[go: up one dir, main page]

WO2019053727A1 - Redifferentiation of cells expanded in vitro from adult human pancreatic islet beta cells, by small-molecule inhibitors of signaling pathway - Google Patents

Redifferentiation of cells expanded in vitro from adult human pancreatic islet beta cells, by small-molecule inhibitors of signaling pathway Download PDF

Info

Publication number
WO2019053727A1
WO2019053727A1 PCT/IL2018/051053 IL2018051053W WO2019053727A1 WO 2019053727 A1 WO2019053727 A1 WO 2019053727A1 IL 2018051053 W IL2018051053 W IL 2018051053W WO 2019053727 A1 WO2019053727 A1 WO 2019053727A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
inhibitor
pathway inhibitor
bet
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IL2018/051053
Other languages
French (fr)
Inventor
Shimon Efrat
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ramot at Tel Aviv University Ltd
Original Assignee
Ramot at Tel Aviv University Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ramot at Tel Aviv University Ltd filed Critical Ramot at Tel Aviv University Ltd
Publication of WO2019053727A1 publication Critical patent/WO2019053727A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0678—Stem cells; Progenitor cells; Precursor cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/065—Modulators of histone acetylation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/22—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from pancreatic cells

Definitions

  • the present invention in some embodiments thereof, relates to a method of increasing insulin content in adult islet beta cells.
  • Type I diabetes is caused by the autoimmune destruction of the pancreatic islet insulin- producing beta cells. Insulin administration does not prevent the long-term complications of the disease, since the optimal insulin dosage is difficult to adjust. Replacement of the damaged cells with regulated insulin-producing cells is considered the ultimate cure for type 1 diabetes.
  • Pancreas transplantation has been successful but is severely limited by the shortage of donors. With the development of new islet isolation and immunosuppression procedures, significant success has been reported using islets from 2-3 donors per recipient (Shapiro AM, Lakey JR, Ryan EA et al. New Engl J Med 2000;343:230-238). This progress underscores the urgent need for developing alternatives to human pancreas donors, namely abundant sources of cultured human ⁇ cells for transplantation.
  • Terminally differentiated, postmitotic islet cells are difficult to expand in tissue culture.
  • the replication span could not be extended by expression of the catalytic subunit of human telomerase (hTERT), which was introduced into the cells with a retrovirus (Halvorsen TL, Beattie GM, Lopez AD, Hayek A, Levine F. J Endocrinol 2000;166: 103-109). Due to massive cell death, this method resulted in a 3-4 expansion of the islet cell mass.
  • the process of beta cell expansion by prolonged culture is accompanied by dedifferentiation of the cells.
  • the dedifferentiation of cells is accompanied by drastic changes in phenotype in which their morphology changes from that of epithelial cells containing extensive cell-cell junctions and cytokeratin filament networks, to cells with a fibroblast or mesenchymal appearance.
  • This process of dedifferentiation is known as an epithelial to mesenchymal transition (EMT) and is believed to be mediated in part by the induction of the zinc finger transcription factor, Snail.
  • EMT epithelial to mesenchymal transition
  • HGF hepatocyte growth factor
  • Glucagon-like peptide 1 (GLP-1), and its more stable analog exendin-4, have been shown to stimulate ⁇ -cell proliferation and to induce insulin gene expression in a pancreatic cell line.
  • GLP-1 epidermal growth factor
  • EGF epidermal growth factor
  • TGFa TGFa
  • betacellulin have also been shown to stimulate ⁇ -cell proliferation and differentiation.
  • Betacellulin is a potent mitogen for a number of cell types, including islet beta ( ⁇ ) cells. It was shown to increase islet neogenesis in alloxan and STZ-treated mice, and accelerate islet- regeneration in 90 %-pancreatectomized rats [Li L, et al., Endocrinology 2001;142:5379-5385].
  • U.S. Patent Application No. 20060292127 teaches dedifferentiating, and not redifferentiating, beta cells by contacting the cells with agents that regulate Snail/slug/slit family of transcription factors.
  • a method of ex-vivo increasing insulin content in pancreatic progenitor cells comprising:
  • pancreatic progenitor cells contacting the pancreatic progenitor cells with a Bromodomain and Extra-Terminal motif (BET) protein (BET) inhibitor and an agent selected from the group consisting of a NOTCH pathway inhibitor, a ⁇ pathway inhibitor and a WNT pathway inhibitor under conditions that increase the insulin content in the pancreatic progenitor cells; and (b) analyzing the amount of insulin in, or secreted by, the pancreatic cells, thereby increasing insulin content in the progenitor cells.
  • BET Bromodomain and Extra-Terminal motif
  • a method of ex-vivo increasing insulin content in pancreatic progenitor cells comprising culturing the pancreatic progenitor cells in CMRL medium, wherein the medium comprises a Bromodomain and Extra- Terminal motif (BET) protein (BET) inhibitor under conditions that increase the insulin content in the pancreatic progenitor cells.
  • CMRL medium comprising a Bromodomain and Extra- Terminal motif (BET) protein (BET) inhibitor under conditions that increase the insulin content in the pancreatic progenitor cells.
  • BET Bromodomain and Extra- Terminal motif
  • an isolated population of cells generated according to the method described herein.
  • a method of treating diabetes in a subject comprising transplanting a therapeutically effective amount of the population of adult islet beta cells described herein into the subject, thereby treating diabetes.
  • a pharmaceutical composition comprising as an active ingredient the population of adult islet beta cells described herein and a pharmaceutically acceptable carrier.
  • a cell medium comprising CMRL and a Bromodomain and Extra- Terminal motif (BET) protein (BET) inhibitor.
  • BET Bromodomain and Extra- Terminal motif
  • the progenitor cells are selected from the group consisting of dedifferentiated adult islet beta cells, mesenchymal stem cells and induced pluripotent stem cells dedifferentiated from beta cells.
  • the progenitor cells comprise dedifferentiated adult islet beta cells.
  • the BET inhibitor is a small molecule inhibitor.
  • the small molecule inhibitor is selected from the group consisting of CPI-0610, DUAL946, GSK525762, I-BET151, JQ1, OTX015, PFI- 1, RVX-208, RVX2135 and TEN-010.
  • the BET pathway inhibitor is I- BET151.
  • the method further comprises contacting the cells with an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor.
  • the method further comprises contacting the cells with a NOTCH pathway inhibitor and a TGFP pathway inhibitor.
  • the agent comprises two agents.
  • the first of the two agents comprises a NOTCH pathway inhibitor and a second of the two agents comprises a TGFP pathway inhibitor.
  • the NOTCH pathway inhibitor targets a protein selected from the group consisting of NICD, ⁇ -secretase and HDAC.
  • the NOTCH pathway inhibitor is a small molecule inhibitor.
  • the small molecule inhibitor is selected from the group consisting of 6-4 pyridine-3-amine, Cyclopiazonic acid, DAPT, LY685458, PF-03084014, ⁇ -Secretase Inhibitor 111. (R (-Flurbiprofen and LBH589.
  • the TGFP pathway inhibitor is a small molecule inhibitor.
  • the small molecule inhibitor is selected from the group consisting of LY573636, LY364947, LY2157299 and ALK5 Inhibitor II.
  • the WNT pathway inhibitor is selected from the group consisting of Niclosamide, Sulindac, Aspirin, Celecoxib and Indomethacin.
  • the NOTCH pathway inhibitor is PF- 03084014 and the TGFp pathway inhibitor is LY2157299.
  • the BET pathway inhibitor is I- BET151.
  • the contacting is effected on an adherent substrate.
  • the adherent substrate comprises matrigel or extracellular matrix component.
  • the extracellular matrix component is selected from the group consisting of collagen, laminin and fibronectin.
  • the dedifferentiated adult islet beta cells are generated by culturing the adult islet beta cells for at least 10 passages.
  • the culturing is effected in CMRL medium.
  • the contacting is effected for at least 3 days.
  • the method further comprises isolating the adult islet beta cells following the contacting.
  • the isolated population is for use in treating diabetes.
  • the isolated population is genetically modified to express a pharmaceutical agent.
  • the BET inhibitor is a small molecule inhibitor.
  • the small molecule inhibitor is selected from the group consisting of CPI-0610, DUAL946, GSK525762, I-BET151, JQl, OTX015, PFI- 1, RVX-208, RVX2135 and TEN-010.
  • the BET pathway inhibitor is I- BET151.
  • the cell medium further comprises an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor.
  • the cell medium further comprises a NOTCH pathway inhibitor and a TGFP pathway inhibitor.
  • the NOTCH pathway inhibitor targets a protein selected from the group consisting of NICD, ⁇ -secretase and HDAC.
  • the NOTCH pathway inhibitor is a small molecule inhibitor.
  • the small molecule inhibitor is selected from the group consisting of 6-4 pyridme-3-amine, Cyclopiazonic acid, DAPT, LY685458, PF-03084014, ⁇ -Secretase Inhibitor III, (R (-Flurbiprofen and LBH589.
  • the TGFP pathway inhibitor is a small molecule inhibitor.
  • the small molecule inhibitor is selected from the group consisting of LY573636, LY364947, LY2157299 and ALK5 Inhibitor II.
  • the WNT pathway inhibitor is selected from the group consisting of Niclosamide, Sulindac, Aspirin, Celecoxib and Indomethacin.
  • the NOTCH pathway inhibitor is PF- 03084014 and the TGFp pathway inhibitor is LY2157299.
  • FIG. 4 Redifferentiation of expanded human islet cells by treatment with NOTCH, TGFp, and BET inhibitors.
  • Cells at passage 7 were treated for 7 days with the indicated compounds in Matrigel-coated plates.
  • P PF-03084014, 1 ⁇ ; L9, LY-2157299, 0.5 ⁇ ; B, I-BET151, 0.6 ⁇ ; C, 1% DMSO control. Column labeled "0.5" marks half of the concentration of all 3 compounds. All values were statistically significant compared with C (p ⁇ 0.05).
  • FIG. 5 Changes in ⁇ -cell transcript levels in cells treated with the 3-compound combination.
  • FIG. 6 is a graph illustrating the effect of compound withdrawal following redifferentiation of expanded human islet cells by treatment with NOTCH, TGFp, and BET inhibitors.
  • FIG. 7 is a bar graph illustrating the effect of the 3 compound combination on ⁇ -cell proliferation.
  • Cells at passage 7 were treated for 7 days with the 3-compound cocktail (PF- 03084014, 1 ⁇ ; LY-2157299, 0.5 ⁇ ; I-BET151, 0.6 ⁇ ) in Matrigel-coated plates.
  • FIGs. 8A-B are graphs and photographs illustrating the expression of other islet hormones following redifferentiation of expanded human islet cells by treatment with NOTCH, TGFp, and BET inhibitors.
  • Cells at passage 7 were treated for 7 days with the 3-compound cocktail (PF- 03084014, 1 ⁇ ; LY-2157299, 0.5 ⁇ ; I-BET151, 0.6 ⁇ ) in Matrigel-coated plates.
  • Hormone- positive cells were quantitated by immuno staining, using IncuCyte instrument. Values are percentages of hormone-positive cells in a total of 30,000 cells from a single donor. The results show that the compound cocktail induces redifferentiation of other hormone-expressing cells, however all hormone-expressing cells are mono-hormonal.
  • the present invention in some embodiments thereof, relates to a method of increasing insulin content in adult islet beta cells.
  • a method of ex- vivo increasing insulin content in pancreatic progenitor cells comprising:
  • BET Bromodomain and Extra-Terminal motif
  • ex-vivo refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube).
  • pancreatic progenitor cells refers to a population of cells which are not fully differentiated into pancreatic cells, yet are committed to differentiating towards at least one type of pancreatic cell - e.g. beta cells that produce insulin; alpha cells that produce glucagon; delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide.
  • the pancreatic progenitor cells of this aspect of the present invention are capable (following differentiation) of expressing at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide.
  • pancreatic progenitor cells express some of the phenotypic markers that are characteristic of pancreatic lineages (e.g. GLUT2, PDX-1 ⁇ 3 ⁇ , PCl/3, Beta2, Nkx2.2 and PC2). Typically, they do not produce progeny of other embryonic germ layers when cultured by themselves in vitro, unless dedifferentiated or reprogrammed. It will be appreciated that it is not implied that each of the cells within the population have the capacity of forming more than one type of progeny, although individual cells that are multipotent pancreatic progenitor cells may be present.
  • the pancreatic progenitor cells according to the invention are generated (ex vivo) from embryonic stem cells, perinatal stem cell, somatic stem cells, and bioengineered stem cells, preferably the stem cells are liESC, or iPSC, in particular hiPSC.
  • stem cells are liESC, or iPSC, in particular hiPSC.
  • Non-limiting examples of human embryonic stem cells lines are for example the cell lines CHB-1 ,CHB-2, CHB-3, CHB-4, CHB-5, CHB-6. CHB-8, CHB-9, CHB-10.
  • human somatic stem cells or “human adult stem cells” as used herein refers to stem cells found throughout the human body after birth. Such cells can thus be obtained from adult tissue samples rather than human embryos, the destruction of which they do not require.
  • “human somatic stem cells” encompass hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, and testicular cells.
  • Cells derived from bone marrow and amniotic fluid which can include both hematopoietic stem cells and mesenchymal stem cells, have been found to differentiate into beta cells with manipulation in an in vitro environment (Jiang et al., Nature, 418:41-4; 2002, and De Coppi et al., Nat Biotechnology, 25: 100-106, 2007).
  • the term “human somatic stem cells” refers to hematopoietic stem cells or mesenchymal stem cells.
  • the term “hematopoietic stem cells” refers herein to a stem cell displaying a hematopoietic stem cell phenotype.
  • hematopoietic stem cells phenotype it is herein meant the expression of at least one hematopoietic stem cells marker, and /or the presence of hematopoietic stem cell morphology.
  • hematopoietic stem cell markers include, without limitation, CD34+,
  • hematopoietic stem cells are non-adherent and rounded cells, with a rounded nucleus and low cytoplasm-to-nucleus ratio. They can further be identified by their small size, lack of lineage (lin) markers, low staining (side population) with vital dyes such as rhodamine 123 (rhodamineDULL, also called rholo) or Hoechst 33342, and presence of various antigenic markers on their surface.
  • Hematopoietic stem cells can be found in bone marrow and bone marrow biological samples.
  • mesenchymal stem cells refers herein to a stem cell displaying a mesenchymal stem cell phenotype.
  • mesenchymal stem cell phenotype it is herein meant the expression of at least one mesenchymal stem cells marker, and/or the presence of a mesenchymal stem cell morphology.
  • mesenchymal stem cell markers include, without limitation, CD73, CD90 and CD105.
  • Mesenchymal stem cells lack the expression of the markers CD1 1 b, CD14, CD19, CD34, CD45, CD79a and HLA-DR.
  • mesenchymal stem cells are characterized by a small cell body with a few cell processes that are long and thin.
  • the cell body contains a large, round nucleus with a prominent nucleolus, which is surrounded by finely dispersed chromatin particles, giving the nucleus a clear appearance.
  • the remainder of the cell body contains a small amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosomes.
  • the cells, which are long and thin, are widely dispersed and the adjacent extracellular matrix is populated by a few reticular fibrils but is devoid of the other types of collagen fibrils.
  • Mesenchymal stem cells can be found for example in placenta, adipose tissue, lung, bone marrow and blood, Wharton's jelly from the umbilical cord, muscle, and teeth (perivascular niche of dental pulp and periodontal ligament).
  • the pancreatic progenitor cells comprise dedifferentiated adult islet beta cells.
  • adult islet beta cells refers to post-natal (e.g., non-embryonic) pancreatic islet endocrine cells which are capable of secreting insulin in response to elevated glucose concentrations and express typical beta cell markers.
  • beta cell markers include, but are not limited to, insulin and PDX.
  • Dedifferentiated adult islet beta cells may be generated ex- vivo from adult islet beta cells.
  • the adult beta cells may be comprised in pancreatic islets or may be isolated from the islets.
  • Pancreatic islets comprise the following: 1) beta cells that produce insulin; 2) alpha cells that produce glucagon; 3) delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide.
