Patent Application
20150322406
The present invention relates to a method for reprogramming a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell. This method may be carried out ex vivo or in vivo. This method has therapeutic applications for the treatment of a subject with a metabolic disorder, such as diabetes, or a subject at risk of developing a metabolic disorder, such as diabetes.
The generation of specific therapeutically relevant cell types is a pre-requisite for cell replacement therapies and an important goal of personalized medicine. Pancreatic β-cells control glucose homeostasis through the production of insulin, and inadequate numbers of insulin-producing β-cells is the major cause of type 1 diabetes mellitus (reviewed in 1). Therefore the replenishment of lost β-cells may allow for improved diabetes treatment2.
The pancreas is composed of an exocrine (ductal and acinar cells) and an endocrine (β-cells, α-cells, δ-cells and pp cells) part, which are distinctively differentiated to secrete enzymes or hormones respectively5. Importantly, β-cells are not regenerated during adulthood. β-cell neogenesis in the adult has only been achieved experimentally with rather artificial approaches. For instance, pancreatic duct ligation6 and pancreatectomy7 experimentally induce adult β-cell neogenesis. Additionally, conversion of other cell types into β-cells by genetic approaches has been demonstrated. Adenoviral overexpression of the three transcription factors Ngn3, Maf1a and Pdx1 is sufficient to convert adult acinar cells into β-cells8, and extreme β-cell loss after diphtheria toxin overexpression or overexpression of Pax4 converts glucagon-producing cells into β-cells9,10. However, the regulation and mechanisms of β-cell neogenesis in the normal adult pancreas remains largely unknown.
Fbw7 is the substrate recognition component of an SCF-type E3 ubiquitin ligase and can regulate the abundance of several transcription factor substrates involved in the control of progenitor cell fate decisions such as Notch1, c-Myc and c-Jun3,4. Fbw7 is frequently inactivated by deletion, mutation or promoter hypermethylation in cancer26. Fbw7 mutations occur in approximately 30% of T-cell acute lymphoblastic leukaemia (T-ALL), 16% of primary endometrial cancer and 11% of colorectal cancer. Overall, approximately 6% of all human primary tumours have mutations in Fbw727.
The present inventors have found that inactivation of fbw7 reprograms differentiated pancreatic ductal cells in adult mice into cells that show hallmarks of β-cells. The induced β-cells express proteins essential for β-cell function and are indistinguishable from islet β-cells in histological morphology and ultrastructure. Ductal cell to β-cell trans-differentiation occurred rapidly within several days, did not require cellular proliferation, and involved an intermediate stage when cells undergoing reprogramming co-expressed ductal and β-cell markers. Thus, inactivation of fbw7 induces direct cell reprogramming of exocrine ductal cells into β-cells without reversion to a progenitor cell state. Our study highlights the plasticity of seemingly differentiated adult cells, and identifies Fbw7 as a master regulator of cell fate decisions in the adult pancreas.
In a first aspect, the invention provides a method for reprogramming a somatic cell to a pancreatic β-cell, the method comprising the step of decreasing expression or activity of Fbw7 in the somatic cell. The somatic cell may, for example, be a pancreatic ductal cell, a skin fibroblast or a keratinocyte. Preferably, the somatic cell is a pancreatic ductal cell. The method may be carried out ex vivo or in vivo. For example, the method may be carried out in a subject with a metabolic disorder, such as diabetes, or in a subject at risk of developing a metabolic disorder, such as diabetes. The method may comprise contacting the somatic cell, e.g. pancreatic ductal cell, with an agent that reduces the expression or activity of Fbw7 in the somatic cell. The activity of Fbw7 may be the phosphorylation-mediated degradation of neurogenin-3 (NGN3). For example, the agent that reduces the expression or activity of Fbw7 may inhibit the phosphorylation of NGN3 by GSK3β.
In a second aspect, the invention provides a method for reprogramming a somatic cell to a pancreatic β-cell, the method comprising the step of decreasing phosphorylation-mediated degradation of NGN3. The somatic cell may, for example, be a pancreatic ductal cell, a skin fibroblast or a keratinocyte. Preferably, the somatic cell is a pancreatic ductal cell. In one embodiment, the method may comprise the step of decreasing phosphorylation of NGN3 by GSK3β. The method may be carried out ex vivo or in vivo. For example, the method may be carried out in a subject with a metabolic disorder, such as diabetes, or in a subject at risk of developing a metabolic disorder, such as diabetes. The method may comprise the step of contacting the somatic cell, e.g. pancreatic ductal cell, with an agent that decreases the phosphorylation of NGN3 by GSK3β, such as an inhibitor of GSK3β kinase activity.
In a third aspect, the invention provides an agent that reduces the expression or activity of Fbw7 for use in a method of reprogramming a somatic cell to a pancreatic β-cell, the method comprising the step of contacting the somatic cell with said agent. The somatic cell may, for example, be a pancreatic ductal cell, a skin fibroblast or a keratinocyte. Preferably, the somatic cell is a pancreatic ductal cell. The activity of Fbw7 may be the phosphorylation-mediated degradation of NGN3. For example, the agent may inhibit the phosphorylation of NGN3 by GSK3β.
