Rnls deficiency does not prevent all forms of cell death and that its protective effect may be limited to specific sources of cellular stress.
  • ER stress resistance was a direct effect of Rnls mutation and not caused by an off-target effect of the Rnls gRNA
  • additional cell lines were generated in which either exons 2 to 4 or exon 5 of the Rnls gene were deleted using different sets of gRNAs.
  • These alternative /rii/.v-deficient beta cell lines were again protected against ER stress-induced cell death (Figs. 11 A-l ID), confirming that ER stress resistance was a direct result of Rnls deletion.
  • Example 5 Rnls overexpression sensitizes beta cells to ER stress and autoimmunity
  • Rnls was overexpressed in NIT-1 beta cells using a lentiviral transgene. While Rnls overexpression alone only marginally increased sensitivity to TG-induced killing (Fig. 12A-12B), it appeared to significantly accelerate the autoimmune killing of beta cells implanted into diabetic mice (Fig. 13A-13C). Re-introduction of Rnls into Rnls mut cells was done using a transgene that carried a synonymous mutation at the gRNA target site to prevent CRISPR-Cas9 targeting. Rnls re-expression restored the sensitivity of Rnls mut cells to ER stress (Fig.
  • Rnls re-expression also accelerated the autoimmune destruction of Rnls mut cells in diabetic NOD mice (Figs. 14A-14B).
  • Figs. 14A-14B Collectively, the data show that Rnls expression modulates the vulnerability of beta cells to ER stress and autoimmunity.
  • Rnls expression was 10-15 fold higher in pancreatic islets of diabetes-prone NOD mice than in diabetes-resistant C57BL/6 mice (Fig. 15). Elevated Rnls expression was not merely a result of pancreas inflammation, because similar Rnls mRNA levels were measured in the islets of both NOD and immuno-deficient NODxc/ri mice.
  • This intriguing observation suggests the Rnls may be a genetically encoded modifier of beta cell vulnerability in both mouse and human, though exactly how Rnls expression is regulated in both species remains to be elucidated.
  • Rnls is a flavoprotein oxidase whose cellular function has not yet been elucidated (26). Its proposed substrates are 2- and 6-dihydroNAD(P) (27), isoforms of b- NAD(P)H, though whether these are physiologically relevant is unknown. However, the crystal structure of human RNLS was solved several years ago (28). The enzyme utilizes an FAD co-factor for catalysis, resembling other oxidases including monoamine-oxidase B (MAO-B). Based on structural similarities, the FDA-approved MAO-B inhibitor pargyline (29) was predicted bind to RNLS (Fig. 5A).
  • RNLS is a modifier of beta cell vulnerability in T1D. This finding may explain why genome variants in the RNLS locus impact the overall risk ( 8 ) and the age of onset (9) of T1D. How disease-associated variants modify RNLS function or expression is unknown. In light of our results, exploring how this candidate T1D risk gene is regulated seems warranted. Rnls was differentially expressed in islets of diabetes- prone NOD mice and diabetes-free C57BL/6 mice, lending further support to the notion that RNLS may be a genetic risk factor for beta cell autoimmunity.
  • RNLS deficiency endows beta cells with the ability to resist autoimmunity suggests a genetic engineering solution to beta cell replacement in T1D that would interfere neither with the identity of the beta cell nor with immunity and immune surveillance.
  • RNLS deletion could be a safe and effective modification in SC-beta cells to overcome autoimmunity in patients with T1D.
  • an FDA-approved drug that replicates the protective effect of RNLS deletion. Its apparent efficacy in protecting beta cells, together with its favorable safety profile, should make pargyline, and other MAO- B inhibitors predicted to target RNLS, worthy of further evaluation for the prevention or treatment of type 1 diabetes.
  • Example 9 Evaluation of MAO inhibitors as RNLS inhibitors
  • RNLS protein Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP. RNLS protein was obtained from the supernatant of cell lysates, followed by purification via Ni Bio-rad column. 2 mM RNLS dissolved in PBS was incubated with pargyline (Sigma-Aldrich, #P8013), rasagiline (Tocris, cat# 4308), or selegiline (Tocris, cat# 1095) at 100 mM for 20 min at 4 °C before addition of SYPRO Orange dye (Invitrogen, #S6650) for the measurement of thermal denaturation.