  • the polypeptide hormones (insulin, glucagon, somatostatin and pancreatic polypeptide) inside these cells are stored in secretary vesicles in the form of secretory granules.
  • islets may be isolated from pancreatic tissue using collagenase and ficoll gradients.
  • the adult islet beta cells of the present invention are dispersed into a single cell suspension - e.g. by the addition of trypsin or by trituration.
  • the adult islet beta cells may be further isolated being substantially free from other substances (e.g., other cells, proteins, nucleic acids, etc.) that are present in its in-vivo environment e.g. by FACs sorting.
  • substances e.g., other cells, proteins, nucleic acids, etc.
  • the adult islet beta cells may be obtained from any autologous or non- autologous (i.e., allogeneic or xenogeneic) mammalian donor.
  • cells may be isolated from a human cadaver.
  • Dedifferentiation of the adult islet beta cells may be effected by expansion (i.e. culturing) for a prolonged number of passages - e.g. in CMRL medium.
  • expanding refers to increasing the number and/or overall mass of adult islet beta cells of the present invention by the process of cell division, rather than simply enlarging by hypertrophy.
  • the cells are expanded in Mesencult XF medium comprising glucose at a concentration of about 10-100 mM glucose, more preferably 10-50 mM, more preferably 10-25 mM, such as for example 25 mM.
  • the cells are preferably expanded for at least 6 passages, 7 passages, 8 passages, 9 passages, 10 passages, 11 passages, 12 passages, 13 passages, 14 passages, 15 passages or at least 16 passages.
  • the expanding is effected under adherent conditions which comprise incubating on an attachment medium selected from the group consisting of laminin, fibronectin, matrigel and Mesencult XF attachment substrate.
  • dedifferentiation of the adult islet beta cells may be effected by transfecting the cells with genes known to generated induced pluripotent stem cells (iPS) cells.
  • iPS induced pluripotent stem cells
  • Oct-3/4 and certain members of the Sox gene family have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible.
  • Additional genes including certain members of the Klf family (Klfl, Klf2, Klf4, and Klf5), the Myc family (C-myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency.
  • Redifferentiation of the cells is effected in the presence of a Bromodomain and Extra- Terminal motif (BET) protein (BET) inhibitor and an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor.
  • BET Bromodomain and Extra- Terminal motif
  • BET inhibitor refers to an agent that inhibits the binding of BET family bromodomains to acetylated lysine residues.
  • BET family bromodomains it is meant a polypeptide comprising two bromodomains and an extraterminal (ET) domain or a fragment thereof having transcriptional regulatory activity or acetylated lysine binding activity.
  • Exemplary BET family members include BRD2, BRD3, BRD4 and BRDT are given in WO 2011/143669. Examples of BET inhibitors include but are not limited to the compounds of the instant invention.
  • the BET inhibitor according to the invention targets BD1 and/or BD2, and is preferentially a small molecule agent.
  • small molecule agents include, but are not limited to BET762, TEN-010, CPI-203, LY29002, RVX8, CPI-0610, DUAL946, GSK525762, 1-BET151, JQ1, OTX015, PFI-1, RVX-208, RVX2135, preferentially BET 151 and JQ1, and even more preferably BET 151.
  • the cell culture medium according to the invention comprises the BET inhibitor in a concentration from ⁇ to ⁇ , preferentially from 0.1 ⁇ to 1 ⁇ , from 0.3 ⁇ to 0.8 ⁇ or from 0.4 ⁇ to 0.7 ⁇ - for example about 0.6 ⁇ .
  • Polynucleotide agents capable of down-regulating expression of BET family members are described herein below.
  • Notch pathway inhibitor refers to an agent that is capable of downregulating activity and/or amount of a component participating in the NOTCH pathway. Exemplary components are described herein below.
  • the Notch signaling pathway is a conserved intercellular signaling mechanism that is essential for proper embryonic development in numerous metazoan organisms.
  • Members of the Notch gene family (NOTCHs) encode transmembrane receptors that are critical for various cell fate decisions. Multiple ligands that activate Notch and related receptors have been identified, including Senate and Delta in Drosophila and JAG1 (MIM.601920) in vertebrates.
  • Notch receptors NOTCH 1 to NOTCH4
  • five ligands Jagged-1 (JAG1) and -2 (JAG2) and Delta-like [DLLs]: DLL1, DLL2 and DLL4
  • JAG1 and -2 JAG2
  • DLL1 and DLL2 Two ligands
  • JAG2 JAG2
  • DLL1 and DLL2 Two ligands
  • the Notch pathway regulates cell fate determination of neighboring cells through lateral inhibitiona, depending on their ability to express either the receptors or the ligands.
  • NOTCHs are activated by a series of cleavages that releases its intracellular domain (NICD).
  • NBD intracellular domain
  • ADAM 17 tumor necrosis factor-alpha converting enzyme or TACE MEVI.603369
  • PSENl MIM.104311 presenilin-1
  • Nuclear translocation of NICD results in transcriptional activation of genes of the HESs family (Hes/E(spl) family) and HEYs family (Hesr/Hey family) through interaction of NICD with RBPSUH (or CBFl MIM.147183), Su(H), and Lag-1, which is also known as the recombination signal sequence-binding protein (RBP)-j (also called Suppressor of Hairless, Su(H)), each of these also falling under the category of a component of a NOTCH pathway.
  • RBPSUH or CBFl MIM.147183
  • Su(H) HEYs family
  • Lag-1 which is also known as the recombination signal sequence-binding protein (RBP)-j (also called Suppressor of Hairless, Su(H))
  • Notch signaling when activated, Notch signaling enables neighboring cells to acquire distinct phenotypes, through a process named lateral inhibition.
  • the Notch receptor is pre-cleaved in the Golgi and is targeted subsequently to the plasma membrane where it interacts with ligands located on neighboring cells.
  • This proteolytic activity enables the Notch intracellular domain (NICD) to translocate to the nucleus where it activates the transcription of target genes (e.g. the Hes and Hey family of transcriptional repressors).
  • TACE Notch intracellular domain
  • Monoubiquitylation (Ub) of the ligand by mindbomb (MIB) induces endocytosis of the ligand and the Notch extracellular domain (NECD) into the ligand cells where additional signaling might be initiated.
  • Notch receptors undergo a complex set of proteolytic processing events in response to ligand activating, which eventually leads to release of the intracellular domain of the receptor.
  • Signal transduction is normally initiated by binding to transmembrane ligands of the Serrate or Delta class, which induces proteolytic release of the intracellular NOTCH domain (NICD).
  • NBD NOTCH domain
  • Free NICD translocates to the nucleus to form a short-lived complex with a Rel-like transcription factor, CSL, and Mastermind-like co-activators that activates lineage- specific programs of gene expression.
  • CSL Rel-like transcription factor
  • the present invention contemplates down-regulating any component of the NOTCH pathway that is up-regulated in B cell dedifferentiation above a predetermined threshold.
  • the component of the NOTCH pathway which is targeted is NICD, ⁇ -secretase and/or HDAC.
  • the NOTCH pathway component is upregulated by at least 1.5 times, more preferably by at least 2 times and more preferably by at least 3 times.
  • RNA level using techniques such as Northern blot analysis, RT-PCR and oligonucleotides microarray
  • protein level using techniques such as ELISA, Western blot analysis, immunohistochemistry and the like, which may be effected using antibodies specific to the NOTCH pathway component.
  • the NOTCH pathway component is Hairy and Enhancer of Split 1 (HES 1; NM.sub.-005524, NP.sub.-005515), NOTCH1 (NM.sub.-017617, NP.sub.-060087.3) NOTCH 2 (NM.sub.-024408, NP.sub.-077719.2) and NOTCH 3 (NM.sub.- -000435, NP.sub.-000426 .2).
  • HES 1 Hairy and Enhancer of Split 1
  • NOTCH1 NM.sub.-017617, NP.sub.-060087.3
  • NOTCH 2 NM.sub.-024408, NP.sub.-077719.2
  • NOTCH 3 NM.sub.- -000435, NP.sub.-000426 .2
  • Exemplary small molecule inhibitors of the NOTCH pathway include 6-4 pyridines- amine. Cyclopiazonic acid, DAPT, LY685458, PF-03084014, ⁇ -Secretase Inhibitor III, (R)- Flurbiprofen and LBH589. Additional inhibitors are set forth in Table 1 below.
  • TGFP pathway inhibitor refers to an agent that is capable of downregulating activity and/or amount of a component participating in the TGFP pathway. Exemplary components are described herein below.
  • TGFP signals are conveyed through two transmembrane serine-threonine kinase receptors (type I and type II TGFP receptors) to the five receptor-regulated (R)-SMAD transcription factors (SMAD1-3, 5, 8), which translocate into the nucleus, recruit transcriptional co-activators and co- repressors, and regulate gene expression.
  • the type I receptor family is comprised of activin-like kinase (ALK) receptors 1 through 7.
  • ALK activin-like kinase
  • Two major pathway branches are activated by TGFP family ligands: SMAD1, 5 and 8 are activated by BMPs through ALK1-3 and 6, whereas SMAD2 and 3 are activated by TGFp, activins, and nodals, through ALK4, 5 and 7.
  • SMAD2 and 3 phosphorylation by ALK5 (also termed TGBRI) is the best-characterized TGFP pathway signaling effect, and the main one associated with EMT, whereas mesenchymal-epithelial transition (MET) is associated primarily with phosphorylation of SMAD1, 5 and 8.
  • the inhibitor targets ALK-5.
  • the TGFP pathway component is upregulated by at least 1.5 times, more preferably by at least 2 times and more preferably by at least 3 times.
  • Methods of analyzing whether a particular component is upregulated during B cell differentiation are known in the art, and may be effected on the RNA level (using techniques such as Northern blot analysis, RT-PCR and oligonucleotides microarray) and/or the protein level (using techniques such as ELISA, Western blot analysis, immunohistochemistry and the like, which may be effected using antibodies specific to the TGFP pathway component).
  • Exemplary small molecule inhibitors of the TGFP pathway include but are not limited to LY573636, LY364947, LY2157299 and ALK5 Inhibitor II. Additional inhibitors are set forth in Table 1 below.
  • Polynucleotide agents capable of down -regulating expression of a component of the TGFP pathway are described herein below.
  • Wnt pathway inhibitor refers to an agent that is capable of downregulating activity and/or amount of a component participating in the Wnt pathway.
  • exemplary components include ⁇ -catenin, Zinc Finger E-Box Binding Homeobox 1 (ZEB-1), TWIST, SNAIL, SOX-2 and SOX-6.
  • the RNA transcript encoding the component of the Wnt pathway is targeted by miRNA-200c.
  • the Wnt pathway component is upregulated by at least 1.5 times, more preferably by at least 2 times and more preferably by at least 3 times.
  • RNA level using techniques such as Northern blot analysis, RT-PCR and oligonucleotides microarray
  • protein level using techniques such as ELISA, Western blot analysis, immunohistochemistry and the like, which may be effected using antibodies specific to the Wnt pathway component.
  • Exemplary small molecule inhibitors of the Wnt pathway include but are not limited to Niclosamide, Sulindac, Aspirin, Celecoxib and Indomethacin. Additional inhibitors are set forth in Table 1 below.
  • Polynucleotide agents capable of down-regulating expression of a component of the Wnt pathway are described herein below.
  • ALK5 Inhibitor II TGFP TGFPRI (ALK5) 1-20 ⁇
  • Particularly effective combinations of inhibitors include: PF-03084014 together with LY2157299; and PF-03084014 together with LY2157299 and I-BET151 (e.g. at the disclosed concentrations).
  • the present inventors also contemplate the use of polynucleotide agents or antibodies that downregulate expression of at least one component of any of the pathways described herein above.
  • the antibody specifically binds at least one epitope of a single component of any one of the pathways described herein.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • antibody as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages.
  • These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of
  • RNA silencing Downregulation of a particular component of any one of the pathways described herein can be also achieved by RNA silencing.
  • RNA silencing refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post- transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein- coding gene.
  • RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
  • RNA silencing agent refers to an RNA which is capable of specifically inhibiting or “silencing” the expression of a target gene.
  • the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism.
  • RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
  • RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
  • the RNA silencing agent is capable of inducing RNA interference.
  • the RNA silencing agent is capable of mediating translational repression.
  • the RNA silencing agent is specific to the target RNA and does not cross inhibit or silence a gene or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene.
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
  • siRNAs short interfering RNAs
  • the corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi.
  • the process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla.
  • Such protection from foreign gene expression may have evolved in response to the production of double- stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single- stranded RNA or viral genomic RNA.
  • dsRNAs double- stranded RNAs
  • RNA-induced silencing complex RISC
  • some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.
  • the dsRNA is greater than 30 bp.
  • dsRNA i.e. dsRNA greater than 30 bp
  • dsRNA i.e. dsRNA greater than 30 bp
  • the use of long dsRNAs can provide numerous advantages in that the cell can select the optimal silencing sequence alleviating the need to test numerous siRNAs; long dsRNAs will allow for silencing libraries to have less complexity than would be necessary for siRNAs; and, perhaps most importantly, long dsRNA could prevent viral escape mutations when used as therapeutics.
  • the invention contemplates introduction of long dsRNA (over 30 base transcripts) for gene silencing in cells where the interferon pathway is not activated (e.g. embryonic cells and oocytes) see for example Billy et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides, Oct. 1, 2003, 13(5): 381-392. doi: 10.1089/154545703322617069.
  • long dsRNA over 30 base transcripts
  • the invention also contemplates introduction of long dsRNA specifically designed not to induce the interferon and PKR pathways for down- regulating gene expression.
  • Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.
  • siRNAs small inhibitory RNAs
  • siRNA refers to small inhibitory RNA duplexes (generally between 18-30 base- pairs) that induce the RNA interference (RNAi) pathway.
  • RNAi RNA interference
  • siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location.
  • RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
  • RNA agent refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11.
  • RNA silencing agent may be a miRNA or miRNA mimic.
  • miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants.
  • the primary transcript (termed the “pri- miRNA”) is processed through various nucleolytic steps to a shorter precursor miRNA, or "pre- miRNA.”
  • the pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually base- pair with the target)
  • the pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex.
  • miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17: 1376-1386).
  • miRNAs bind to transcript sequences with only partial complementarity (Zeng et al., 2002, Molec. Cell 9: 1327-1333) and repress translation without affecting steady-state RNA levels (Lee et al., 1993, Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862). Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (Hutvagner et al., 2001, Science 293:834-838; Grishok et al., 2001, Cell 106: 23-34; Ketting et al., 2001, Genes Dev.
  • miRNA capable of downregulating ZEB-1, SOX-2 and SOX-6 is miR-200. It will be appreciated from the description provided herein above that contacting cells with a miRNA may be effected by transfecting the cells with e.g. the mature double stranded miRNA, the pre-miRNA or the pri-miRNA.
  • the pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
  • the pri-miRNA sequence may comprise from 45-30,000, 50-25,000, 100-20,000, 1,000- 1,500 or 80-100 nucleotides.
  • the term "microRNA”, “miRNA”, and “miR” are synonymous and refer to a collection of non-coding single- stranded RNA molecules of about 19-28 nucleotides in length, which regulate gene expression. miRNAs are found in a wide range of organisms and have been shown to play a role in development, homeostasis, and disease etiology.