In a fourth aspect, the invention provides an agent that decreases the phosphorylation-mediated degradation of NGN3 for use in a method of reprogramming a somatic cell to a pancreatic β-cell, the method comprising the step of contacting the somatic cell with said agent. The somatic cell may, for example, be a pancreatic ductal cell, a skin fibroblast or a keratinocyte. Preferably, the somatic cell is a pancreatic ductal cell. The agent may inhibit the phosphorylation of NGN3 by GSK3β. For example, the agent may be an inhibitor of GSK3β kinase activity.
In a fifth aspect, the invention provides a method of treating a subject with a metabolic disorder or a subject at risk of developing a metabolic disorder, the method comprising the step of administering to the subject pancreatic β-cells obtained by the method of the first or second aspect above. This method of treating a subject may include the step of reprogramming a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell using the method of the first or second aspect above followed by the step of administering the pancreatic β-cells obtained by this method to the subject. The metabolic disorder is preferably diabetes.
In a sixth aspect, the invention provides pancreatic β-cells obtained by the method of the first or second aspect above for use in a method for treatment of a subject with a metabolic disorder or a subject at risk of developing a metabolic disorder, said method comprising administering said pancreatic β-cells to the subject. The metabolic disorder is preferably diabetes.
In a seventh aspect, the invention provides a screening method for identifying a candidate agent for reprogramming a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell, the method comprising determining the ability of the agent to reduce expression or activity of Fbw7, wherein an agent that reduces expression or activity of Fbw7 is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell. The activity of Fbw7 may be the phosphorylation-mediated degradation of NGN3, for example the phosphorylation of NGN3 by GSK3β.
In an eighth aspect, the invention provides a screening method for identifying a candidate agent for reprogramming a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell, the method comprising determining the ability of the agent to decrease phosphorylation-mediated degradation of NGN3, wherein an agent that decreases phosphorylation-mediated degradation of NGN3 is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell. The method may comprise the step of determining the ability of the agent to decrease phosphorylation of NGN3 by GSK3β, wherein an agent that decreases phosphorylation of NGN3 by GSK3β is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell.
These and other aspects of the invention are described in further detail below.
The present invention is based on the finding that inactivation of fbw7 reprograms differentiated somatic cells, e.g. pancreatic ductal cells, into cells that show hallmarks of pancreatic β-cells. The invention is also based on the identification of NGN3 as a novel substrate for Fbw7 and the finding that Fbw7 targets NGN3 for ubiquitin-mediated destruction by the proteasome.
In a first aspect, the invention provides a method for reprogramming a somatic cell to a pancreatic β-cell, the method comprising the step of decreasing expression or activity of Fbw7 in the somatic cell. The somatic cell may, for example, be a pancreatic ductal cell, a skin fibroblast or a keratinocyte. Preferably, the somatic cell is a pancreatic ductal cell. The method may be carried out ex vivo or in vivo. For example, the method may be carried out in a subject with a metabolic disorder, such as diabetes, or in a subject at risk of developing a metabolic disorder, such as diabetes. Alternatively, the method may be carried out in an experimental animal, such as a mouse or rat.
The method may comprise contacting the somatic cell, such as a pancreatic ductal cell, with an agent that reduces the expression or activity of Fbw7 in the somatic cell. The activity of Fbw7 may be the phosphorylation-mediated degradation of NGN3. For example, the agent that reduces the expression or activity of Fbw7 may inhibit the phosphorylation of NGN3 by GSK3β.
Pancreatic ductal cells form the epithelial lining of the branched tubes that deliver enzymes produced by pancreatic acinar cells into the duodenum. In addition, these cells secrete bicarbonate that neutralizes stomach acidity. During development, epithelium of endodermal origin evaginates from the future duodenum area and invades the mesenchyme to form a complex branched network. All endocrine, acinar and ductal cells arise from common precursors in this epithelial structure. Adult ductal cells share some similarities with embryonic primitive ducts and may retain the ability to generate endocrine cells in the adult28. Pancreatic ductal cells may, for example, be obtained from pancreatic biopsies, e.g. from a human pancreatic biopsy, or from a full post-mortem pancreas in the case of an experimental animal. Pancreatic tissue may, for example, be digested using collagenase and single cell suspensions of pancreatic ductal cells cultured as described in detail below.
The pancreatic ductal cells have preferably been obtained from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. For example, these cells may have been obtained from a pancreatic biopsy taken from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. The subject is preferably a mammal, and most preferably a human.
Fbw7 is an ubiquitin E3 ligase substrate adaptor. Ubiquitin E3 ligases can physically bind to and add ubiquitin chains to a target protein to result in its recognition and degradation in an ATP-dependent manner by the proteasome. Fbw7 (or FBW7) is also known as F-box/WD repeat-containing protein 7 isoform 1, CDC4, Ago (or AGO), hSel10, Fbxw7, FLJ16457, FBW6, SEL-10, CDC4, DKFZp686F23254, FBX30, FBXO30, FBXW6, or SEL1.
The amino acid and nucleotide sequences of human and mouse Fbw7 are shown in Tables 1, 2a and 2b below.