  • pargyline Sigma-Aldrich, #P8013
  • rasagiline Tocris, cat# 4308
  • selegiline Tocris, cat# 1095
  • the thermal shift assay was performed using the QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) with an initial temperature hold at 25 °C for 2 min, followed by a temperature ramp up to 95°C at a rate of 1°C / s, and a final temperature hold at 95°C for 2 min. Results were collected at 0.25 °C increments.
  • Example 10 In vivo effects of pargyline treatment in diabetes prevention models
  • PD-1 blockade has also been shown to cause rapid onset of diabetes in NOD mice (See Ansari MJ et al J Exp Med 2003, 198: 63), and immune checkpoint inhibition (including PD-1 inhibition) for the treatment of various cancers can cause autoimmune diabetes in some patients (reviewed in Clotman K et al J Clin Endocrinol Metab 2018, 103: 3144).
  • STZ streptozotocin
  • Example 11 Screen for additional MAOI that bind to RNLS
  • Nonobese diabetic mice NOD.scid mice
  • C57BL/6J C57BL/6J mice
  • Animals were housed in pathogen-free facilities at the Joslin Diabetes Center and all experimental procedures were approved and performed in accordance with institutional guidelines and regulations.
  • mice GeCKO-v2 Genetic-Scale CRISPR Knock-Out
  • Addgene Additionalgene
  • #1000000052 A lentiviral pooled library was obtained from Addgene (Addgene, #1000000052), targeting 19050 with 3 gRNAs/gene (30) and was prepared as previously described (31).
  • NIT-1 cells were transplanted subcutaneously into 8-week-old female NOD.sc/ri mice, and 10 7 of diabetic NOD splenocytes in 200 m ⁇ sterile PBS were injected intravenously at the same time to induce autoimmunity.
  • NOD.sc/ri mice with subcutaneously transplanted mutant NIT-1 cells but without diabetic NOD splenocytes injection were used as control (non-autoimmune group).
  • Diabetic NOD splenocytes were isolated from spontaneously diabetic female NOD mice as described previously (32). The screen was terminated at 8 weeks post-injection and the remaining grafts were retrieved from both the autoimmune group and the non-autoimmune group of mice.
  • Genomic DNA was extracted from the grafts (Quick-gDNA midiprep kit, Zymo Research), the NGS (Next Generation Sequencing) libraries were prepared as previously described (33), and subjected to NGS sequencing analysis (Novogene, CA).
  • the gRNA sequences from the NGS sequencing data were extracted using standard bioinformatics methods, and the distribution of gRNAs were calculated as Count Per Million (CPM).
  • NIT-1 (#CRL-2055) and 293FT (#R7007) cell lines were obtained from ATCC and Thermo Fisher Scientific respectively. Cells were maintained in DMEM (Gibco, 10313039), supplemented with 10% fetal bovine serum (FBS, Gibco), glutagro and penicillin/streptomycin (Coming), in a 37° C incubator with 5% CCh.
  • DMEM Gibco, 10313039
  • FBS fetal bovine serum
  • Coming penicillin/streptomycin
  • non-targeting (NT) gRNA 5’ TAAAAACGCTGGCGGCCTAG 3’, MGLibA_67395 (SEQ ID NO: 2)
  • mdRnls gRNA 5’ CTACTCCTCTCGCTATGCTC 3’, MGLibA_46009 (SEQ ID NO: 1)
  • NT or Rnls gRNA containing lentivirus was used to establish these cell lines, respectively.
  • Rnls mutation in Rnls mut cells was confirmed by deep sequencing analysis (MGH DNA Core Facility, Cambridge, MA).
  • the Rnls overexpressing NIT-1 cell line was generated by lentiviral infection of wild-type (WT) NIT-1 cells with EFla promoter-driven full-length mouse Rnls (Of note, the full-length mouse Rnls that we cloned and used is based on the annotation from the NCBI database in late 2017 that included 300aa, the Rnls annotation in NCBI was updated in March 2019 and now encodes a protein with 42 additional amino acids at the N-terminus).