  • miRNA mimic refers to synthetic non-coding RNAs that are capable of entering the RNAi pathway and regulating gene expression. miRNA mimics imitate the function of endogenous miRNAs and can be designed as mature, double stranded molecules or mimic precursors (e.g., or pre-miRNAs). miRNA mimics can be comprised of modified or unmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acid chemistries (e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (EN A)).
  • nucleic acid chemistries e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (EN A)
  • the length of the duplex region can vary between 13-33, 18-24 or 21-23 nucleotides.
  • the miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
  • the sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA.
  • the sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
  • Preparation of miRNAs mimics can be effected by any method known in the art such as chemical synthesis or recombinant methods.
  • RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the selected mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245].
  • UTRs untranslated regions
  • siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90% decrease in cellular GAPDH mRNA and completely abolished protein level (www(dot)ambion(dot)com/techlib/tn/91/912(dot)html).
  • potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
  • sequence alignment software such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/).
  • Qualifying target sequences are selected as template for siRNA synthesis.
  • Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%.
  • Several target sites are preferably selected along the length of the target gene for evaluation.
  • a negative control is preferably used in conjunction.
  • Negative control siRNA preferably includes the same nucleotide composition as the siRNAs but lack significant homology to the genome.
  • a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • the RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically- modified nucleotides and non-nucleotides.
  • the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide.
  • a "cell-penetrating peptide” is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell.
  • the cell- penetrating peptide used in the membrane-permeable complex of some embodiments of the invention preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double- stranded ribonucleic acid that has been modified for such linkage.
  • Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference.
  • the cell- penetrating peptides of some embodiments of the invention preferably include, but are not limited to, penetratin, transportan, pisl, TAT(48-60), pVEC, MTS, and MAP.
  • Another agent capable of downregulating a component of any of the pathways described herein is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or pathway.
  • DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262)
  • a general model (the " 10-23" model) for the DNAzyme has been proposed.
  • " 10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each.
  • DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL.
  • Downregulation of at least one component can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the component.
  • Ribozyme molecule capable of specifically cleaving an mRNA transcript encoding the component.
  • Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)].
  • the possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
  • Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double- stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDS) and non-homologous end-joining (NFfEJ).
  • HDS homology directed repair
  • NFfEJ non-homologous end-joining
  • HDR utilizes a homologous sequence as a template for regenerating the missing DNA sequence at the break point.
  • a DNA repair template containing the desired sequence must be present during HDR.
  • Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location.
  • restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location.
  • ZFNs Zinc finger nucleases
  • TALENs transcription-activator like effector nucleases
  • CRISPR/Cas system CRISPR/Cas system.
  • CRISPR-Cas system Many bacteria and archaea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) genes that produce RNA components and CRISPR associated (Cas) genes that encode protein components.
  • CRISPR RNAs crRNAs
  • crRNAs contain short stretches of homology to specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen.
  • RNA/protein complex RNA/protein complex and together are sufficient for sequence- specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821.) ⁇ It was further demonstrated that a synthetic chimeric guide RNA (gRNA) composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary to the crRNA in vitro.
  • gRNA synthetic chimeric guide RNA
  • transient expression of Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double- stranded brakes in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a,b; Jinek et al., 2013; Mali et al., 2013).
  • the CRIPSR/Cas system for genome editing contains two distinct components: a gRNA and an endonuclease e.g. Cas9.
  • the gRNA is typically a 20 nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript.
  • the gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA.
  • the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence.
  • PAM Protospacer Adjacent Motif
  • the binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break.
  • the double-stranded brakes produced by CRISPR/Cas can undergo homologous recombination or NHEJ.
  • the Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA.
  • CRISPR/Cas A significant advantage of CRISPR/Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs enables multiple genes to be targeted simultaneously. In addition, the majority of cells carrying the mutation present biallelic mutations in the targeted genes.
  • 'nickases' Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called 'nickases' .
  • the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or 'nick'.
  • a single-strand break, or nick is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template.
  • two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a double-strand break, in what is often referred to as a 'double nick' CRISPR system.
  • a double-nick can be repaired by either NHEJ or HDR depending on the desired effect on the gene target.
  • using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off- target effect as either gRNA alone will result in nicks that will not change the genomic DNA.
  • dCas9 Modified versions of the Cas9 enzyme containing two inactive catalytic domains
  • dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains.
  • the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.
  • both gRNA and Cas9 should be expressed in a target cell.
  • the insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids.
  • CRISPR plasmids are commercially available such as the px330 plasmid from Addgene.
  • the present invention also contemplates culturing the cells in a medium comprising additional differentiation factors under conditions to allow differentiation of the cells into insulin producing cells.
  • the cells are redifferentiated (together with the inhibitory agents disclosed herein) in a culture medium comprising nicotinamide, exendin-4, activin A and 10-50 mM glucose, the culture medium being devoid of serum.
  • Exemplary culture media contemplated by the present inventors which may be used to redifferentiate the progenitor cells include CMRL-1066, DMEM, RPMI etc.
  • Exemplary concentration ranges of nicotinamide include 1-100 mM, more preferably 1-50 mM, more preferably 1-20 mM, such as for example 10 mM.
  • Exemplary concentration ranges of exendin 4 include 1-100 nM, more preferably 1-50 nM, more preferably 1-20 nM, such as for example 8 nM.
  • Exemplary concentration ranges of activin A include 1-100 nM, more preferably 1-50 nM, more preferably 1-20 nM, such as for example 8 nM.
  • Exemplary concentration ranges of glucose include 10-100 mM, more preferably 10-50 mM, more preferably 10-25 mM, such as for example 25 mM.
  • the cells are cultured on an adherent substrate, such as matrigel or an extracellular matrix component.
  • adherent substrate such as matrigel or an extracellular matrix component.
  • extracellular matrix components contemplated by the present invention include, but are not limited to collagen, laminin and fibronectin.
  • the differentiating agents described herein are preferably included in the differentiation medium.
  • Cells may be cultured in the differentiation medium (i.e. with the agents) for at least one day, at least two days, at least 3 days or more.
  • the cells are not cultured in the differentiating medium for more than 3 weeks, more than two weeks or even more than 1 week.
  • the cells may be contacted with the differentiating agents for 3 days- 10 days (e.g. one week).
  • Cells obtained using the methods described herein are capable of secreting insulin in a glucose responsive manner and typically express beta cell specific genes (e.g. PDX-1).
  • beta cell specific genes e.g. PDX-1
  • At least 10 % of the cells of the isolated populations generated according to the methods described herein are cells redifferentiated from beta cells.
  • At least 20 % of the cells of the isolated populations generated according to the methods described herein are cells redifferentiated from beta cells.
  • At least 30 % of the cells of the isolated populations generated according to the methods described herein are cells redifferentiated from beta cells.
  • At least 40 % of the cells of the isolated populations generated according to the methods described herein are cells redifferentiated from beta cells.
  • At least 20 % of the cells of the isolated populations generated according to the methods described herein secrete insulin.
  • At least 30 % of the cells of the isolated populations generated according to the methods described herein secrete insulin.
  • At least 40 % of the cells of the isolated populations generated according to the methods described herein secrete insulin.
  • At least 50 % of the cells of the isolated populations generated according to the methods described herein secrete insulin.
  • a majority of the cells in the populations described herein express PDX1, NKX2.2, NKX6.1, IAPP and PC 1/3 and additional proteins essential for beta cell function.
  • the cells express an increase amount of beta cell transcription factors (e.g. HBLX9, NEUROD, NKX2.2, and NKX6.1), as compared to non-redifferentiated beta cells, as measured by RT-PCR.
  • beta cell transcription factors e.g. HBLX9, NEUROD, NKX2.2, and NKX6.1
  • genes which may be upregulated in the isolated populations described herein include KIR6.2, SUR1 and GCK.
  • the population of adult islet beta cells of the present invention may be further modified (e.g. genetic modification) to express a pharmaceutical agent such as a therapeutic agent, a telomerase gene, an agent that reduces immune mediated rejection or a marker gene.
  • a pharmaceutical agent such as a therapeutic agent, a telomerase gene, an agent that reduces immune mediated rejection or a marker gene.
  • therapeutic agents such as antimetabolites (e.g., purine analogs, pyrimidine analogs), enzyme inhibitors and peptidomimetics may be generally useful in the present invention.
  • An example of a gene that may reduce immune mediated rejection is the uteroglobin gene. Uteroglobin is a protein expressed during pregnancy that confers immunologic tolerance and prevents inflammatory reactions.
  • the beta cells may be isolated from other pancreatic cells present in the islet (if this hasn't been carried out prior to the redifferentiation process). This may be effected using zinc binding dyes such as Newport green (see Parnaud G, et al. Proliferation of sorted human and rat beta cells. Diabetologia 2008. 51:91- 100) or with anti-NCAM antibodies (see Banerjee M, Otonkoski T. A simple two-step protocol for the purification of human pancreatic beta cells. Diabetologia 2009. 52:621-625.).
  • zinc binding dyes such as Newport green (see Parnaud G, et al. Proliferation of sorted human and rat beta cells. Diabetologia 2008. 51:91- 100) or with anti-NCAM antibodies (see Banerjee M, Otonkoski T. A simple two-step protocol for the purification of human pancreatic beta cells. Diabetologia 2009. 52:621-625.).
  • the adult islet pancreatic cells of the present invention store and secrete insulin in a glucose responsive manner, they may be used for treating a disease which is associated with insulin deficiency such as diabetes.
  • diabetes refers to a disease resulting either from an absolute deficiency of insulin (type 1 diabetes) due to a defect in the biosynthesis or production of insulin, or a relative deficiency of insulin in the presence of insulin resistance (type 2 diabetes), i.e., impaired insulin action, in an organism.
  • type 1 diabetes an absolute deficiency of insulin
  • type 2 diabetes a relative deficiency of insulin in the presence of insulin resistance
  • the diabetic patient thus has absolute or relative insulin deficiency, and displays, among other symptoms and signs, elevated blood glucose concentration, presence of glucose in the urine and excessive discharge of urine.
  • treating refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition in an individual suffering from, or diagnosed with, the disease, disorder or condition.
  • Those of skill in the art will be aware of various methodologies and assays which can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays which can be used to assess the reduction, remission or regression of a disease, disorder or condition.
  • transplanting refers to providing the redifferentiated adult islet beta cells of the present invention, using any suitable route.
  • beta cell therapy is effected by injection using a catheter into the portal vein of the liver, although other methods of administration are envisaged.
  • the adult islet beta cells of the present invention can be derived from either autologous sources or from allogeneic sources such as human cadavers or donors. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non- autologous cells in immune-isolating, semipermeable membranes before transplantation.
  • Human islet cell expansion Human islet cells were expanded in culture as described
  • Expanded islet cells at passages 6-7 were seeded in Matrigel-coated 12-well plates at 7 X 10 4 cells/well. They were then treated with the indicated small-molecule combinations for 7 days, and compared to cells treated with 1% DMSO (solvent negative control).
  • FIG. 4 illustrates the synergistic effect of the combination of PF-03084014, LY-2157299 and I-BET151 over each of the agents individually on human pancreatic beta cells at passage 7.
  • Figure 2 shows an image of cells re-differentiated with this 3-compound combination.
  • FIG. 3 illustrates an additional analysis of changes in ⁇ -cell transcript levels in cells treated with this 3-compound combination showing a large increase in transcripts encoding other ⁇ -cell expressed proteins - IAPP, NKX2.2, NKX 6.1, ABCC8, GCK, PCSK1 and NeuroDl.
  • Figure 6 is a graph illustrating the effect of compound withdrawal following redifferentiation of expanded human islet cells by treatment with NOTCH, ⁇ , and BET inhibitors. The results show that redifferentiation is stable upon compound removal. SFM may induce further differentiation.
  • Figure 7 is a graph illustrating the effect of the 3 compound combination on ⁇ -cell proliferation. The results show that redifferentiation is accompanied by growth arrest.
  • Figures 8A-B are graphs and photographs illustrating the expression of other islet hormones following redifferentiation of expanded human islet cells by treatment with NOTCH, TGFp, and BET inhibitors. The results show that the compound cocktail induces redifferentiation of other hormone-expressing cells, however all hormone-expressing cells are mono-hormonal.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'augmentation ex vivo de la teneur en insuline dans des cellules progénitrices pancréatiques. Le procédé consiste à (a) mettre en contact lesdites cellules progénitrices pancréatiques avec un inhibiteur de la protéine BET à motif de bromodomaine et domaine extraterminal (BET) et avec un agent choisi dans le groupe constitué par un inhibiteur de la voie NOTCH, un inhibiteur de la voie du TGF-bêta et un inhibiteur de la voie WNT dans des conditions qui augmentent la teneur en insuline dans lesdites cellules progénitrices pancréatiques; et (b) analyser la quantité d'insuline dans, ou sécrétée par, lesdites cellules pancréatiques.The present invention relates to a method for ex vivo increase of insulin content in pancreatic progenitor cells. The method comprises (a) contacting said pancreatic progenitor cells with a BRE mod amenity and extraterminal domain (BET) motif inhibitor and with an agent selected from the group consisting of an inhibitor of the NOTCH pathway, an inhibitor the TGF-beta pathway and an inhibitor of the WNT pathway under conditions that increase the insulin content in said pancreatic progenitor cells; and (b) analyzing the amount of insulin in, or secreted by, said pancreatic cells.