When carried out ex vivo, the method may comprise the step of decreasing expression or activity of Fbw7 in a somatic cell, such as a pancreatic ductal cell, obtained from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. The method may comprise contacting the somatic cell, such as a pancreatic ductal cell, with an agent that reduces the expression or activity of Fbw7 in the somatic cell. The activity of Fbw7 may be the phosphorylation-mediated degradation of NGN3. For example, the agent that reduces the expression or activity of Fbw7 may inhibit the phosphorylation of NGN3 by GSK3β.
When carried out in vivo, the method may comprise the step of decreasing expression or activity of Fbw7 in a somatic cell, such as a pancreatic ductal cell, present in a subject with a metabolic disorder, such as diabetes, in a subject at risk of developing a metabolic disorder, such as diabetes, or in an experimental animal, such as a mouse or a rat. For example, the method may comprise the step of administering to the subject or experimental animal an agent that reduces the expression or activity of Fbw7 in a somatic cell, such as a pancreatic ductal cell. The activity of Fbw7 may be the phosphorylation-mediated degradation of NGN3. For example, the agent that reduces the expression or activity of Fbw7 may inhibit the phosphorylation of NGN3 by GSK3β. The subject is preferably a mammal, and most preferably a human.
The agent that reduces the expression or activity of Fbw7 may be administered to the subject or experimental animal by any suitable route of administration. For example, the agent may be administered by intraductal, intraperitoneal or intravenous injection or orally.
Decreased or reduced expression of Fbw7 may be achieved at the nucleic acid level or at the protein level. For example, the transcription of the Fbw7 gene into Fbw7 mRNA may be decreased or reduced, or the translation of Fbw7 mRNA into Fbw7 protein may be decreased or reduced.
Alternatively, decreased or reduced Fbw7 expression or activity may be achieved by decreasing or reducing Fbw7 protein stability or by inhibiting Fbw7 dimerisation.
The activity of Fbw7 includes its downstream effects on its substrates such as c-Myc, Cyclin E, c-jun, c-Myb, KLF5, Mcl-1, mTOR, Notch1, SREBP1, presenilin and Notch intracellular domain 1 (NICD1). The inventors have also identified NGN3 as a novel substrate for Fbw7 and have found that Fbw7 targets NGN3 for ubiquitin-mediated destruction by the proteasome, thus reducing its level in cells. Specifically, Fbw7 recognises phosphorylated NGN3 and targets it for ubiquitin-mediated destruction. Therefore, reducing the activity of Fbw7 may include reducing its downstream activity, e.g. by reducing ubiquitin-mediated destruction of NGN3 by Fbw7. For example, reducing the activity of Fbw7 may include reducing the phosphorylation of NGN3. This reduces its targeting for ubiquitin-mediated destruction by Fbw7, thus reducing the downstream activity of Fbw7. For example, the agent that reduces the expression or activity of Fbw7 may inhibit the phosphorylation of NGN3, e.g. by GSK3β. Therefore, the agent that reduces the expression or activity of Fbw7 may be an inhibitor of GSK3β. Examples of agents that reduces the expression or activity of Fbw7 by inhibiting the phosphorylation of NGN3 by GSK3β include lithium chloride, CHIR-99021 (CT99021), SB 216763, TWS119, CHIR-98014, SB 415286, tideglusib, LY2090314 and TDZD-8.
NGN3 (neurogenin-3) is a regulator of pancreatic islet differentiation and regeneration29. NGN3 is also known as Neurog3, Atoh5, MGC129292, MGC129293, Math4B, bHLHa7, Atonal protein homolog 5, Helix-loop-helix protein mATH-4B, MATH4B), atonal homolog 5, protein atonal homolog 5.
The amino acid and nucleotide sequences of human and mouse NGN3 are shown in Tables 3 and 4 below.
The agent that reduces the expression or activity of Fbw7 for use in the invention may, for example, be a small molecule, a nucleotide sequence, a protein (e.g. an antibody), a peptide, a modified peptide (e.g. a stapled peptide), an aptamer or an RNA interference (RNAi) agent (e.g. an siRNA). For example, the agent may be a small molecule inhibitor of NGN3 phosphorylation by GSK3β (as discussed above), e.g. lithium chloride, CHIR-99021 (CT99021), SB 216763, TWS119, CHIR-98014, SB 415286, tideglusib, LY2090314 or TDZD-8; an anti-Fbw7 antibody; or an siRNA molecule that reduces the expression of Fbw7. The agent may also reduce or decrease the stability of Fbw7 or inhibit its dimerization. Exemplary siRNA sequences that can be used to reduce the expression of Fbw7 include:
As mentioned above and as demonstrated by the Examples below, the inventors have identified NGN3 as a novel substrate for Fbw7 and have found that Fbw7 targets NGN3 for ubiquitin-mediated destruction by the proteasome, thus reducing its level in cells. Specifically, Fbw7 recognises phosphorylated NGN3 and targets it for ubiquitin-mediated destruction (see
Therefore, in a second aspect, the invention provides a method for reprogramming a pancreatic ductal cell to a pancreatic β-cell, the method comprising the step of decreasing phosphorylation-mediated degradation of NGN3. For example, the method may comprise the step of decreasing phosphorylation of NGN3 by GSK3β. The method may be carried out ex vivo or in vivo. For example, the method may be carried out in a subject with a metabolic disorder, such as diabetes, in a subject at risk of developing a metabolic disorder, such as diabetes, or in an experimental animal. The method may comprise the step of contacting the somatic cell, such as a pancreatic ductal cell, with an agent that decreases the phosphorylation of NGN3 by GSK3β, such as an inhibitor of GSK3β kinase activity.