  • Rnls mutant NIT-1 cells were transduced with lentivirus carrying a CRISPR-immune EFla promoter-driven full- length mouse Rnls ( CiRnls ) carrying a synonymous mutation in the Rnls gRNA target site.
  • CiRnls CRISPR-immune EFla promoter-driven full- length mouse Rnls
  • the modified gRNA targeting site sequence used in CiRnls was 5’
  • TTATAGTAGCCGGTACGCA 3’ (SEQ ID NO: 3).
  • the /rii/.v-deficient NIT-1 cell lines (Rnls AEx2/4 and Rnls AEx5) were generated following previously published protocols (36). Two gRNAs were designed to target the 5’- and 3 ’-end of Rnls exon 2-4 or exon 5 genomic DNA sequences.
  • gRNA sequences for exon 2-4 were 5’CGTCTGGGAAGTCTTGGTCG 3’ and 5’ CGGGACTCATCCCATTGTCG 3’ (SEQ ID NOs: 4-5); gRNA sequences for exon 5 were 5 ’ GGGGAGTGAGGATAGGATAG 3’ and 5’ TCCGTAGTGGTTTTAGAGTG 3’ (SEQ ID NOs: 6-7).
  • the lenti-multi-CRISPR plasmid (Addgene, #85402) was used to express two single gRNAs cassettes for the deletion of exons 2-4 or exon 5 of Rnls.
  • the two gRNAs cassettes were amplified by Phusion High-Fidelity PCR kit: 40 cycles: 98°C, 15 sec; 60°C,
  • PCR products were digested with Bbsl (Invitrogen) and sub-cloned into the pSpCas9(BB)-2A-Puro (PX459) v2.0 vector (Addgene, #62988). NIT-1 cells were then transfected with these plasmids by polyethylenimine (Fisher Scientific), followed by puromycin selection. All plasmid sequences were verified by Sanger sequencing before transduction and transfection.
  • Islets were prepared and purified as described previously (35) from 8 week old female NOD.sc/ri mice.
  • Purified NOD.sc/ri islets were immediately cultured in a low-attachment plate in RMPI 1640 medium (Gibco), supplemented with 10% FBS and penicillin/streptomycin.
  • Lentivirus encoding a NT or Rnls gRNA together with Cas9 endonuclease under the control of the rat insulin promoter (RIP) was added to the culture media for overnight infection. The next day, islets were washed with culture media twice and -300 islets were transplanted under each kidney capsule of 8 week old of female NOD.sc/ri mice.
  • mice were left to recover from surgery for two weeks, then mice were randomly assigned to non-autoimmune and autoimmune groups. Mice in the autoimmune group were injected intravenously with 10 7 splenocytes purified from spontaneously diabetic female NOD mice. Splenocytes were prepared as described previously (32). At day 25 post splenocyte injection, islet grafts were retrieved for gene expression analysis by quantitative real-time PCR (qPCR).
  • qPCR quantitative real-time PCR
  • gPCR Quantitative real-time PCR
  • RNA extraction was reverse-transcribed into cDNA using the Superscript IV first-strand synthesis kit (Invitrogen). Insulin 1 (MmO 1259683_g 1 ), Glucagon (Mm01269055_ml) mdHprtl (Mm0302475_ml) probes for TaqMan assays were purchased from Thermo Fisher Scientific. Gene expression levels of Chop and Txnip were analyzed by SYBR green PowerUp qPCR assays (Applied Biosystems).
  • Cells were seeded in 96-well white plate (50,000 cells / well) for overnight culture with or without thapsigargin, tunicamycin, streptozotocin (Sigma- Aldrich), mitomycin C (Fisher Scientific) or hydrogen peroxide (H2O2, Fisher Scientific) at the indicated concentrations. Cell viability was assessed after 24 h using the CellTiter-Glo luminescence Cell Viability Assay (Promega).
  • NIT-1 cell lines were engineered to constitutively express the firefly luciferase gene (Luc2) driven by the EFla promoter via lentiviral delivery, except in Fig. 7, where a CMV-Luc2 construct was used.