Description

REDIFFERENTIATION OF CELLS EXPANDED IN VITRO FROM ADULT HUMAN PANCREATIC ISLET BETA CELLS, BY SMALL-MOLECULE INHIBITORS OF
SIGNALING PATHWAY FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments thereof, relates to a method of increasing insulin content in adult islet beta cells.
Type I diabetes is caused by the autoimmune destruction of the pancreatic islet insulin- producing beta cells. Insulin administration does not prevent the long-term complications of the disease, since the optimal insulin dosage is difficult to adjust. Replacement of the damaged cells with regulated insulin-producing cells is considered the ultimate cure for type 1 diabetes. Pancreas transplantation has been successful but is severely limited by the shortage of donors. With the development of new islet isolation and immunosuppression procedures, significant success has been reported using islets from 2-3 donors per recipient (Shapiro AM, Lakey JR, Ryan EA et al. New Engl J Med 2000;343:230-238). This progress underscores the urgent need for developing alternatives to human pancreas donors, namely abundant sources of cultured human β cells for transplantation.
Terminally differentiated, postmitotic islet cells are difficult to expand in tissue culture. Adult and fetal human islet cells grown on HTB-9 matrix in RPMI 1640 medium containing 11 mM glucose, and supplemented with 10 % FBS and hepatocyte growth factor, were shown to proliferate at the most for 10-15 population doublings, after which they underwent senescence. The replication span could not be extended by expression of the catalytic subunit of human telomerase (hTERT), which was introduced into the cells with a retrovirus (Halvorsen TL, Beattie GM, Lopez AD, Hayek A, Levine F. J Endocrinol 2000;166: 103-109). Due to massive cell death, this method resulted in a 3-4 expansion of the islet cell mass.
International Application WO2006/054305 teaches expansion of islet cells in CMRL-1066 medium.
The process of beta cell expansion by prolonged culture is accompanied by dedifferentiation of the cells.
In many instances, the dedifferentiation of cells is accompanied by drastic changes in phenotype in which their morphology changes from that of epithelial cells containing extensive cell-cell junctions and cytokeratin filament networks, to cells with a fibroblast or mesenchymal appearance. This process of dedifferentiation is known as an epithelial to mesenchymal transition (EMT) and is believed to be mediated in part by the induction of the zinc finger transcription factor, Snail.
A number of factors have been shown to promote both β-cell proliferation and differentiation in tissue culture. Members of the growth hormone family, including placental lactogen (PL), growth hormone (GH) and prolactin (PRL), induce replication in neonatal rat islet cells. Significant mitogenic effects of hepatocyte growth factor (HGF) have been observed on human fetal and adult islets and mouse islets. In the presence of activin A or nicotinamide, HGF has been shown to stimulate β-cell differentiation in cultured fetal pancreatic islets as well as a pancreatic cell line. Glucagon-like peptide 1 (GLP-1), and its more stable analog exendin-4, have been shown to stimulate β-cell proliferation and to induce insulin gene expression in a pancreatic cell line. Members of the epidermal growth factor (EGF) family, including EGF, TGFa and betacellulin, have also been shown to stimulate β-cell proliferation and differentiation. Betacellulin is a potent mitogen for a number of cell types, including islet beta (β) cells. It was shown to increase islet neogenesis in alloxan and STZ-treated mice, and accelerate islet- regeneration in 90 %-pancreatectomized rats [Li L, et al., Endocrinology 2001;142:5379-5385].
International Application WO2006/054305 teaches redifferentiation of expanded islet beta cells in a medium comprising betacellulin and/or ngn-3.
International Application WO2009/078012 teaches redifferentiation of expanded islet beta cells by downregulation of the NOTCH pathway.
International Application WO2012/035539 teaches redifferentiation of expanded islet beta cells by downregulating the protein SLUG.
U.S. Patent Application No. 20060292127 teaches dedifferentiating, and not redifferentiating, beta cells by contacting the cells with agents that regulate Snail/slug/slit family of transcription factors.
Additional background art includes US Patent Application No. 20170107487,
WO2018002290 and Fu et al. eLife 2014;3:e04631. DOI: 10.7554/eLife.04631.
SUMMARY OF THE INVENTION
According to an aspect of the present invention there is provided a method of ex-vivo increasing insulin content in pancreatic progenitor cells, comprising:
(a) contacting the pancreatic progenitor cells with a Bromodomain and Extra-Terminal motif (BET) protein (BET) inhibitor and an agent selected from the group consisting of a NOTCH pathway inhibitor, a ΤΰΡβ pathway inhibitor and a WNT pathway inhibitor under conditions that increase the insulin content in the pancreatic progenitor cells; and (b) analyzing the amount of insulin in, or secreted by, the pancreatic cells, thereby increasing insulin content in the progenitor cells.
According to an aspect of the present invention there is provided a method of ex-vivo increasing insulin content in pancreatic progenitor cells, comprising culturing the pancreatic progenitor cells in CMRL medium, wherein the medium comprises a Bromodomain and Extra- Terminal motif (BET) protein (BET) inhibitor under conditions that increase the insulin content in the pancreatic progenitor cells.
According to an aspect of the present invention there is provided an isolated population of cells generated according to the method described herein.
According to an aspect of the present invention there is provided a method of treating diabetes in a subject, comprising transplanting a therapeutically effective amount of the population of adult islet beta cells described herein into the subject, thereby treating diabetes.
According to an aspect of the present invention there is provided a pharmaceutical composition comprising as an active ingredient the population of adult islet beta cells described herein and a pharmaceutically acceptable carrier.
According to an aspect of the present invention there is provided a cell medium comprising CMRL and a Bromodomain and Extra- Terminal motif (BET) protein (BET) inhibitor.
According to embodiments of the present invention, the progenitor cells are selected from the group consisting of dedifferentiated adult islet beta cells, mesenchymal stem cells and induced pluripotent stem cells dedifferentiated from beta cells.
According to embodiments of the present invention, the progenitor cells comprise dedifferentiated adult islet beta cells.
According to embodiments of the present invention, the BET inhibitor is a small molecule inhibitor.
According to embodiments of the present invention, the small molecule inhibitor is selected from the group consisting of CPI-0610, DUAL946, GSK525762, I-BET151, JQ1, OTX015, PFI- 1, RVX-208, RVX2135 and TEN-010.
According to embodiments of the present invention, the BET pathway inhibitor is I- BET151.
According to embodiments of the present invention, the method further comprises contacting the cells with an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor.
According to embodiments of the present invention, the method further comprises contacting the cells with a NOTCH pathway inhibitor and a TGFP pathway inhibitor. According to embodiments of the present invention, the agent comprises two agents.
According to embodiments of the present invention, the first of the two agents comprises a NOTCH pathway inhibitor and a second of the two agents comprises a TGFP pathway inhibitor.
According to embodiments of the present invention, the NOTCH pathway inhibitor targets a protein selected from the group consisting of NICD, γ-secretase and HDAC.
According to embodiments of the present invention, the NOTCH pathway inhibitor is a small molecule inhibitor.
According to embodiments of the present invention, the small molecule inhibitor is selected from the group consisting of 6-4 pyridine-3-amine, Cyclopiazonic acid, DAPT, LY685458, PF-03084014, γ-Secretase Inhibitor 111. (R (-Flurbiprofen and LBH589.
According to embodiments of the present invention, the TGFP pathway inhibitor is a small molecule inhibitor.
According to embodiments of the present invention, the small molecule inhibitor is selected from the group consisting of LY573636, LY364947, LY2157299 and ALK5 Inhibitor II.
According to embodiments of the present invention, the WNT pathway inhibitor is selected from the group consisting of Niclosamide, Sulindac, Aspirin, Celecoxib and Indomethacin.
According to embodiments of the present invention, the NOTCH pathway inhibitor is PF- 03084014 and the TGFp pathway inhibitor is LY2157299.
According to embodiments of the present invention, the BET pathway inhibitor is I- BET151.
According to embodiments of the present invention, the contacting is effected on an adherent substrate.
According to embodiments of the present invention, the adherent substrate comprises matrigel or extracellular matrix component.
According to embodiments of the present invention, the extracellular matrix component is selected from the group consisting of collagen, laminin and fibronectin.
According to embodiments of the present invention, the dedifferentiated adult islet beta cells are generated by culturing the adult islet beta cells for at least 10 passages.
According to embodiments of the present invention, the culturing is effected in CMRL medium.
According to embodiments of the present invention, the contacting is effected for at least 3 days.
According to embodiments of the present invention, the method further comprises isolating the adult islet beta cells following the contacting. According to embodiments of the present invention, the isolated population is for use in treating diabetes.
According to embodiments of the present invention, the isolated population is genetically modified to express a pharmaceutical agent.
According to embodiments of the present invention, the BET inhibitor is a small molecule inhibitor.
According to embodiments of the present invention, the small molecule inhibitor is selected from the group consisting of CPI-0610, DUAL946, GSK525762, I-BET151, JQl, OTX015, PFI- 1, RVX-208, RVX2135 and TEN-010.
According to embodiments of the present invention, the BET pathway inhibitor is I- BET151.
According to embodiments of the present invention, the cell medium further comprises an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor.
According to embodiments of the present invention, the cell medium further comprises a NOTCH pathway inhibitor and a TGFP pathway inhibitor.
According to embodiments of the present invention, the NOTCH pathway inhibitor targets a protein selected from the group consisting of NICD, γ-secretase and HDAC.
According to embodiments of the present invention, the NOTCH pathway inhibitor is a small molecule inhibitor.
According to embodiments of the present invention, the small molecule inhibitor is selected from the group consisting of 6-4 pyridme-3-amine, Cyclopiazonic acid, DAPT, LY685458, PF-03084014, γ-Secretase Inhibitor III, (R (-Flurbiprofen and LBH589.
According to embodiments of the present invention, the TGFP pathway inhibitor is a small molecule inhibitor.
According to embodiments of the present invention, the small molecule inhibitor is selected from the group consisting of LY573636, LY364947, LY2157299 and ALK5 Inhibitor II.
According to embodiments of the present invention, the WNT pathway inhibitor is selected from the group consisting of Niclosamide, Sulindac, Aspirin, Celecoxib and Indomethacin.
According to embodiments of the present invention, the NOTCH pathway inhibitor is PF- 03084014 and the TGFp pathway inhibitor is LY2157299. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
FIG. l. Redifferentiation of expanded human islet cells by treatment with NOTCH+TGFP inhibitors, I-BET151, and Matrigel. Cells at passage 6 were treated for 7 days with the indicated compounds. C-peptide-positive and live cells were quantitated by immunostaining and DRAQ5 nuclear staining, respectively, using IncuCyte instrument. Values are mean+SD (n=3 donors) of percentages in a total of 30,000 cells counted in each sample. P, PF-03084014, 1 μΜ; F, (R)- Flurbiprofen, 300 μΜ; L9, LY-2157299, 0.5 μΜ; A, ALK5 Inh. II, 10 μΜ; L7, LY364947, 0.25 HM;C, 1% DMSO control.
FIG. 2. Redifferentiation of expanded human islet cells following treatment with PF- 03084014, LY-2157299 and I-BET151. Cells were treated and stained as detailed in the legend to Figure 1. Bar=30 μιτι.
FIG. 3. Changes in β-cell transcript levels in cells treated with the 3-compound combination. Expanded human islet cells at passage 6 were treated for 7 days with PF-03084014, LY-2157299, and I-BET151 in Matrigel-coated plates. RNA was analysed by qPCR. Values are mean+SD (n=3 donors) and normalized to RPLPO. Values are relative to expanded untreated cells (RQ=1).
FIG. 4. Redifferentiation of expanded human islet cells by treatment with NOTCH, TGFp, and BET inhibitors. Cells at passage 7 were treated for 7 days with the indicated compounds in Matrigel-coated plates. C-peptide-positive cells were quantitated by immunostaining, using IncuCyte instrument. Values are mean+SD (n=3 donors) of percentages in a total of 30,000 cells counted in each sample. P, PF-03084014, 1 μΜ; L9, LY-2157299, 0.5 μΜ; B, I-BET151, 0.6 μΜ; C, 1% DMSO control. Column labeled "0.5" marks half of the concentration of all 3 compounds. All values were statistically significant compared with C (p<0.05).
FIG. 5. Changes in β-cell transcript levels in cells treated with the 3-compound combination. RT-PCR analysis of RNA extracted from cells at passage 7 treated for 7 days with the 3-compound cocktail (PF-03084014, 1 μΜ; LY-2157299, 0.5 μΜ; I-BET151, 0.6 μΜ) in Matrigel-coated plates. Values are mean+SD (n=3 donors) relative to expanded untreated cells (RQ=1) and normalized to RPLPO. Values in uncultured human islets are shown for comparison. FIG. 6 is a graph illustrating the effect of compound withdrawal following redifferentiation of expanded human islet cells by treatment with NOTCH, TGFp, and BET inhibitors. Cells at passage 7 were treated for 7 days with the 3-compound cocktail (PF-03084014, 1 μΜ; LY- 2157299, 0.5 μΜ; I-BET151, 0.6 μΜ) in Matrigel-coated plates. They were then transferred to serum- free medium (SFM) without the compounds for 7 additional days. C-peptide-positive cells were quantitated by immuno staining, using IncuCyte instrument. Values are mean+SD (n=3 donors) of percentages in a total of 30,000 cells counted in each sample. The results show that redifferentiation is stable upon compound removal. SFM may induce further differentiation.
FIG. 7 is a bar graph illustrating the effect of the 3 compound combination on β-cell proliferation. Cells at passage 7 were treated for 7 days with the 3-compound cocktail (PF- 03084014, 1 μΜ; LY-2157299, 0.5 μΜ; I-BET151, 0.6 μΜ) in Matrigel-coated plates. Expositive cells were quantitated by immuno staining, using IncuCyte instrument. Values are mean+SD (n=3 donors) of percentages in a total of 30,000 cells counted in each sample. The results show that redifferentiation is accompanied by growth arrest.
FIGs. 8A-B are graphs and photographs illustrating the expression of other islet hormones following redifferentiation of expanded human islet cells by treatment with NOTCH, TGFp, and BET inhibitors. Cells at passage 7 were treated for 7 days with the 3-compound cocktail (PF- 03084014, 1 μΜ; LY-2157299, 0.5 μΜ; I-BET151, 0.6 μΜ) in Matrigel-coated plates. Hormone- positive cells were quantitated by immuno staining, using IncuCyte instrument. Values are percentages of hormone-positive cells in a total of 30,000 cells from a single donor. The results show that the compound cocktail induces redifferentiation of other hormone-expressing cells, however all hormone-expressing cells are mono-hormonal.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The present invention, in some embodiments thereof, relates to a method of increasing insulin content in adult islet beta cells.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
In-vitro expansion of functional β-cells from adult human islets is an attractive approach for generating an abundant source of β-cells for cell replacement therapy of diabetes. However, attempts at expanding human islet β-cells in tissue culture result in loss of cell phenotype. The present inventors now disclose the use of small molecule inhibitors for re- differentiating expanded human islet β-cells. Based on a preliminary screen of multiple compounds, the present inventors focused on a compound combination which induced the most effective differentiation, as judged by the percent of C -pep tide-positive cells - Figures 1 and 4. This approach allows for generation of abundant human insulin-producing cells from single donors for transplantation into multiple recipients.
Thus, according to one aspect of the present invention, there is provided a method of ex- vivo increasing insulin content in pancreatic progenitor cells, comprising:
(a) contacting the pancreatic progenitor cells with a Bromodomain and Extra-Terminal motif (BET) protein (BET) inhibitor and an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor under conditions that increase the insulin content in the pancreatic progenitor cells; and
(b) analyzing the amount of insulin in, or secreted by, the pancreatic cells, thereby increasing insulin content in the progenitor cells.
As used herein the phrase "ex-vivo" refers to cells which are removed from a living organism and cultured outside the organism (e.g., in a test tube).
The phrase "pancreatic progenitor cells" refers to a population of cells which are not fully differentiated into pancreatic cells, yet are committed to differentiating towards at least one type of pancreatic cell - e.g. beta cells that produce insulin; alpha cells that produce glucagon; delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide. Thus, the pancreatic progenitor cells of this aspect of the present invention are capable (following differentiation) of expressing at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide.