The pancreatic ductal cells have preferably been obtained from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. For example, these cells may have been obtained from a pancreatic biopsy taken from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. The subject is preferably a mammal, and most preferably a human.
When carried out ex vivo, the method may comprise the step of decreasing phosphorylation-mediated degradation of NGN3 in a somatic cell, such as a pancreatic ductal cell, obtained from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. The method may comprise contacting the somatic cell, such as a pancreatic ductal cell, with an agent that decreases the phosphorylation-mediated degradation of NGN3 in the somatic cell. For example, the agent that decreases phosphorylation-mediated degradation of NGN3 may inhibit phosphorylation of NGN3 by GSK3β. The subject is preferably a mammal, and most preferably a human.
When carried out in vivo, the method may comprise the step of decreasing phosphorylation-mediated degradation of NGN3 in a somatic cell, such as a pancreatic ductal cell, present in a subject with a metabolic disorder, such as diabetes, in a subject at risk of developing a metabolic disorder, such as diabetes, or in an experimental animal, such as a mouse or rat. For example, the method may comprise the step of administering to the subject or experimental animal an agent that decreases the phosphorylation-mediated degradation of NGN3 in the somatic cell, such as a pancreatic ductal cell. For example, the agent that decreases phosphorylation-mediated degradation of NGN3 may inhibit phosphorylation of NGN3 by GSK3β. The subject is preferably a mammal, and most preferably a human.
The agent that decreases the phosphorylation-mediated degradation of NGN3 for use in the invention may, for example, be a small molecule, a nucleotide sequence, a protein (e.g. an antibody), a peptide, a modified peptide (e.g. a stapled peptide), an aptamer or an RNA interference (RNAi) agent (e.g. an siRNA). For example, the agent may be a small molecule inhibitor of NGN3 phosphorylation by GSK3β (e.g. lithium chloride, CHIR-99021 (CT99021), SB 216763, TWS119, CHIR-98014, SB 415286, tideglusib, LY2090314 or TDZD-8), an anti-GSK3β antibody or an siRNA molecule that reduces the expression of GSK3β.
In a third aspect, the invention provides an agent that reduces the expression or activity of Fbw7 for use in a method of reprogramming a somatic cell to a pancreatic β-cell, the method comprising the step of contacting the somatic cell with said agent. For example, the somatic cell may be a pancreatic ductal cell, a skin fibroblast or a keratinocyte. The somatic cell is preferably a pancreatic ductal cell. The activity of Fbw7 may be the phosphorylation-mediated degradation of NGN3. For example, the agent may inhibit the phosphorylation of NGN3 by GSK3β.
The pancreatic ductal cells have preferably been obtained from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. For example, these cells may have been obtained from a pancreatic biopsy taken from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. The subject is preferably a mammal, and most preferably a human.
As set out above, Fbw7 (or FBW7) is also known as F-box/WD repeat-containing protein 7 isoform 1, CDC4, Ago (or AGO), hSel10, Fbxw7, FLJ16457, FBW6, SEL-10, CDC4, DKFZp686F23254, FBX30, FBXO30, FBXW6, or SEL1. The amino acid and nucleotide sequences of human and mouse Fbw7 are shown in Table 1.
The method of reprogramming the somatic cell, such as a pancreatic ductal cell, may be carried out ex vivo or in vivo. When the method is carried out ex vivo, the method may include the step of obtaining a somatic cell, such as a pancreatic ductal cell, from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes, and then contacting the somatic cell, such as a pancreatic ductal cell, with said agent ex vivo. When the method is carried out in vivo, the method may include the step of contacting a somatic cell, such as a pancreatic ductal cell, present in a subject with a metabolic disorder, such as diabetes, or in a subject at risk of developing a metabolic disorder, such as diabetes, with said agent. For example, the method may include administering said agent to a subject with a metabolic disorder, such as diabetes, or in a subject at risk of developing a metabolic disorder, such as diabetes. The subject is preferably a mammal, and most preferably a human.
Decreased or reduced expression of Fbw7 may be achieved at the nucleic acid level or at the protein level. For example, transcription of the Fbw7 gene into Fbw7 mRNA may be decreased or reduced, or translation of Fbw7 mRNA into Fbw7 protein may be decreased or reduced.
Alternatively, decreased or reduced Fbw7 expression or activity may be achieved by decreasing or reducing Fbw7 protein stability or by inhibiting Fbw7 dimerisation.