  • Mice transplanted with luciferase-expressing cells were injected with D-luciferin intraperitoneally at a dose of 150 mg/kg for bioluminescence imaging.
  • D-luciferin (Gold Biotechnology, Cat# LUCK) solution was prepared in sterile DPBS (without calcium or magnesium) at a concentration of 15 mg/ml and filtered (0.22 pm) prior to injection.
  • Luminescence was measured using an IVIS Spectrum imaging system (PerkinElmer). Xbpl splicing assay
  • PCR products oiXbpl were sized at 473bp iXbplu) and 447bp ( Xbpls ) and segregated by electrophoresis using a 3% agarose gel. Ratio of Xbpls /Xbplu was measured by Adobe Photoshop CC 2019. Primer sequences used iorXbpl were forward - 5’ AAACAGAGTAGCAGCGCAGACTGC 3’ (SEQ ID NO: 12) and reverse - 5’ TCCTTCTGGGTAGACCTCTGGGAG 3’ (SEQ ID NO: 13).
  • Cell lysates were collected on ice in RIPA lysis buffer containing proteinase and phosphatase inhibitors (complete proteinase inhibitor cocktail, Sigma- Aldrich; Pierce phosphatase inhibitor, Thermo Scientific Fisher). Protein concentrations were measured by Pierce BCA protein assay (Thermo Scientific Fisher). 40 pg denatured cell lysate protein were used for SDS-PAGE electrophoresis (4-20% TGX gel, Bio-Rad).
  • PERK Cell signaling, #3192
  • phospho-PERK Thr980, Cell signaling, #3179
  • ATF4 Cell signaling, #11815
  • ATF6 Novus Biologicals, #NBP1-40256SS
  • eIF2a Cell signaling, #2103
  • phosphor-eIF2a Ser51, Cell signaling, #3597
  • IREla Cell signaling, #3294
  • phosphor-IREla Ser724, Novus Biologicals, #NB100-2323SS
  • Txnip ML, K0205-3
  • cleaved Caspase 3 Cell signaling, #9664S
  • NRF2 Anta Cruz, #SC365949
  • actin actin
  • CRISPR/Cas9 editing in Rnls""" cells was detected by T7 endonuclease I mismatch cleavage assay.
  • Genomic DNA gDNA was purified from control and Rnls mut NIT-1 cells using Quick-gDNA miniprep kit (Zymo Research).
  • the Rnls gRNA targeting site was amplified using the Phusion high-fidelity PCR kit (Thermo Fisher Scientific).
  • Primers for Rnls gRNA site PCR were: forward 5’ TGCTATAGACAGTTGGGACTTGTTT 3’ (SEQ ID NO: 14); reverse 5’ AT ATT GC GTT C T ATT AT C A AT GGAGAT GA AGC 3’ (SEQ ID NO: 15).
  • the PCR products (-200 ng) were used to form heteroduplexes by denaturing at 95 °C for 5 min and then re-annealing the products in a thermocycler using the following protocol: ramp down to 85°C at -2°C/sec; ramp down to 25°C at -0.1°C/sec; hold at 4°C. 10 units T7 endonuclease I was added to the annealed PCR products and the reaction was incubated at 37°C for 15 min. The digestion reaction was stopped by 1 m ⁇ 0.5M EDTA and immediately applied to a 1.5% agarose gel to visualize digested and undigested products by electrophoresis.
  • CD25 CD4 + T cells were isolated from BDC2.5-TCR transgenic (Tg)
  • NOD mice using a CD25 + regulatory T cell isolation kit (Miltenyi, 130-091-041). Purified CD4 + T cells were maintained in culture for three weeks prior to being cultured with NIT-1 cells by weekly stimulation with 1 mM BDC2.5 mimotope in the presence of irradiated splenocytes from NOD.sc/ri mice and 20 U/mL IL-2 (Peprotech, 212-12-20UG). The day prior to T cell and NIT-1 co-culture, NIT-1 cell lines were incubated with or without 1 mM Thapsigargin for 5 h. NIT-1 cells were washed extensively with complete DMEM after incubation.