Typically, pancreatic progenitor cells express some of the phenotypic markers that are characteristic of pancreatic lineages (e.g. GLUT2, PDX-1 Ηηί3β, PCl/3, Beta2, Nkx2.2 and PC2). Typically, they do not produce progeny of other embryonic germ layers when cultured by themselves in vitro, unless dedifferentiated or reprogrammed. It will be appreciated that it is not implied that each of the cells within the population have the capacity of forming more than one type of progeny, although individual cells that are multipotent pancreatic progenitor cells may be present.
In one embodiment, the pancreatic progenitor cells according to the invention are generated (ex vivo) from embryonic stem cells, perinatal stem cell, somatic stem cells, and bioengineered stem cells, preferably the stem cells are liESC, or iPSC, in particular hiPSC. Non-limiting examples of human embryonic stem cells lines are for example the cell lines CHB-1 ,CHB-2, CHB-3, CHB-4, CHB-5, CHB-6. CHB-8, CHB-9, CHB-10. CHB-1 1, CHB-12, Rockefeller University Embryonic Stem Cell Line 1 (RUES 1), Rockefeller University Embryonic Stem Cell Line 2 (RUES2), HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 26, HUES 27, HUES 28, CyT49, Rockefeller University Embryonic Stem Cell Line 3 (RUES3), WA01 (HI), UCSF4, NYUES 1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, HUES 48, HUES 49, HUES 53, HUES 65, HUES 66, UCLA 1 , UCLA 2, UCLA 3, WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14), HUES 62, HUES 63, HUES 64, CTl , CT2, CT3, CT4, MA135, Endeavour-2, WIBR1 , WIBR2, HUES 45, Shef 3, Shef 6, WIBR3, WIBR4, WIBR5, WIBR6, BJNheml9, BJNhem20, SA001, SA002, UCLA 4, UCLA 5, UCLA 6, HUES, ESI-014, ESI-017, WA15, WA17, WA18, WA19, WA20, WA21, WA22, WA23, WA24, CSES2, CSES4, CSES7, CSES8, CSES 1 1, CSES 12, CSES 13, CSES 14, CSES 15, CSES 17, CSES 19, CSES20, CSES21, CSES22, CSES23, CSES24, CSES25, HAD-C 100, HAD-C 102, HAD-C 106, ESI-035, ESI -049, ESI-051 , ESI-053, CSES5, CSES6, CSES 18, CA1, CA2, MEL-1, MEL- 2, MEL- 3, MEL-4, UCLA 8, UCLA 9, UCLA 10, UM4-6, GENEA002, GENEA048, Elf, HUES 42, HUES 44, NMR-1, UM14-1, UM14-2, HUES 68, HUES 70, HUES 69, HUES PGD 10, UCLA 11, UCLA 12, WA25, WA26, WA27, HS346, HS401, HS420, TE03, 14 (TE04), 16 (TE06), UM22-2, CR-4, KCLOl 1, GENEA015, GENEA016, GENEA047, GENEA042, GENEA043, GENEA057, GENEA052, SA121 .
The phrase "human somatic stem cells" or "human adult stem cells" as used herein refers to stem cells found throughout the human body after birth. Such cells can thus be obtained from adult tissue samples rather than human embryos, the destruction of which they do not require. According to the invention, "human somatic stem cells" encompass hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, and testicular cells.
Cells derived from bone marrow and amniotic fluid, which can include both hematopoietic stem cells and mesenchymal stem cells, have been found to differentiate into beta cells with manipulation in an in vitro environment (Jiang et al., Nature, 418:41-4; 2002, and De Coppi et al., Nat Biotechnology, 25: 100-106, 2007).
Preferably, the term "human somatic stem cells" refers to hematopoietic stem cells or mesenchymal stem cells. According to the invention, the term "hematopoietic stem cells" refers herein to a stem cell displaying a hematopoietic stem cell phenotype. By "hematopoietic stem cells phenotype it is herein meant the expression of at least one hematopoietic stem cells marker, and /or the presence of hematopoietic stem cell morphology.
Examples of typical hematopoietic stem cell markers include, without limitation, CD34+,
CD59+, Thyl /CD90+, CD381o/ - , and C-kit/CDl 17+.
As regards their morphology, hematopoietic stem cells are non-adherent and rounded cells, with a rounded nucleus and low cytoplasm-to-nucleus ratio. They can further be identified by their small size, lack of lineage (lin) markers, low staining (side population) with vital dyes such as rhodamine 123 (rhodamineDULL, also called rholo) or Hoechst 33342, and presence of various antigenic markers on their surface.
Hematopoietic stem cells can be found in bone marrow and bone marrow biological samples.
According to the invention, the term "mesenchymal stem cells" refers herein to a stem cell displaying a mesenchymal stem cell phenotype.
By "mesenchymal stem cell phenotype" it is herein meant the expression of at least one mesenchymal stem cells marker, and/or the presence of a mesenchymal stem cell morphology.
Examples of typical mesenchymal stem cell markers include, without limitation, CD73, CD90 and CD105. Mesenchymal stem cells lack the expression of the markers CD1 1 b, CD14, CD19, CD34, CD45, CD79a and HLA-DR.
As regards their morphology, mesenchymal stem cells are characterized by a small cell body with a few cell processes that are long and thin. The cell body contains a large, round nucleus with a prominent nucleolus, which is surrounded by finely dispersed chromatin particles, giving the nucleus a clear appearance. The remainder of the cell body contains a small amount of Golgi apparatus, rough endoplasmic reticulum, mitochondria, and polyribosomes. The cells, which are long and thin, are widely dispersed and the adjacent extracellular matrix is populated by a few reticular fibrils but is devoid of the other types of collagen fibrils.
Mesenchymal stem cells can be found for example in placenta, adipose tissue, lung, bone marrow and blood, Wharton's jelly from the umbilical cord, muscle, and teeth (perivascular niche of dental pulp and periodontal ligament).
According to a particular embodiment, the pancreatic progenitor cells comprise dedifferentiated adult islet beta cells.
As used herein, the phrase "adult islet beta cells" refers to post-natal (e.g., non-embryonic) pancreatic islet endocrine cells which are capable of secreting insulin in response to elevated glucose concentrations and express typical beta cell markers. Examples of beta cell markers include, but are not limited to, insulin and PDX.
Dedifferentiated adult islet beta cells may be generated ex- vivo from adult islet beta cells. The adult beta cells may be comprised in pancreatic islets or may be isolated from the islets. Pancreatic islets comprise the following: 1) beta cells that produce insulin; 2) alpha cells that produce glucagon; 3) delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide. The polypeptide hormones (insulin, glucagon, somatostatin and pancreatic polypeptide) inside these cells are stored in secretary vesicles in the form of secretory granules.
Methods of isolating islets are well known in the art. For example, islets may be isolated from pancreatic tissue using collagenase and ficoll gradients. Preferably the adult islet beta cells of the present invention are dispersed into a single cell suspension - e.g. by the addition of trypsin or by trituration.
The adult islet beta cells may be further isolated being substantially free from other substances (e.g., other cells, proteins, nucleic acids, etc.) that are present in its in-vivo environment e.g. by FACs sorting.
The adult islet beta cells may be obtained from any autologous or non- autologous (i.e., allogeneic or xenogeneic) mammalian donor. For example, cells may be isolated from a human cadaver.
Dedifferentiation of the adult islet beta cells may be effected by expansion (i.e. culturing) for a prolonged number of passages - e.g. in CMRL medium.
As used herein, the term "expanding" refers to increasing the number and/or overall mass of adult islet beta cells of the present invention by the process of cell division, rather than simply enlarging by hypertrophy.
According to another embodiment, the cells are expanded in Mesencult XF medium comprising glucose at a concentration of about 10-100 mM glucose, more preferably 10-50 mM, more preferably 10-25 mM, such as for example 25 mM.
The cells are preferably expanded for at least 6 passages, 7 passages, 8 passages, 9 passages, 10 passages, 11 passages, 12 passages, 13 passages, 14 passages, 15 passages or at least 16 passages.
According to a particular embodiment, the expanding is effected under adherent conditions which comprise incubating on an attachment medium selected from the group consisting of laminin, fibronectin, matrigel and Mesencult XF attachment substrate.
According to another embodiment, dedifferentiation of the adult islet beta cells may be effected by transfecting the cells with genes known to generated induced pluripotent stem cells (iPS) cells. Oct-3/4 and certain members of the Sox gene family (Soxl, Sox2, Sox3, and Soxl5) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the Klf family (Klfl, Klf2, Klf4, and Klf5), the Myc family (C-myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency.
Redifferentiation of the cells is effected in the presence of a Bromodomain and Extra- Terminal motif (BET) protein (BET) inhibitor and an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor.
The term "BET inhibitor" refers to an agent that inhibits the binding of BET family bromodomains to acetylated lysine residues. By "BET family bromodomains" it is meant a polypeptide comprising two bromodomains and an extraterminal (ET) domain or a fragment thereof having transcriptional regulatory activity or acetylated lysine binding activity. Exemplary BET family members include BRD2, BRD3, BRD4 and BRDT are given in WO 2011/143669. Examples of BET inhibitors include but are not limited to the compounds of the instant invention. Advantageously, the BET inhibitor according to the invention targets BD1 and/or BD2, and is preferentially a small molecule agent. Examples of such small molecule agents include, but are not limited to BET762, TEN-010, CPI-203, LY29002, RVX8, CPI-0610, DUAL946, GSK525762, 1-BET151, JQ1, OTX015, PFI-1, RVX-208, RVX2135, preferentially BET 151 and JQ1, and even more preferably BET 151.
In a preferred embodiment, the cell culture medium according to the invention, comprises the BET inhibitor in a concentration from ΙΟηΜ to ΙΟμΜ, preferentially from 0.1 μΜ to 1 μΜ, from 0.3 μΜ to 0.8 μΜ or from 0.4 μΜ to 0.7 μΜ - for example about 0.6 μΜ.
Polynucleotide agents capable of down-regulating expression of BET family members are described herein below.
The term "Notch pathway inhibitor" refers to an agent that is capable of downregulating activity and/or amount of a component participating in the NOTCH pathway. Exemplary components are described herein below.
The Notch signaling pathway is a conserved intercellular signaling mechanism that is essential for proper embryonic development in numerous metazoan organisms. Members of the Notch gene family (NOTCHs) encode transmembrane receptors that are critical for various cell fate decisions. Multiple ligands that activate Notch and related receptors have been identified, including Senate and Delta in Drosophila and JAG1 (MIM.601920) in vertebrates.
Four different Notch receptors (NOTCHs: NOTCH 1 to NOTCH4) and five ligands (Jagged-1 (JAG1) and -2 (JAG2) and Delta-like [DLLs]: DLL1, DLL2 and DLL4) have been characterized in mammalian cells. These transmembrane receptors and ligands are expressed in different combinations in most, if not all, cell types. The Notch pathway regulates cell fate determination of neighboring cells through lateral inhibitiona, depending on their ability to express either the receptors or the ligands.
Following ligand binding, NOTCHs are activated by a series of cleavages that releases its intracellular domain (NICD). This processing requires the activity of two proteases, namely ADAM 17 (tumor necrosis factor-alpha converting enzyme or TACE MEVI.603369) and presenilin-1 (PSENl MIM.104311), both of which also fall under the category of a component of a NOTCH pathway.
Nuclear translocation of NICD results in transcriptional activation of genes of the HESs family (Hes/E(spl) family) and HEYs family (Hesr/Hey family) through interaction of NICD with RBPSUH (or CBFl MIM.147183), Su(H), and Lag-1, which is also known as the recombination signal sequence-binding protein (RBP)-j (also called Suppressor of Hairless, Su(H)), each of these also falling under the category of a component of a NOTCH pathway.
Overall, when activated, Notch signaling enables neighboring cells to acquire distinct phenotypes, through a process named lateral inhibition. The Notch receptor is pre-cleaved in the Golgi and is targeted subsequently to the plasma membrane where it interacts with ligands located on neighboring cells. Receptor— ligand interaction results in a conformational change in the receptor, thus enabling additional cleavages by TACE and the.gamma.-secretase complex. This proteolytic activity enables the Notch intracellular domain (NICD) to translocate to the nucleus where it activates the transcription of target genes (e.g. the Hes and Hey family of transcriptional repressors).
Monoubiquitylation (Ub) of the ligand by mindbomb (MIB) induces endocytosis of the ligand and the Notch extracellular domain (NECD) into the ligand cells where additional signaling might be initiated.
Notch receptors undergo a complex set of proteolytic processing events in response to ligand activating, which eventually leads to release of the intracellular domain of the receptor. Signal transduction is normally initiated by binding to transmembrane ligands of the Serrate or Delta class, which induces proteolytic release of the intracellular NOTCH domain (NICD).
Free NICD translocates to the nucleus to form a short-lived complex with a Rel-like transcription factor, CSL, and Mastermind-like co-activators that activates lineage- specific programs of gene expression. As mentioned, the present invention contemplates down-regulating any component of the NOTCH pathway that is up-regulated in B cell dedifferentiation above a predetermined threshold. Preferably, the component of the NOTCH pathway which is targeted is NICD, γ-secretase and/or HDAC.
According to one embodiment the NOTCH pathway component is upregulated by at least 1.5 times, more preferably by at least 2 times and more preferably by at least 3 times.
Methods of analyzing whether a particular component is upregulated during B cell differentiation are known in the art, and may be effected on the RNA level (using techniques such as Northern blot analysis, RT-PCR and oligonucleotides microarray) and/or the protein level (using techniques such as ELISA, Western blot analysis, immunohistochemistry and the like, which may be effected using antibodies specific to the NOTCH pathway component).
According to another embodiment, the NOTCH pathway component is Hairy and Enhancer of Split 1 (HES 1; NM.sub.-005524, NP.sub.-005515), NOTCH1 (NM.sub.-017617, NP.sub.-060087.3) NOTCH 2 (NM.sub.-024408, NP.sub.-077719.2) and NOTCH 3 (NM.sub.- -000435, NP.sub.-000426 .2).
Exemplary small molecule inhibitors of the NOTCH pathway include 6-4 pyridines- amine. Cyclopiazonic acid, DAPT, LY685458, PF-03084014, γ-Secretase Inhibitor III, (R)- Flurbiprofen and LBH589. Additional inhibitors are set forth in Table 1 below.
Polynucleotide agents capable of down-regulating expression of a component of the NOTCH pathway are described herein below.
The term "TGFP pathway inhibitor" refers to an agent that is capable of downregulating activity and/or amount of a component participating in the TGFP pathway. Exemplary components are described herein below.
TGFP signals are conveyed through two transmembrane serine-threonine kinase receptors (type I and type II TGFP receptors) to the five receptor-regulated (R)-SMAD transcription factors (SMAD1-3, 5, 8), which translocate into the nucleus, recruit transcriptional co-activators and co- repressors, and regulate gene expression. The type I receptor family is comprised of activin-like kinase (ALK) receptors 1 through 7. Two major pathway branches are activated by TGFP family ligands: SMAD1, 5 and 8 are activated by BMPs through ALK1-3 and 6, whereas SMAD2 and 3 are activated by TGFp, activins, and nodals, through ALK4, 5 and 7. SMAD2 and 3 phosphorylation by ALK5 (also termed TGBRI) is the best-characterized TGFP pathway signaling effect, and the main one associated with EMT, whereas mesenchymal-epithelial transition (MET) is associated primarily with phosphorylation of SMAD1, 5 and 8. According to a particular embodiment, the inhibitor targets ALK-5.
According to one embodiment the TGFP pathway component is upregulated by at least 1.5 times, more preferably by at least 2 times and more preferably by at least 3 times. Methods of analyzing whether a particular component is upregulated during B cell differentiation are known in the art, and may be effected on the RNA level (using techniques such as Northern blot analysis, RT-PCR and oligonucleotides microarray) and/or the protein level (using techniques such as ELISA, Western blot analysis, immunohistochemistry and the like, which may be effected using antibodies specific to the TGFP pathway component).
Exemplary small molecule inhibitors of the TGFP pathway include but are not limited to LY573636, LY364947, LY2157299 and ALK5 Inhibitor II. Additional inhibitors are set forth in Table 1 below.
Polynucleotide agents capable of down -regulating expression of a component of the TGFP pathway are described herein below.
The term "Wnt pathway inhibitor" refers to an agent that is capable of downregulating activity and/or amount of a component participating in the Wnt pathway. Exemplary components include β-catenin, Zinc Finger E-Box Binding Homeobox 1 (ZEB-1), TWIST, SNAIL, SOX-2 and SOX-6. According to a particular embodiment, the RNA transcript encoding the component of the Wnt pathway is targeted by miRNA-200c.
According to one embodiment the Wnt pathway component is upregulated by at least 1.5 times, more preferably by at least 2 times and more preferably by at least 3 times.
Methods of analyzing whether a particular component is upregulated during B cell differentiation are known in the art, and may be effected on the RNA level (using techniques such as Northern blot analysis, RT-PCR and oligonucleotides microarray) and/or the protein level (using techniques such as ELISA, Western blot analysis, immunohistochemistry and the like, which may be effected using antibodies specific to the Wnt pathway component).