The activity of Fbw7 includes its downstream effects on its substrates such as c-Myc, Cyclin E, c-jun, c-Myb, KLF5, Mcl-1, mTOR, Notch1, SREBP1 and presenilin. As described herein, the inventors have also identified NGN3 as a novel substrate for Fbw7 and have found that Fbw7 targets NGN3 for ubiquitin-mediated destruction by the proteasome, thus reducing its level in cells. Specifically, Fbw7 recognises phosphorylated NGN3 and targets it for ubiquitin-mediated destruction. Therefore, reducing the activity of Fbw7 includes reducing its downstream activity, e.g. by reducing ubiquitin-mediated destruction of NGN3 by Fbw7. For example, reducing the activity of Fbw7 may include reducing the phosphorylation of NGN3. This reduces its targeting for ubiquitin-mediated destruction by Fbw7, thus reducing the downstream activity of Fbw7. For example, the agent that reduces the expression or activity of Fbw7 may inhibit the phosphorylation of NGN3, e.g. by GSK3β. Therefore, the agent that reduces the expression or activity of Fbw7 may be an inhibitor of GSK3β. Examples of agents that reduce the expression or activity of Fbw7 by inhibiting the phosphorylation of NGN3 by GSK3β include lithium chloride, CHIR-99021 (CT99021), SB 216763, TWS119, CHIR-98014, SB 415286, tideglusib, LY2090314 and TDZD-8.
The agent that reduces the expression or activity of Fbw7 for use in the invention may, for example, be a small molecule, a nucleotide sequence, a protein (e.g. an antibody), a peptide, a modified peptide (e.g. a stapled peptide), an aptamer or an RNA interference (RNAi) agent (e.g. an siRNA). For example, the agent may be a small molecule inhibitor of NGN3 phosphorylation by GSK3β (as discussed above), e.g. lithium chloride, CHIR-99021 (CT99021), SB 216763, TWS119, CHIR-98014, SB 415286, tideglusib, LY2090314 or TDZD-8; an anti-Fbw7 antibody; an anti-Fbw7 antibody; or an siRNA molecule that reduces the expression of Fbw7. Exemplary siRNA sequences that can be used to reduce the expression of Fbw7 include:
In a fourth aspect, the invention provides an agent that decreases the phosphorylation-mediated degradation of NGN3 for use in a method of reprogramming a pancreatic ductal cell to a pancreatic β-cell, the method comprising the step of contacting the pancreatic ductal cell with said agent. The agent may inhibit the phosphorylation of NGN3 by GSK3β. For example, the agent may be an inhibitor of GSK3β kinase activity.
The somatic cells, such as pancreatic ductal cells, have preferably been obtained from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes. For example, these cells may have been obtained from a pancreatic biopsy taken from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes.
As set out above, NGN3 is also known as Neurog3, Atoh5, MGC129292, MGC129293, Math4B, bHLHa7, Atonal protein homolog 5, Helix-loop-helix protein mATH-4B, MATH4B), atonal homolog 5, protein atonal homolog 5.
The amino acid and nucleotide sequences of human and mouse NGN3 are shown in Tables 3 and 4.
The method of reprogramming the somatic cell, such as a pancreatic ductal cell, may be carried out ex vivo or in vivo. When the method is carried out ex vivo, the method may include the step of obtaining a somatic cell, such as a pancreatic ductal cell, from a subject with a metabolic disorder, such as diabetes, or from a subject at risk of developing a metabolic disorder, such as diabetes, and then contacting the somatic cell, such as a pancreatic ductal cell, with said agent ex vivo. The subject is preferably a mammal, and most preferably a human. When the method is carried out in vivo, the method may include the step of contacting a somatic cell, such as a pancreatic ductal cell, present in a subject with a metabolic disorder, such as diabetes, or in a subject at risk of developing a metabolic disorder, such as diabetes, with said agent. For example, the method may include administering said agent to a subject with a metabolic disorder, such as diabetes, or in a subject at risk of developing a metabolic disorder, such as diabetes. The subject is preferably a mammal, and most preferably a human.
The agent that decreases the phosphorylation-mediated degradation of NGN3 for use in the invention may, for example, be a small molecule, a nucleotide sequence, a protein (e.g. an antibody), a peptide, a modified peptide (e.g. a stapled peptide), an aptamer or an RNA interference (RNAi) agent (e.g. an siRNA). For example, the agent may be a small molecule inhibitor of NGN3 phosphorylation by GSK3β (as discussed above), e.g. lithium chloride, an anti-GSK3β antibody or an siRNA molecule that reduces the expression of GSK3β. Exemplary siRNA sequences that can be used to reduce the expression of Fbw7 include:
In a fifth aspect, the invention provides a method of treating a subject with a metabolic disorder or a subject at risk of developing a metabolic disorder, the method comprising the step of administering to the subject pancreatic β-cells obtained by the method of the first or second aspect above. This method of treating a subject may include the step of reprogramming a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell using the method of the first or second aspect above followed by the step of administering the pancreatic β-cells obtained by this method to the subject. The metabolic disorder is preferably diabetes. The subject is preferably a mammalian subject and most preferably a human subject. Pancreatic β-cells may be administered to the subject by any suitable method, such as intra-portal vein (liver) islet transplantation or kidney capsule transplantation in humans, and kidney capsule, intrapancreatic or intrahepatic transplantation in mice.
In a sixth aspect, the invention provides pancreatic β-cells obtained by the method of the first or second aspect above for use in a method for treatment of a subject with a metabolic disorder or a subject at risk of developing a metabolic disorder, said method comprising administering said pancreatic β-cells to the subject. The metabolic disorder is preferably diabetes. The subject is preferably a mammalian subject and most preferably a human subject. Pancreatic β-cells may be administered to the subject by any suitable method, such as intra-portal vein (liver) islet transplantation or kidney capsule transplantation in humans, and kidney capsule, intrapancreatic or intrahepatic transplantation in mice.