  • NIT-1 cells 5xl0 4 NIT-1 cells were then seeded in each well of a 96-well plate in 100 pL culture medium. The next day, 10 5 BDC2.5-Tg CD4 + T cells and 5 x 10 5 NOD.sc/ri splenocytes re-suspended in 100pL RPMI medium were added to NIT-1 cultures. Cells were co-cultured in a 37° C incubator with 5% CCh for 24 hours. Cells were treated with BD Golgi plug (diluted 1 in 1,000; BD bioscience, #555029) for the last 5 h of culture.
  • BV785 CD3 (clone 17A2, #100231)
  • PE-Cy7 CD4 (clone GK1.5, #100421)
  • PE anti-TNF-a (clone MP6-XT22, #506306)
  • PE rat IgGl kappa isotype control (clone RTK2071, #400407)
  • APC IFN-g (clone XMG1.2, #505810)
  • APC rat IgGl kappa isotype control (clone RTK2071, #400411).
  • Cells were treated with or without 1.25 mM Thapsigargin in a 37° C incubator with 5% CCh for 24 hours, collected and stained with the following antibodies, all of which were purchased from BioLegend; APC anti-mouse H-2Kd (clone SFl-1.1, #116620), APC mouse IgG2a kappa isotype control (clone MOPC-173, #400219), PE anti mouse IA k (Ap k ) (clone 10-3.6, #109908) which cross-reacts with mouse I-Ag 7 , PE mouse IgG2a kappa isotype control (clone MOPC-173, #400213). Flow cytometry was performed as described above.
  • T cells were re-suspended in 100 pL complete RPMI medium. NIT-1 cells and CD8 + T cells were then mixed at a 1 : 1 ratio in the antibody-coated 96-well plate and co-cultured for 24 hours in a 37° C incubator with 5% CO2. Cells were discarded and the plate was washed with PBS-0.1% Tween20 washing buffer three times. 2 pg/mL detection antibody (Biotinylated Anti-mouse IFN- g, BD Biosciences, #51-1818KA) was added to each well and the plate was incubated for 2 hours at room temperature.
  • detection antibody Biotinylated Anti-mouse IFN- g, BD Biosciences, #51-1818KA
  • hRNLS uses flavin adenine dinucleotide (FAD) as a co-factor for catalysis. Therefore, to find compounds that could potentially inhibit hRNLS, the Protein Data Bank was searched for protein-inhibitor complex structures that had FAD. MAO-B in complex with inhibitors that covalently attached to FAD were identified through the search. Structural alignments and analysis of the hRNLS crystal structure with these complex structures based on FAD suggested that these inhibitors, for instance pargyline, may inhibit hRNLS as well.
  • FAD flavin adenine dinucleotide
  • the model of full-length hRNLS in complex with pargyline was built based on the crystal structures of human renalase (PDB: 3QJ4) (36) and MAO-B rasagiline complex (PDB: 2C65) (37).
  • the model was first optimized using the Protein Preparation Wizard (38) from Schrodinger at pH 7.0 and energy minimized with gradually reduced restraints (1000, 5, 0 force constant) on backbone and solute heavy atoms.
  • a multi-stage 100 ns molecular dynamics (MD) simulation using Desmond (39) was performed afterwards. The final frame of the MD simulation was used as the final model in Figure 6 A.
  • RNLS protein Human recombinant RNLS protein was generated by GenScript USA Inc., using the E. Coli expression vector pET28a-MBP. RNLS protein was obtained from the supernatant of cell lysates, followed by purification via Ni Bio-rad column. 2 mM RNLS dissolved in PBS was incubated with pargyline (Sigma-Aldrich, #P8013) at concentrations of 0, 0.1, 1, 10, 25, 50, 100 mM for 20 min at 4 °C before addition of SYPRO Orange dye (Invitrogen, #S6650) for the measurement of thermal denaturation.
  • pargyline Sigma-Aldrich, #P8013
  • SYPRO Orange dye Invitrogen, #S6650
  • the thermal shift assay was performed using the QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) with an initial temperature hold at 25°C for 2 min, followed by a temperature ramp up to 95°C at a rate of 1°C / s, and a final temperature hold at 95°C for 2 min. Results were collected at 0.25 °C increments.