Exemplary small molecule inhibitors of the Wnt pathway include but are not limited to Niclosamide, Sulindac, Aspirin, Celecoxib and Indomethacin. Additional inhibitors are set forth in Table 1 below.
Polynucleotide agents capable of down-regulating expression of a component of the Wnt pathway are described herein below.
As mentioned, additional inhibitors that may be used during the redifferentiation process are disclosed in Table 1, herein below. Table 1
Compound Pathway Target Concentration
6-4 pyridine- 3 -amine NOTCH NCID 0.5-10 Li
Cyclopia/onic acid NOTCH NCID 1 - 10 LI M DAPT NOTCH γ-secretase 1-25 μΜ
LY685458 NOTCH γ-secretase 1-20 μΜ
PF-03G84014 NOTCH γ-secretase 0.3- 10 μΜ γ-Secretase Inhibitor III NOTCH γ-secretase 10-90 μΜ
(R (-Flurbiprofen NOTCH γ-secretase 0.1 -2.7 ηιΜ
LBH589 NOTCH HDAC 12.5-200 ηΜ
LY573636 TGFP TGFPRI (ALK5) 1-100 μΜ
LY364947 TGFP TGFPRI (ALK5) 50-2500 ηΜ
LY2157299 TGFP TGFPRI (ALK5) 50-6250 ηΜ
ALK5 Inhibitor II TGFP TGFPRI (ALK5) 1-20 μΜ
Niclosamide WNT DVL, LRP5/6 1-75 μΜ
Sulindac WNT DVL 50-500 μΜ
Aspirin WNT β-catenin degradation 1-100 mM
Celecoxib WNT β-catenin degradation 20-2500 μΜ
Indomethacin WNT β-catenin degradation 100-6250 μΜ
Particularly effective combinations of inhibitors include: PF-03084014 together with LY2157299; and PF-03084014 together with LY2157299 and I-BET151 (e.g. at the disclosed concentrations).
As mentioned, the present inventors also contemplate the use of polynucleotide agents or antibodies that downregulate expression of at least one component of any of the pathways described herein above.
Preferably, the antibody specifically binds at least one epitope of a single component of any one of the pathways described herein.
As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
Downregulation of a particular component of any one of the pathways described herein can be also achieved by RNA silencing.
As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post- transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein- coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
As used herein, the term "RNA silencing agent" refers to an RNA which is capable of specifically inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.
According to an embodiment of the invention, the RNA silencing agent is specific to the target RNA and does not cross inhibit or silence a gene or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene.
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). The corresponding process in plants is commonly referred to as post-transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The process of post-transcriptional gene silencing is thought to be an evolutionarily-conserved cellular defense mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora and phyla. Such protection from foreign gene expression may have evolved in response to the production of double- stranded RNAs (dsRNAs) derived from viral infection or from the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single- stranded RNA or viral genomic RNA.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs). Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single- stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
Accordingly, some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.
According to one embodiment, the dsRNA is greater than 30 bp. The use of long dsRNAs
(i.e. dsRNA greater than 30 bp) has been very limited owing to the belief that these longer regions of double stranded RNA will result in the induction of the interferon and PKR response. However, the use of long dsRNAs can provide numerous advantages in that the cell can select the optimal silencing sequence alleviating the need to test numerous siRNAs; long dsRNAs will allow for silencing libraries to have less complexity than would be necessary for siRNAs; and, perhaps most importantly, long dsRNA could prevent viral escape mutations when used as therapeutics.
Various studies demonstrate that long dsRNAs can be used to silence gene expression without inducing the stress response or causing significant off-target effects— see for example [Strat et al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res. Protoc. 2004; 13: 115-125; Diallo M., et al., Oligonucleotides. 2003; 13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002; 99: 1443-1448; Tran N., et al., FEBS Lett. 2004; 573: 127- 134].
In particular, the invention according to some embodiments thereof contemplates introduction of long dsRNA (over 30 base transcripts) for gene silencing in cells where the interferon pathway is not activated (e.g. embryonic cells and oocytes) see for example Billy et al., PNAS 2001, Vol 98, pages 14428-14433. and Diallo et al, Oligonucleotides, Oct. 1, 2003, 13(5): 381-392. doi: 10.1089/154545703322617069.
The invention according to some embodiments thereof also contemplates introduction of long dsRNA specifically designed not to induce the interferon and PKR pathways for down- regulating gene expression. For example, Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.
Another method of evading the interferon and PKR pathways in mammalian systems is by introduction of small inhibitory RNAs (siRNAs) either via transfection or endogenous expression.
The term "siRNA" refers to small inhibitory RNA duplexes (generally between 18-30 base- pairs) that induce the RNA interference (RNAi) pathway. Typically, siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location. The observed increased potency obtained using longer RNAs in triggering RNAi is theorized to result from providing Dicer with a substrate (27mer) instead of a product (21mer) and that this improves the rate or efficiency of entry of the siRNA duplex into RISC.
It has been found that position of the 3'-overhang influences potency of an siRNA and asymmetric duplexes having a 3 '-overhang on the antisense strand are generally more potent than those with the 3'-overhang on the sense strand. This can be attributed to asymmetrical strand loading into RISC, as the opposite efficacy patterns are observed when targeting the antisense transcript.
The strands of a double- stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). Thus, as mentioned the RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
The term "shRNA", as used herein, refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11.
According to another embodiment the RNA silencing agent may be a miRNA or miRNA mimic.
miRNAs are small RNAs made from genes encoding primary transcripts of various sizes. They have been identified in both animals and plants. The primary transcript (termed the "pri- miRNA") is processed through various nucleolytic steps to a shorter precursor miRNA, or "pre- miRNA." The pre-miRNA is present in a folded form so that the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually base- pair with the target) The pre-miRNA is a substrate for a form of dicer that removes the miRNA duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (Parizotto et al. (2004) Genes & Development 18:2237-2242 and Guo et al. (2005) Plant Cell 17: 1376-1386).
Unlike, siRNAs, miRNAs bind to transcript sequences with only partial complementarity (Zeng et al., 2002, Molec. Cell 9: 1327-1333) and repress translation without affecting steady-state RNA levels (Lee et al., 1993, Cell 75:843-854; Wightman et al., 1993, Cell 75:855-862). Both miRNAs and siRNAs are processed by Dicer and associate with components of the RNA-induced silencing complex (Hutvagner et al., 2001, Science 293:834-838; Grishok et al., 2001, Cell 106: 23-34; Ketting et al., 2001, Genes Dev. 15:2654-2659; Williams et al., 2002, Proc. Natl. Acad. Sci. USA 99:6889-6894; Hammond et al., 2001, Science 293: 1146-1150; Mourlatos et al., 2002, Genes Dev. 16:720-728). A recent report (Hutvagner et al., 2002, Sciencexpress 297:2056-2060) hypothesizes that gene regulation through the miRNA pathway versus the siRNA pathway is determined solely by the degree of complementarity to the target transcript. It is speculated that siRNAs with only partial identity to the mRNA target will function in translational repression, similar to an miRNA, rather than triggering RNA degradation.
An exemplary miRNA capable of downregulating ZEB-1, SOX-2 and SOX-6 is miR-200. It will be appreciated from the description provided herein above that contacting cells with a miRNA may be effected by transfecting the cells with e.g. the mature double stranded miRNA, the pre-miRNA or the pri-miRNA.
The pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
The pri-miRNA sequence may comprise from 45-30,000, 50-25,000, 100-20,000, 1,000- 1,500 or 80-100 nucleotides. The term "microRNA", "miRNA", and "miR" are synonymous and refer to a collection of non-coding single- stranded RNA molecules of about 19-28 nucleotides in length, which regulate gene expression. miRNAs are found in a wide range of organisms and have been shown to play a role in development, homeostasis, and disease etiology.
The term "microRNA mimic" or "miRNA mimic" refers to synthetic non-coding RNAs that are capable of entering the RNAi pathway and regulating gene expression. miRNA mimics imitate the function of endogenous miRNAs and can be designed as mature, double stranded molecules or mimic precursors (e.g., or pre-miRNAs). miRNA mimics can be comprised of modified or unmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acid chemistries (e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (EN A)). For mature, double stranded miRNA mimics, the length of the duplex region can vary between 13-33, 18-24 or 21-23 nucleotides. The miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides. The sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA. The sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
Preparation of miRNAs mimics can be effected by any method known in the art such as chemical synthesis or recombinant methods.
Synthesis of RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the selected mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245]. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90% decrease in cellular GAPDH mRNA and completely abolished protein level (www(dot)ambion(dot)com/techlib/tn/91/912(dot)html).
Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55%. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably includes the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene. It will be appreciated that the RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically- modified nucleotides and non-nucleotides.
In some embodiments, the RNA silencing agent provided herein can be functionally associated with a cell-penetrating peptide." As used herein, a "cell-penetrating peptide" is a peptide that comprises a short (about 12-30 residues) amino acid sequence or functional motif that confers the energy-independent (i.e., non-endocytotic) translocation properties associated with transport of the membrane-permeable complex across the plasma and/or nuclear membranes of a cell. The cell- penetrating peptide used in the membrane-permeable complex of some embodiments of the invention preferably comprises at least one non-functional cysteine residue, which is either free or derivatized to form a disulfide link with a double- stranded ribonucleic acid that has been modified for such linkage. Representative amino acid motifs conferring such properties are listed in U.S. Pat. No. 6,348,185, the contents of which are expressly incorporated herein by reference. The cell- penetrating peptides of some embodiments of the invention preferably include, but are not limited to, penetratin, transportan, pisl, TAT(48-60), pVEC, MTS, and MAP. Another agent capable of downregulating a component of any of the pathways described herein is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or pathway.
DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences (Breaker, R. R. and Joyce, G. Chemistry and Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 1997; 943:4262) A general model (the " 10-23" model) for the DNAzyme has been proposed. " 10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl, Acad. Sci. USA 199; for rev of DNAzymes see Khachigian, L M [Curr Opin Mol Ther 4: 119-21 (2002))].
Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double- stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174 to Joyce et al. DNAzymes of similar design directed against the human Urokinase receptor were recently observed to inhibit Urokinase receptor expression, and successfully inhibit colon cancer cell metastasis in vivo (Itoh et al., 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www(dot)asgt(dot)org). In another application, DNAzymes complementary to bcr-abl oncogenes were successful in inhibiting the oncogenes expression in leukemia cells, and lessening relapse rates in autologous bone marrow transplant in cases of CML and ALL. Downregulation of at least one component can also be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding the component.
Another agent capable of downregulating the component is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding the component. Ribozymes are being increasingly used for the sequence-specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest [Welch et al., Curr Opin Biotechnol. 9:486-96 (1998)]. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications.
Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double- stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDS) and non-homologous end-joining (NFfEJ). NFfEJ directly joins the DNA ends in a double- stranded break, while HDR utilizes a homologous sequence as a template for regenerating the missing DNA sequence at the break point. In order to introduce specific nucleotide modifications to the genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location. To overcome this challenge and create site-specific single- or double- stranded breaks, several distinct classes of nucleases have been discovered and bioengineered to date. These include the meganucleases, Zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and CRISPR/Cas system.
CRISPR-Cas system - Many bacteria and archaea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) genes that produce RNA components and CRISPR associated (Cas) genes that encode protein components. The CRISPR RNAs (crRNAs) contain short stretches of homology to specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen. Studies of the type II CRISPR/Cas system of Streptococcus pyogenes have shown that three components form an RNA/protein complex and together are sufficient for sequence- specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821.)· It was further demonstrated that a synthetic chimeric guide RNA (gRNA) composed of a fusion between crRNA and tracrRNA could direct Cas9 to cleave DNA targets that are complementary to the crRNA in vitro. It was also demonstrated that transient expression of Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double- stranded brakes in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a,b; Jinek et al., 2013; Mali et al., 2013).
The CRIPSR/Cas system for genome editing contains two distinct components: a gRNA and an endonuclease e.g. Cas9.
The gRNA is typically a 20 nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript. The gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence. The binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break. Just as with ZFNs and TALENs, the double-stranded brakes produced by CRISPR/Cas can undergo homologous recombination or NHEJ.
The Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA.
A significant advantage of CRISPR/Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs enables multiple genes to be targeted simultaneously. In addition, the majority of cells carrying the mutation present biallelic mutations in the targeted genes.
However, apparent flexibility in the base-pairing interactions between the gRNA sequence and the genomic DNA target sequence allows imperfect matches to the target sequence to be cut by Cas9.
Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called 'nickases' . With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or 'nick'. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a double-strand break, in what is often referred to as a 'double nick' CRISPR system. A double-nick can be repaired by either NHEJ or HDR depending on the desired effect on the gene target. Thus, if specificity and reduced off-target effects are crucial, using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off- target effect as either gRNA alone will result in nicks that will not change the genomic DNA.
Modified versions of the Cas9 enzyme containing two inactive catalytic domains (dead Cas9, or dCas9) have no nuclease activity while still able to bind to DNA based on gRNA specificity. The dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains. For example, the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.
There are a number of publically available tools available to help choose and/or design target sequences as well as lists of bioinformatically determined unique gRNAs for different genes in different species such as the Feng Zhang lab's Target Finder, the Michael Boutros lab's Target Finder (E-CRISP), the RGEN Tools: Cas-OFFinder, the CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes and the CRISPR Optimal Target Finder.
In order to use the CRISPR system, both gRNA and Cas9 should be expressed in a target cell. The insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids. CRISPR plasmids are commercially available such as the px330 plasmid from Addgene.
The present invention also contemplates culturing the cells in a medium comprising additional differentiation factors under conditions to allow differentiation of the cells into insulin producing cells.
Exemplary mediums have been disclosed in U.S. Pat. No. 8,728,813, U.S. Patent Application Publication Nos. 20080014182, 20120003739, each of which are incorporated herein by reference.