In a seventh aspect, the invention provides a screening method for identifying a candidate agent for reprogramming a somatic cell to a pancreatic β-cell, the method comprising determining the ability of the agent to reduce expression or activity of Fbw7, wherein an agent that reduces expression or activity of Fbw7 is likely to be able to reprogram the somatic cell to a pancreatic β-cell. For example, the somatic cell may be a pancreatic ductal cell, a skin fibroblast or a keratinocyte. The somatic cell is preferably a pancreatic ductal cell. The activity of Fbw7 may be the phosphorylation-mediated degradation of NGN3, for example the phosphorylation of NGN3 by GSK33.
The screening method may be carried out in vitro or in vivo.
The candidate agent may reduce expression of Fbw7 at the nucleic acid level or at the protein level. For example, the candidate agent may reduce transcription of the Fbw7 gene into Fbw7 mRNA or the candidate agent may reduce translation of Fbw7 mRNA into Fbw7 protein may be reduced.
The activity of Fbw7 includes its downstream effects on its substrates such as c-Myc, Cyclin E, c-jun, c-Myb, KLF5, Mcl-1, mTOR, Notch1, SREBP1 and presenilin. As described herein, reducing the activity of Fbw7 may include reducing ubiquitin-mediated destruction of NGN3 by Fbw7. For example, reducing the activity of Fbw7 may include reducing its downstream activity, e.g. by reducing the phosphorylation of NGN3. This reduces its targeting for ubiquitin-mediated destruction by Fbw7, thus reducing the downstream activity of Fbw7. For example, the method may comprise determining the ability of the agent to reduce phosphorylation of NGN3 (e.g. by GSK3β), wherein an agent that reduces phosphorylation of NGN3 is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell.
The method may comprise the step of contacting a cell expressing Fbw7 with the candidate agent and determining the level of Fbw7 mRNA in the cell before and after the contacting step, wherein a decrease in Fbw7 mRNA following the contacting step indicates that the agent is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell. The level of Fbw7 mRNA may be determined by any suitable method known in the art, such as quantitative PCR.
The method may comprise the step of contacting a cell co-expressing NGN3 and Fbw7 with the candidate agent and determining ubiquitination of NGN3 by Fbw7 in the absence or presence of the agent, wherein a reduction in ubiquitination of NGN3 following the step of contacting the cell with the agent indicated that the agent is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell.
The method may comprise the step of contacting a cell expressing Fbw7 with the candidate agent and determining the level of Fbw7 protein expression or expression of a substrate of Fbw7, such as NGN3, c-Jun or Notch intracellular domain 1 (NICD-1), in the cell before and after the contacting step, wherein a decrease in Fbw7 protein expression or expression of a substrate of Fbw7 following the contacting step indicates that the agent is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell. The level of Fbw7 protein or a substrate of FBw7 may be determined by any suitable method known in the art, such as immunohistochemistry.
In an eighth aspect, the invention provides a screening method for identifying a candidate agent for reprogramming a somatic cell to a pancreatic β-cell, the method comprising determining the ability of the agent to decrease phosphorylation-mediated degradation of NGN3, wherein an agent that decreases phosphorylation-mediated degradation of NGN3 is likely to be able to reprogram a somatic cell to a pancreatic β-cell. For example, the somatic cell may be a pancreatic ductal cell, a skin fibroblast or a keratinocyte. The somatic cell is preferably a pancreatic ductal cell. The method may comprise the step of determining the ability of the agent to decrease phosphorylation of NGN3 by GSK3β, wherein an agent that decreases phosphorylation of NGN3 by GSK3β is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell.
The step of determining whether an agent decreases phosphorylation of NGN3 by GSK3β may be carried out by any suitable method known in the art, such as phospho-specific NGN3 antibody development. Phosphorylation of NGN3 may also be determined indirectly by determining its ability to interact with Fbw7, wherein an agent that decreases the ability of NGN3 to interact with Fbw7 is likely to be able to reprogram a somatic cell, such as a pancreatic ductal cell, to a pancreatic β-cell.
Further aspects and embodiments of the invention will be apparent to those skilled in the art given the present disclosure including the following experimental exemplification.
The Pdx1-cre21, R26-LSL-YFP22, R26-CreERT23 and Fbw7f/f 24 mouse lines have been previously described.
Mice were either injected intraperitoneally with tamoxifen (TM) or intrapancreatically injected with 4-OH-tamoxifen as indicated. Short and long term histological analyses were performed at short. (5 or 13 days) or long (60 or 82 days) post-injections. Pancreata were excised, fixed in cold 4% paraformaldehyde for 12 h, embedded in paraffin, cut into 7-10 μm sections and staining was performed as described below.
Mice were euthanized by cervical dislocation and pancreas dissected out into ice-cold PBS. The pancreas was fixed overnight in 10% neutral buffered formalin, briefly washed with PBS and transferred into 70% ethanol, processed and embedded into paraffin. Sections were cut at 7 μm for Haematoxylin & Eosin staining.