  • the melting temperature (Tm) of RNLS in the presence and absence of pargyline was calculated by the first derivative of the fluorescence emission as a function of temperature (- dF/dT).
  • ICR 8-week old CD 1 mice
  • Islets were transduced with lentivirus encoding a non-targeting (NT) or Rnls gRNA together with rat insulin promoter- driven Cas9 endonuclease.
  • NT non-targeting
  • Rnls gRNA lentivirus encoding a non-targeting (NT) or Rnls gRNA together with rat insulin promoter- driven Cas9 endonuclease.
  • KRB Krebs Ringer Bicarbonate HEPES
  • 0.8 ml KRB was taken out, saved for insulin measurement and replaced with 0.8 ml of 20.2 mM glucose KRB buffer for a final glucose concentration of 16.8 mM for another 1 hour incubation at 37 °C.
  • the KRB buffer was again sampled for insulin, then islets were incubated with 30 mM KC1 along with 16.8 mM glucose for 1 hour at 37 °C before the final insulin sampling.
  • Genomic DNA was purified from islets for normalization of insulin levels to DNA content. Insulin levels were assessed by ultra sensitive mouse insulin ELISA kit (Crystal Chem, #90080).
  • G. R. Moran The catalytic function of renalase: A decade of phantoms. Biochim. Biophys. Acta - Proteins Proteomics . 1864, 177-186 (2016). 27.
  • the term about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated.
  • the term about generally refers to a range of numerical values (e.g., +/-5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).
  • the terms modify all of the values or ranges provided in the list.
  • the term about may include numerical values that are rounded to the nearest significant figure.

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Abstract

L'invention concerne des compositions destinées à être utilisées dans des méthodes de réduction du glucose sanguin, d'augmentation de la sécrétion d'insuline en réponse au glucose, de prévention de la mort de cellules bêta pancréatiques ou de cellules de type bêta, et de prévention du développement du diabète de type 1.
PCT/US2020/066560 2019-12-23 2020-12-22 Inhibiteurs de monoamine oxydase en tant que modificateurs de la vulnérabilité des cellules bêta dans le diabète de type 1 Ceased WO2021133777A1 (fr)

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AU2020412477A AU2020412477A1 (en) 2019-12-23 2020-12-22 Monoamine oxidase inhibitors as modifiers of beta cell vulnerability in type 1 diabetes
CA3174673A CA3174673A1 (fr) 2019-12-23 2020-12-22 Inhibiteurs de monoamine oxydase en tant que modificateurs de la vulnerabilite des cellules beta dans le diabete de type 1
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WO2023245004A1 (fr) * 2022-06-14 2023-12-21 Joslin Diabetes Center Transdifférenciation de cellules canalaires pancréatiques en cellules de type bêta

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US20190376042A1 (en) * 2017-02-28 2019-12-12 Joslin Diabetes Center Protection of Beta Cells from Immune Attack

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US20070078172A1 (en) * 2005-06-16 2007-04-05 Jenrin Discovery Mao-b inhibitors useful for treating obesity
EP2506717B1 (fr) * 2009-12-04 2015-03-25 PerkinElmer, Inc. Procédé d'utilisation d'inhibiteurs du recaptage de dopamine et de leurs analogues pour traiter les symptômes du diabète et retarder ou prévenir les états pathologiques associés au diabète

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Title
See also references of EP4081197A4 *
VENETSANAKI V., BOUTIS ANASTASIOS, CHRISOULIDOU A., PAPAKOTOULAS P.: "Diabetes Mellitus Secondary to Treatment with Immune Checkpoint Inhibitors", CURRENT ONCOLOGY, vol. 26, no. 1, 1 February 2019 (2019-02-01), pages 111 - 114, XP055838120, DOI: 10.3747/co.26.4151 *

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* Cited by examiner, † Cited by third party
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WO2023245004A1 (fr) * 2022-06-14 2023-12-21 Joslin Diabetes Center Transdifférenciation de cellules canalaires pancréatiques en cellules de type bêta

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