According to one embodiment, the cells are redifferentiated (together with the inhibitory agents disclosed herein) in a culture medium comprising nicotinamide, exendin-4, activin A and 10-50 mM glucose, the culture medium being devoid of serum.
Exemplary culture media contemplated by the present inventors which may be used to redifferentiate the progenitor cells include CMRL-1066, DMEM, RPMI etc.
Exemplary concentration ranges of nicotinamide include 1-100 mM, more preferably 1-50 mM, more preferably 1-20 mM, such as for example 10 mM. Exemplary concentration ranges of exendin 4 include 1-100 nM, more preferably 1-50 nM, more preferably 1-20 nM, such as for example 8 nM.
Exemplary concentration ranges of activin A include 1-100 nM, more preferably 1-50 nM, more preferably 1-20 nM, such as for example 8 nM. Exemplary concentration ranges of glucose include 10-100 mM, more preferably 10-50 mM, more preferably 10-25 mM, such as for example 25 mM.
In one embodiment, the cells are cultured on an adherent substrate, such as matrigel or an extracellular matrix component. Examples of extracellular matrix components contemplated by the present invention include, but are not limited to collagen, laminin and fibronectin.
The differentiating agents described herein are preferably included in the differentiation medium. Cells may be cultured in the differentiation medium (i.e. with the agents) for at least one day, at least two days, at least 3 days or more. Preferably, the cells are not cultured in the differentiating medium for more than 3 weeks, more than two weeks or even more than 1 week. Thus, for example the cells may be contacted with the differentiating agents for 3 days- 10 days (e.g. one week).
Cells obtained using the methods described herein are capable of secreting insulin in a glucose responsive manner and typically express beta cell specific genes (e.g. PDX-1).
According to a particular embodiment, at least 10 % of the cells of the isolated populations generated according to the methods described herein are cells redifferentiated from beta cells.
According to a particular embodiment, at least 20 % of the cells of the isolated populations generated according to the methods described herein are cells redifferentiated from beta cells.
According to a particular embodiment, at least 30 % of the cells of the isolated populations generated according to the methods described herein are cells redifferentiated from beta cells.
According to a particular embodiment, at least 40 % of the cells of the isolated populations generated according to the methods described herein are cells redifferentiated from beta cells.
According to still another embodiment, at least 20 % of the cells of the isolated populations generated according to the methods described herein secrete insulin.
According to still another embodiment, at least 30 % of the cells of the isolated populations generated according to the methods described herein secrete insulin.
According to still another embodiment, at least 40 % of the cells of the isolated populations generated according to the methods described herein secrete insulin.
According to still another embodiment, at least 50 % of the cells of the isolated populations generated according to the methods described herein secrete insulin. Preferably, a majority of the cells in the populations described herein express PDX1, NKX2.2, NKX6.1, IAPP and PC 1/3 and additional proteins essential for beta cell function.
Preferably, the cells express an increase amount of beta cell transcription factors (e.g. HBLX9, NEUROD, NKX2.2, and NKX6.1), as compared to non-redifferentiated beta cells, as measured by RT-PCR.
Other genes which may be upregulated in the isolated populations described herein include KIR6.2, SUR1 and GCK.
The population of adult islet beta cells of the present invention may be further modified (e.g. genetic modification) to express a pharmaceutical agent such as a therapeutic agent, a telomerase gene, an agent that reduces immune mediated rejection or a marker gene. It is contemplated that therapeutic agents such as antimetabolites (e.g., purine analogs, pyrimidine analogs), enzyme inhibitors and peptidomimetics may be generally useful in the present invention. An example of a gene that may reduce immune mediated rejection is the uteroglobin gene. Uteroglobin is a protein expressed during pregnancy that confers immunologic tolerance and prevents inflammatory reactions. Methods of genetically modifying the adult islet beta cells of the present invention are described hereinabove.
According to one embodiment, following redifferentiation the beta cells may be isolated from other pancreatic cells present in the islet (if this hasn't been carried out prior to the redifferentiation process). This may be effected using zinc binding dyes such as Newport green (see Parnaud G, et al. Proliferation of sorted human and rat beta cells. Diabetologia 2008. 51:91- 100) or with anti-NCAM antibodies (see Banerjee M, Otonkoski T. A simple two-step protocol for the purification of human pancreatic beta cells. Diabetologia 2009. 52:621-625.).
Since the adult islet pancreatic cells of the present invention store and secrete insulin in a glucose responsive manner, they may be used for treating a disease which is associated with insulin deficiency such as diabetes.
As used herein "diabetes" refers to a disease resulting either from an absolute deficiency of insulin (type 1 diabetes) due to a defect in the biosynthesis or production of insulin, or a relative deficiency of insulin in the presence of insulin resistance (type 2 diabetes), i.e., impaired insulin action, in an organism. The diabetic patient thus has absolute or relative insulin deficiency, and displays, among other symptoms and signs, elevated blood glucose concentration, presence of glucose in the urine and excessive discharge of urine.
The phrase "treating" refers to inhibiting or arresting the development of a disease, disorder or condition and/or causing the reduction, remission, or regression of a disease, disorder or condition in an individual suffering from, or diagnosed with, the disease, disorder or condition. Those of skill in the art will be aware of various methodologies and assays which can be used to assess the development of a disease, disorder or condition, and similarly, various methodologies and assays which can be used to assess the reduction, remission or regression of a disease, disorder or condition.
As used herein, "transplanting" refers to providing the redifferentiated adult islet beta cells of the present invention, using any suitable route. Typically, beta cell therapy is effected by injection using a catheter into the portal vein of the liver, although other methods of administration are envisaged.
As mentioned hereinabove, the adult islet beta cells of the present invention can be derived from either autologous sources or from allogeneic sources such as human cadavers or donors. Since non-autologous cells are likely to induce an immune reaction when administered to the body several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non- autologous cells in immune-isolating, semipermeable membranes before transplantation.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
MATERIALS AND METHODS
Human islet cell expansion: Human islet cells were expanded in culture as described
(Friedman-Mazursky O et al., Sci Rep (2016), 6:20698).
Cell redifferentiation: Expanded islet cells at passages 6-7 were seeded in Matrigel-coated 12-well plates at 7 X 104 cells/well. They were then treated with the indicated small-molecule combinations for 7 days, and compared to cells treated with 1% DMSO (solvent negative control).
Immunostaining: Cells were fixed in 4% paraformaldehyde and stained with mouse-anti- human C-peptide (1:500) and FITC-conjugated goat-anti-mouse IgG (1: 1000). DNA was stained with DRAQ5. Fluorescent cells were quantitated using IncuCyte instrument. RNA analysis: RNA was extracted from the cells and analyzed by RT-PCR as described (Friedman-Mazursky O et al., Sci Rep (2016), 6:20698). Relative quantitation was compared to untreated cells (RQ=1) and normalized to RPLPO of GAPDH. RESULTS
A number of small molecules which inhibit NOTCH, TGFp, and WNT pathways were tested on expanded and dedifferentiated human islet cells to evaluate their redifferentiation potential, as judged by activation of C-peptide expression. None of the compounds assayed alone showed a significant effect. Combinations of two inhibitors blocking NOTCH+WNT and NOTCH+TGFP pathways were then assayed. The dual pathway inhibition induced a significant increase in the percent of C -pep tide-positive cells with all the compound combinations tested, up to about 10% of total cells.
At this stage an inhibitor of Bromodomain and Extra Terminal domain (BET) proteins, I- BET151, which has been suggested to have an effect on blocking mesenchymal gene expression during direct conversion of fibroblasts into neuronal cells, was evaluated. In addition, Matrigel- coated plates were evaluated for possible contribution to cell redifferentiation. These agents were assayed with several combinations of NOTCH+TGFP inhibitors (Figures 1 and 4). In Figure 1, it can be seen that at passage 6, I-BET151 doubled the percent of C-peptide-positive cells for all 6 combinations of NOTCH+TGFP inhibitors, while Matrigel increased the fraction of C-peptide- positive cells by 29-65% for 5 out of 6 combinations of NOTCH+TGFP inhibitors. The top combination of PF-03084014, LY-2157299 and I-BET151 resulted in redifferentiation of about 17% of total cells. Given that BCD cells constitute about 40% of expanded islet cells, this represents redifferentiation of over 42% of BCD cells. Figure 4 illustrates the synergistic effect of the combination of PF-03084014, LY-2157299 and I-BET151 over each of the agents individually on human pancreatic beta cells at passage 7. Figure 2 shows an image of cells re-differentiated with this 3-compound combination. Analysis of changes in β-cell transcript levels in cells treated with this 3-compound combination showed a great increase in transcripts encoding the β-cell transcription factors PDX1 and MAFA, to a level representing about 30% of that of normal islets (Figure 3). INS transcripts were induced to a level representing about 18% of that of normal islets. Figure 5 illustrates an additional analysis of changes in β-cell transcript levels in cells treated with this 3-compound combination showing a large increase in transcripts encoding other β-cell expressed proteins - IAPP, NKX2.2, NKX 6.1, ABCC8, GCK, PCSK1 and NeuroDl.
Figure 6 is a graph illustrating the effect of compound withdrawal following redifferentiation of expanded human islet cells by treatment with NOTCH, ΤΰΡβ, and BET inhibitors. The results show that redifferentiation is stable upon compound removal. SFM may induce further differentiation.
Figure 7 is a graph illustrating the effect of the 3 compound combination on β-cell proliferation. The results show that redifferentiation is accompanied by growth arrest.
Figures 8A-B are graphs and photographs illustrating the expression of other islet hormones following redifferentiation of expanded human islet cells by treatment with NOTCH, TGFp, and BET inhibitors. The results show that the compound cocktail induces redifferentiation of other hormone-expressing cells, however all hormone-expressing cells are mono-hormonal.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:
1. A method of ex-vivo increasing insulin content in pancreatic progenitor cells, comprising:
(a) contacting said pancreatic progenitor cells with a Bromodomain and Extra-Terminal motif (BET) protein (BET) inhibitor and an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor under conditions that increase the insulin content in said pancreatic progenitor cells; and
(b) analyzing the amount of insulin in, or secreted by, said pancreatic cells, thereby increasing insulin content in the progenitor cells.
2. A method of ex-vivo increasing insulin content in pancreatic progenitor cells, comprising culturing said pancreatic progenitor cells in CMRL medium, wherein said medium comprises a Bromodomain and Extra- Terminal motif (BET) protein (BET) inhibitor under conditions that increase the insulin content in said pancreatic progenitor cells.
3. The method of claim 1 or 2, wherein said progenitor cells are selected from the group consisting of dedifferentiated adult islet beta cells, mesenchymal stem cells and induced pluripotent stem cells dedifferentiated from beta cells.
4. The method of claims 1 or 2, wherein said progenitor cells comprise dedifferentiated adult islet beta cells.
5. The method of claims 1 or 2, wherein said BET inhibitor is a small molecule inhibitor.
6. The method of claim 5, wherein said small molecule inhibitor is selected from the group consisting of CPI-0610, DUAL946, GSK525762, I-BET151, JQ1, OTX015, PFI-1, RVX- 208, RVX2135 and TEN-010.
7. The method of claims 1 or 2, wherein said BET pathway inhibitor is I-BET151.
8. The method of any one of claims 2-7, further comprising contacting said cells with an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor.
9. The method of any one of claims 2-7, further comprising contacting said cells with a NOTCH pathway inhibitor and a TGFP pathway inhibitor.
10. The method of claim 1, wherein said agent comprises two agents.
11. The method of claim 1, wherein a first of said two agents comprises a NOTCH pathway inhibitor and a second of said two agents comprises a TGFP pathway inhibitor.
12. The method of claims 1 or 8, wherein said NOTCH pathway inhibitor targets a protein selected from the group consisting of NICD, γ-secretase and HDAC.
13. The method of claim 12, wherein said NOTCH pathway inhibitor is a small molecule inhibitor.
14. The method of claim 13, wherein said small molecule inhibitor is selected from the group consisting of 6-4 pyridine-3-amine, Cyclopiazonic acid, DAPT, LY685458, PF-03084014, γ-Secretase Inhibitor III. (R)-Flurbiprofen and LBH589.
15. The method of claims 1 or 8, wherein said TGFP pathway inhibitor is a small molecule inhibitor.
16. The method of claim 15, wherein said small molecule inhibitor is selected from the group consisting of LY573636, LY364947, LY2157299 and ALK5 Inhibitor II.
17. The method of claims 1 or 8, wherein said WNT pathway inhibitor is selected from the group consisting of Niclosamide, Sulindac, Aspirin, Celecoxib and Indomethacin.
18. The method of claims 9 or 10, wherein said NOTCH pathway inhibitor is PF- 03084014 and said TGFp pathway inhibitor is LY2157299.
19. The method of claim 18, wherein said BET pathway inhibitor is I-BET151.
20. The method of any one of claims 1-18, wherein said contacting is effected on an adherent substrate.
21. The method of claim 20, wherein said adherent substrate comprises matrigel or extracellular matrix component.
22. The method of claim 21, wherein said extracellular matrix component is selected from the group consisting of collagen, laminin and fibronectin.
23. The method of claim 4, wherein said dedifferentiated adult islet beta cells are generated by culturing said adult islet beta cells for at least 10 passages.
24. The method of claim 23, wherein said culturing is effected in CMRL medium.
25. The method of any one of claims 1-24, wherein said contacting is effected for at least 3 days.
26. The method of any one of claims 1-25, further comprising isolating said adult islet beta cells following said contacting.
27. An isolated population of cells generated according to the method of any of claims
1-26.
28. A method of treating diabetes in a subject, comprising transplanting a therapeutically effective amount of the population of adult islet beta cells of claim 27 into the subject, thereby treating diabetes.
29. The isolated population of claim 27, for use in treating diabetes.
30. The isolated population adult islet beta cells of claim 27, being genetically modified to express a pharmaceutical agent.
31. A pharmaceutical composition comprising as an active ingredient the population of adult islet beta cells of claim 27 and a pharmaceutically acceptable carrier.
32. A cell medium comprising CMRL and a Bromodomain and Extra-Terminal motif (BET) protein (BET) inhibitor.
33. The cell medium of claim 32, wherein said BET inhibitor is a small molecule inhibitor.
34. The cell medium of claim 33, wherein said small molecule inhibitor is selected from the group consisting of CPI-0610, DUAL946, GSK525762, I-BET151, JQl, OTX015, PFI-1, RVX-208, RVX2135 and TEN-010.
35. The cell medium of claim 32, wherein said BET pathway inhibitor is I-BET151.
36. The cell medium of any one of claims 32-35, further comprising an agent selected from the group consisting of a NOTCH pathway inhibitor, a TGFP pathway inhibitor and a WNT pathway inhibitor.
37. The cell medium of any one of claims 32-35, further comprising a NOTCH pathway inhibitor and a TGFP pathway inhibitor.
38. The cell medium of claim 36, wherein said NOTCH pathway inhibitor targets a protein selected from the group consisting of NICD, γ-secretase and HDAC.
39. The cell medium of claim 36, wherein said NOTCH pathway inhibitor is a small molecule inhibitor.
40. The cell medium of claim 39, wherein said small molecule inhibitor is selected from the group consisting of 6-4 pyridine-3-amine, Cyclopiazonic acid, DAPT, LY685458, PF- 03084014, γ-Secretase Inhibitor III, < ^-Flurbiprofen and LBH589.
41. The cell medium of claim 36, wherein said TGFP pathway inhibitor is a small molecule inhibitor.
42. The cell medium of claim 41, wherein said small molecule inhibitor is selected from the group consisting of LY573636, LY364947, LY2157299 and ALK5 Inhibitor II.
43. The cell medium of claim 36, wherein said WNT pathway inhibitor is selected from the group consisting of Niclosamide, Sulindac, Aspirin, Celecoxib and Indomethacin.
44. The cell medium of claim 37, wherein said NOTCH pathway inhibitor is PF- 03084014 and said TGFp pathway inhibitor is LY2157299.
PCT/IL2018/051053 2017-09-18 2018-09-17 Redifferentiation of cells expanded in vitro from adult human pancreatic islet beta cells, by small-molecule inhibitors of signaling pathway Ceased WO2019053727A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762559687P 2017-09-18 2017-09-18
US62/559,687 2017-09-18