Immunofluorescence and immunohistochemistry staining was performed as described before13 using antibodies against GFP (Roche Applied Science), glut2 (Millipore), insulin (Sigma), amylase (Santa Cruz) and glucagon (Sigma) were used. The percentage of GFP positive cells per cell type (see
Quantification of number of ducts per area, number of isolated insulin clusters per area, number of insulin clusters in close proximity to a duct per area and number of insulin clusters within a duct (see
EM analysis was done by the Electron Microscopy service at London Research Institute (Cancer Research UK). Briefly, dissected pancreas was fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 2 h at room temperature (24° C.). For conventional transmission electron microscopy, samples were further fixed by osmium tetroxide, embedded in Epon resin and sectioned at 60-80 nm. Images were obtained with a Tecnai G2 Spirit BioTWIN transmission electron microscope.
Pancreata were dissected out and lysates were homogenized in RIPA lysis buffer supplemented with protease inhibitor (Sigma). Immunoblots were carried out as previously described25. The gels were transferred to nitrocellulose membranes, and the membranes were immunoblotted with various antibodies as indicated: NICD-1 (Abcam), p-c-Junser73 (Santa Cruz), p-Myc (Santa Cruz), p-cyclin E (Santa Cruz) and actin (Sigma).
Statistical evaluation was performed by Student's unpaired t test. Data are presented as mean±SEM. **p≦0.001 was considered highly statistically significant; ***p≦0.0001 was considered very highly statistically significant.
HEK293T cells were cultured in DMEM and 10% FBS. Cells were plated at subconfluence and transfected with Lipofectamine Reagent (Invitrogen) following the manufacturer instructions.
Pancreas isolated from the mice with the indicated genotypes was digested in collagenase (1.6 mg/ml—Worthington Biochemical Corporation) at 37° C. during 30 min at constant shaking. Pancreas tissue homogenate after digestion was passed through a Nylon mesh (70 μm). Eluted cells were cultured under self-renewal conditions in Neurobasal Medium (Invitrogen) supplemented with 1% Penicillin/Streptomycin (10000 U/ml; Invitrogen), 1% L-glutamine (200 mM; Invitrogen), 2% B27 supplement (Invitrogen), human epidermal growth factor (EGF; 20 ng/ml; PeproTech) and fibroblast growth factor (FGF-basic; 20 ng/ml; PeproTech). Cells were plated in non-adherent or adherent 24-well plates as indicated. For differentiation, the primary spheres were mechanically dissociated and replated in 24 well plates.
PCR generated NGN3-HA was cloned into pDEST-Flag using Gateway® technology. All the phosphomutants forms were generated by PCR-direct mutagenesis and cloned in pDEST-Flag using Gateway® technology. For the generation of the construct to generate the NGN3-AA transgenic mouse, the PCR product (NGN3-AA) was cloned into pRosa26-DV1 plasmid using Gateway® technology.
Cells transfected with the indicated plasmids were treated with proteasome inhibitor for 3 hours. Cells were then lysed for 30 min at 4° C. in 500 μl of immunoprecipitation (IP) buffer containing 0.5% Triton X-100, 1 mM EDTA, 100 μM Na-orthovanadate, 0.25 mM PMSF, and protease inhibitor mixture (Sigma) in PBS. After centrifugation, supernatants were incubated for 3 h at 4° C. with 1 μg of the Flag or HA antibody (Sigma) coupled to Protein A-Sepharose beads. IP products and whole lysates were run in a 12% gel, transferred to a nitrocellulose membrane and immunoblotted with anti-HA or anti-Flag antibodies.
HEK293T cells lysates or tissues were homogenized in RIPA lysis buffer supplemented with protease inhibitor (Sigma). Immunoblots were carried out as previously described4. The gels were transferred to nitrocellulose membranes, and the membranes were immunoblotted with various antibodies as indicated: anti-NGN3, anti-flag, anti-HA, and anti-Myc.
For qRT-PCRs, cDNA was synthesised using Invitrogen Superscript reagents according to the manufacturer's instructions. Quantitative real-time PCR was accomplished with SYBRGreen incorporation (Platinum® Quantitative PCR SuperMix-UDG w/ROX, Invitrogen) using an ABI7900HT (Applied Bioscience), and the data were analyzed using the SDS 2.3 software. Results were normalized to those obtained with β-actin and results are presented as fold induction over control mice.
The sequences of the primers used for Q-PCR analysis were:
Mice were euthanised by cervical dislocation and the pancreas was dissected out. The pancreas was fixed overnight in 10% neutral buffered formalin, briefly washed with PBS, transferred into 70% ethanol, processed and embedded into paraffin. Sections were cut at 4 μm for immunohistochemistry. For immunohistochemistry, an antibody against insulin was used.
Sphere derived differentiated cells were washed twice with cold PBS and fixed with 4% PFA at room temperature for 15 minutes. Cells were washed with PBS and permeabilized with PBS+0.1% Triton for 10 min. Cells were washed with PBS and blocked in 1% BSA/PBS before addition of primary antibodies diluted 1:1000 in blocking buffer. Cells were washed three times for 5 minutes with blocking buffer followed by the addition of secondary antibodies diluted 1:400 in blocking buffer for 1 hour. Cells were washed with PBS and incubated with DAPI prior to visualising on a Zeiss Axioplan 2 upright microscope using AxioVision 4.1 imaging software.