Publications (1)

Publication Number Publication Date
WO2019053727A1 true WO2019053727A1 (en) 2019-03-21

Family

ID=65722473

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2018/051053 Ceased WO2019053727A1 (en) 2017-09-18 2018-09-17 Redifferentiation of cells expanded in vitro from adult human pancreatic islet beta cells, by small-molecule inhibitors of signaling pathway

Country Status (1)

Country Link
WO (1) WO2019053727A1 (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020237141A1 (en) * 2019-05-22 2020-11-26 The Cleveland Clinic Foundation Generating dorsal foregut, and anterior domain, endoderm cells
WO2021029854A1 (en) * 2019-08-09 2021-02-18 Pfizer Inc. Solid state forms of (s)-2-(((s)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-n-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1h-imidazol-4-yl)pentanamide and uses thereof
US10941118B2 (en) 2019-08-09 2021-03-09 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
US11013752B2 (en) 2016-06-03 2021-05-25 Hough Ear Institute Combination therapies for inner ear sensory hair cell regeneration/replacement
CN114317401A (en) * 2021-12-07 2022-04-12 浙江大学 Method for promoting long-term stable passage of islet precursor cells
US11504354B1 (en) 2021-09-08 2022-11-22 SpringWorks Therapeutics Inc.. Chlorinated tetralin compounds and pharmaceutical compositions
EP4063487A4 (en) * 2020-04-09 2023-01-04 Shanghai Celliver Biotechnology Co., Ltd. AMPLIFICATION AND DIFFERENTIATION METHOD FOR PANCREATIC CELLS AND USE
US11884634B2 (en) 2019-08-09 2024-01-30 Pfizer Inc. Compositions of solid forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4- tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide
US12110277B2 (en) 2021-09-01 2024-10-08 Springworks Therapeutics, Inc. Synthesis of nirogacestat

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006054305A2 (en) * 2004-11-22 2006-05-26 Ramot At Tel Aviv University Ltd. Populations of expanded and re-differentiated adult islet beta cells capable of producing insulin and methods of generating same
WO2015175307A1 (en) * 2014-05-16 2015-11-19 Janssen Biotech, Inc. Use of small molecules to enhance mafa expression in pancreatic endocrine cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006054305A2 (en) * 2004-11-22 2006-05-26 Ramot At Tel Aviv University Ltd. Populations of expanded and re-differentiated adult islet beta cells capable of producing insulin and methods of generating same
WO2015175307A1 (en) * 2014-05-16 2015-11-19 Janssen Biotech, Inc. Use of small molecules to enhance mafa expression in pancreatic endocrine cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FU, WENXIAN ET AL.: "Epigenetic modulation of type-1 diabetes via a dual effect on pancreatic macrophages and β cells.", ELIFE, vol. 3, 19 November 2014 (2014-11-19), pages 1 - 20, XP055334353, Retrieved from the Internet <URL:https://elifesciences.org/articles/04631> [retrieved on 20181205] *
TOREN-HARITAN, GINAT ET AL.: "TGF-beta pathway inhibition redifferentiates human pancreatic islet beta cells expanded in vitro", PLOS ONE, vol. 10, no. 9, 29 September 2015 (2015-09-29), pages e0139168, XP055583258, Retrieved from the Internet <URL:https://journals.plos.org/ plosone/article?id=10.1371/journal.pone.0139168> [retrieved on 20181204] *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11013752B2 (en) 2016-06-03 2021-05-25 Hough Ear Institute Combination therapies for inner ear sensory hair cell regeneration/replacement
US12435315B2 (en) 2019-05-22 2025-10-07 The Cleveland Clinic Foundation Generating dorsal foregut, and anterior domain, endoderm cells
WO2020237141A1 (en) * 2019-05-22 2020-11-26 The Cleveland Clinic Foundation Generating dorsal foregut, and anterior domain, endoderm cells
US11820748B2 (en) 2019-08-09 2023-11-21 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
US11884635B2 (en) 2019-08-09 2024-01-30 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
KR20220062510A (en) * 2019-08-09 2022-05-17 화이자 인코포레이티드 (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1 Solid state forms of -(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
CN114555562A (en) * 2019-08-09 2022-05-27 辉瑞公司 Solid state forms of (S) -2- (((S) -6, 8-difluoro-1, 2,3, 4-tetrahydronaphthalen-2-yl) amino) -N- (1- (2-methyl-1- (neopentylamino) propan-2-yl) -1H-imidazol-4-yl) pentanamide and uses thereof
WO2021029854A1 (en) * 2019-08-09 2021-02-18 Pfizer Inc. Solid state forms of (s)-2-(((s)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-n-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1h-imidazol-4-yl)pentanamide and uses thereof
US12297177B2 (en) 2019-08-09 2025-05-13 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
US11566006B2 (en) 2019-08-09 2023-01-31 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
KR102772889B1 (en) 2019-08-09 2025-02-26 화이자 인코포레이티드 Solid-state form of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
TWI867022B (en) * 2019-08-09 2024-12-21 美商輝瑞大藥廠 Solid state forms of (s)-2-(((s)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-n-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1h-imidazol-4-yl)pentanamide and uses thereof
US10941118B2 (en) 2019-08-09 2021-03-09 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
US11845732B2 (en) 2019-08-09 2023-12-19 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
JP2024149803A (en) * 2019-08-09 2024-10-18 ファイザー・インク Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
US11884634B2 (en) 2019-08-09 2024-01-30 Pfizer Inc. Compositions of solid forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4- tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide
US11905255B1 (en) 2019-08-09 2024-02-20 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
US12116347B2 (en) 2019-08-09 2024-10-15 Pfizer Inc. Solid state forms of (S)-2-(((S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide and uses thereof
EP4063487A4 (en) * 2020-04-09 2023-01-04 Shanghai Celliver Biotechnology Co., Ltd. AMPLIFICATION AND DIFFERENTIATION METHOD FOR PANCREATIC CELLS AND USE
US12110277B2 (en) 2021-09-01 2024-10-08 Springworks Therapeutics, Inc. Synthesis of nirogacestat
US12234210B2 (en) 2021-09-01 2025-02-25 Springworks Therapeutics, Inc. Synthesis of nirogacestat
US12247012B2 (en) 2021-09-01 2025-03-11 Springworks Therapeutics, Inc. Synthesis of nirogacestat
US11612588B1 (en) 2021-09-08 2023-03-28 Spring Works Therapeutics, Inc. Chlorinated tetralin compounds and pharmaceutical compositions
US11504354B1 (en) 2021-09-08 2022-11-22 SpringWorks Therapeutics Inc.. Chlorinated tetralin compounds and pharmaceutical compositions
CN114317401A (en) * 2021-12-07 2022-04-12 浙江大学 Method for promoting long-term stable passage of islet precursor cells
WO2023104015A1 (en) * 2021-12-07 2023-06-15 浙江大学 Method for promoting long-term stable passage of pancreatic islet precursor cells

Similar Documents

Publication Publication Date Title
WO2019053727A1 (en) Redifferentiation of cells expanded in vitro from adult human pancreatic islet beta cells, by small-molecule inhibitors of signaling pathway
Wang et al. Genome-wide analysis of PDX1 target genes in human pancreatic progenitors
US10752883B2 (en) Generation of neural stem cells and motor neurons
JP6491661B2 (en) Stem cell microparticles and miRNA
EP3401393B1 (en) Micrornas for the generation of astrocytes
AU2011311344B2 (en) Short RNA molecules
US20150017134A1 (en) Emt-inducing transcription factors cooperate with sox9
US20250129339A1 (en) Generation of glucose-responsive beta cells
Vasanthan et al. Differential expression of basal micro RNA s’ patterns in human dental pulp stem cells
Bai et al. MicroRNAs can effectively induce formation of insulin‐producing cells from mesenchymal stem cells
US20170009211A1 (en) DOWNREGULATION OF miR-7 FOR PROMOTION OF BETA CELL DIFFERENTIATION AND INSULIN PRODUCTION
Shaer et al. miR-375 induces human decidua basalis-derived stromal cells to become insulin-producing cells
US10568913B2 (en) Method for generating mature β-like cells
US8728813B2 (en) Methods of expanding and redifferentiating islet beta cells
WO2017100498A1 (en) Isolation of fusion-competent myoblasts and therapeutic applications thereof related to muscular dystrophy
US20100278789A1 (en) Methods of generating expanded and re-differentiated adult islet beta cells capable of producing insulin
Peng et al. Involvement of gene methylation changes in the differentiation of human amniotic epithelial cells into islet-like cell clusters
US20160017290A1 (en) Methods of increasing insulin content in cells
Wang et al. Identification and action of N-myc downstream regulated gene 4 A2 in rat pancreas

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18856023

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18856023

Country of ref document: EP

Kind code of ref document: A1