Transgenic mouse generation was performed by the transgenic unit at the Cancer Research UK London Research Institute.
To assess the role of fbw7 during pancreatic cell-type specification, mice in which fbw7 was deleted in pdx1-positive pancreatic progenitor cells were analysed during embryonic development by generating Pdx1-Cre; fbw7f/f mice. While pancreatic organ size and gross morphology appeared normal, histological analysis revealed some scattered cells in the Pdx1-Cre; fbw7f/f ducts with altered morphology when compared with fbw7f/f ducts (see FIG. 1a-d). These mutant cells were characterized by enlarged cytoplasm and a nuclear shape different from surrounding ductal cells (see
One of the key features of functional β-cells is the expression of the glucose transporter 2 (glut2). In control animals, glut2 was co-expressed with insulin in islet β-cells (see
To analyse the induced β-cells, double immunofluorescence for markers of pancreatic cell types was performed. In fbw7f/f control adult pancreas, cytokeratin-19 (ck19), a bona fide marker of ductal cells, and insulin were never co-expressed (see
During embryonic development, Pdx1-expressing progenitors give rise to all the pancreatic lineages12. Thus, the emergence of insulin-positive cells found in Pdx1-Cre; fbw7f/f ducts may reflect a developmental-specific defect. To test whether fbw7 deletion was sufficient to induce β-cell neogenesis in adulthood, and to clarify the cells of origin giving rise to the induced β-cells, fbw7 inactivation was combined with lineage tracing. To this end, the R26-CreERT line, which expresses a tamoxifen-inducible form of cre recombinase from the ubiquitous ROSA26 (R26) promoter, was employed in combination with R26-LSL-YFP, in which Cre-mediated recombination leads to the permanent expression of YFP (see
Adult (12 week old) R26-CreERT; R26-LSL-YFP (referred to as RY hereafter) and R26-CreERT; fbw7f/f; R26-LSL-YFP (referred to as RFY hereafter) mice were injected intraperitoneally with tamoxifen and histological analysis of the pancreas was performed 2 and 12 weeks post injection. CreERT mediated recombination was detected in all the pancreatic cell types, but with different efficiencies. While almost all acinar cells showed YFP positivity (97%), recombination occurred much less frequently in islet (12%) and ductal cells (5%) (see
Fbw7 targets many proteins involved in proliferation and differentiation such as N-terminally phosphorylated c-Jun, Notch intracellular domain 1 (NICD1), phosphorylated c-Myc and phosphorylated CyclinE for proteasomal degradation3. It has been shown that SCF(Fbw7)-mediated degradation of its substrates is tissue specific4,13-16. Pdx1-Cre; fbw7f/f pancreas showed increased p-Junser73 and NICD1 protein levels when compared to fbw7f/f as detected by western blot while—Myc and CyclinE protein levels were unaffected (see
Ectopic expression of combinations of transcription factors can induce changes of cellular fates (reviewed in 19), however examples of reprogramming in vivo by loss of only a single molecule are rare. pax5 inactivation converts differentiated B cell into other hematopoietic cell lineages20. In the present study, it has been shown that fbw7 controls the differentiation of a subset of ductal cells into β-cells and other pancreatic lineages, implying an unexpected plasticity and heterogeneity of pancreatic ductal cells, which might be therapeutically exploitable.
The conversion of pancreatic ductal cells into β-cells observed in the RFY mice in vivo could be due to up-regulation of substrates involved in beta cell neogenesis in the adult pancreas. One of such factors is NGN3, which has been shown to be required for proper differentiation of beta-cells from pancreatic progenitors6. To test whether Fbw7 had any effect on NGN3 protein stability (see
To test this possibility, a mouse line that would allow conditional overexpression NGN3-AA in the pancreas was generated (see
Accordingly, NGN3-AA protein was detected in the pancreas but not in the liver (see
To address the differentiation potential of pancreatic spheres in RFY and RY mice injected with tamoxifen, an in vitro culture system that enriches for ductal cells was optimized. Cells isolated and cultured as described in the Methods section were monitored over time. As early as day 4 post-plating, pancreatic spheres were observed in the cultures and remained constant until 15 days post-plating (see
To explore further the functionality of the newly formed β-cells upon fbw7 deletion, mRNA expression profiling of GFP+ sorted cells from tamoxifen injected CY and CFY mice was carried out and the results were compared to those from GFP+ cells from MIP-GFP mice as a positive control for β-cells (
An important hallmark of β-cell function is the ability to release insulin upon glucose stimulation. To test this in induced β-cells, GFP+ cells from CY and CFY mouse pancreas were sorted and challenged with glucose treatment in vitro (
A glucose tolerance test was performed in R26-LSL-NGN3-AA and R26-LSL-NGN3-AA; Pdx1-Cre mice. Mice were fasted for 12 h before glucose injection. Glucose levels in the blood were measured at the indicated times. The data suggest that NGN3-AA transgenic mice show improved glucose tolerance compared to control mice (
sapiens)
musculus)
sapiens)
musculus)
sapiens)
musculus)
| Number | Date | Country | Kind |
|---|---|---|---|
| 1211606.7 | Jun 2012 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2013/051649 | 6/24/2013 | WO | 00 |
