Patent Application
20190282710
The present invention relates to β-cell-mimetic cells. Methods for producing β-cell-mimetic cells as well as methods of use of β-cell-mimetic cells as a medicament and methods of use of β-cell-mimetic cells for the prevention, delay of progression or treatment of a metabolic disease in a subject are also provided.
Diabetes mellitus is a complex and progressive disease with a pathophysiology involving metabolic impairments that can lead to many clinical complications. Diabetes mellitus is currently estimated to affect at least 415 million people (1 in 11 adults) worldwide (Diabetes Atlas 7th Edition, International Diabetes Federation, 2015), a number already exceeding the value for 2025 predicted a decade ago. The most characteristic feature of diabetic patients is a chronically elevated blood-glucose level, known as hyperglycaemia, that results from either an absolute loss of pancreatic insulin-producing β-cells (type-1 diabetes, T1D) or a progressive exhaustion of active β-cells due to environmental factors such as a sedentary lifestyle, malnutrition, or obesity (type-2 diabetes, T2D). Unless sufficiently treated in time, sustained hyperglycaemia can initiate many pathologic cascades that result in more severe disorders such as cardiovascular disease, renal failure, the metabolic syndrome, neuropathic pain, hormone dysfunction and cancer. Therefore, improved glycaemic control by a therapeutic intervention that either enables tightly controlled insulin delivery or restores a patient's β-cell function will be of utmost importance in diabetes treatment.
Because T1D patients suffer from complete insulin deficiency due to a selective autoimmune destruction of β-cells treatment options focus on a disciplined or automated supply of exogenous insulin. By contrast, the number of possible drug targets for T2D therapy is higher due to the progressive and multifactorial nature of this disease type. For example, incretin hormones (e.g., GLP-1-analogues) improve the efficiency of the exhausting β-cells to secrete insulin upon glucose stimulation. In recent years, studies capitalizing on the high capacity of mammalian cells to produce insulinogenic components within a patient have gained increased attention because they promise effective drug production, delivery and dosage. For example, the regeneration of functional glucose-responsive insulin-secreting β-cells from stem cells (Pagliuca F W et al., Cell 159, 428-439 (2014)) represents a major breakthrough for treating T1D: transplantation of these ex vivo reprogrammed cells into T1 D patients would directly restore their defective glucose-stimulated insulin expression. Approaches based on the delivery of glucose-responsive insulin expression elements into extrapancreatic mammalian cell types (Han J et al., WJG 18, 6420-6426 (2012)) can protect against fundamental diabetic vulnerabilities such as autoimmune (re)-targeting in T1D (Aguayo-Mazzucato C and Bonner-Weir S, Nat Rev Endocrinol 6, 139-148 (2010)) and metabolic stress-induced β-cell apoptosis in T2D (Marzban L et al., Diabetes 55, 2192-2201 (2006)). Recently, synthetic biology-inspired rational circuit design has led to the engineering of immunoprotective implants that enable trigger-inducible insulin- (Stanley S et al., Nat Med 21, 92-98 (2015)) or GLP1-expression (Ye H et al., Science 332, 1565-1568 (2011)) with traceless and non-invasive signals. However, neither of these approaches combines direct glucose sensing, endogenous real-time control of therapeutic dosage, and straightforward engineering of non-stem-cell human cells.
The invention provides therapeutically applicable β-cell-mimetic cells and methods for producing such β-cell-mimetic cells. The β-cell-mimetic cells of the present invention comprise a carbohydrate-inducible transcriptional system that directly senses extracellular carbohydrate concentrations and is capable to coordinate the dose-dependent transcription of therapeutic proteins such as e.g. insulin and GLP-1 The system mimics core functions of pancreatic β-cells, which sense carbohydrates as glucose via a mechanism that combines glycolysis and stimulus-secretion coupling. Implanted β-cell-mimetic cells corrected insulin deficiency and self-sufficiently abolished persistent hyperglycaemia in T1D mice. Similarly, glucose-inducible GLP-1 transcription improved endogenous glucose-stimulated insulin release and glucose tolerance in T2D mice. The β-cell-mimetic cells of the present invention are useful for the treatment of metabolic diseases such as e.g. metabolic diseases selected from the group consisting of T1D, T2D, metabolic syndrome and cardiovascular disease.
Thus, in a first aspect, the invention relates to a recombinant cell comprising
a nucleic acid construct comprising a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein.
In a further aspect, the invention relates to an encapsulated cell comprising a recombinant cell.
In a further aspect, the invention relates to a recombinant cell or an encapsulated recombinant cell for use as a medicament.
In a further aspect, the invention relates to a recombinant cell or an encapsulated recombinant cell for use in a method for the prevention, delay of progression or treatment of a metabolic disease in a subject.
In a further aspect, the invention relates to a method of producing a recombinant cell expressing a therapeutic protein, said method comprising the steps of:
(a) obtaining a population of cells;
(b) transfecting said population of cells with a nucleic acid construct comprising a promoter which is responsive to a product of the carbohydrate metabolism of said cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein;
(c) incubating the population of transfected cell in the presence of carbohydrates for a sufficient time to permit the transfected cells to express a therapeutic protein.
In a further aspect, the invention relates to a method to deliver a nucleic acid construct to a cell, wherein the nucleic acid construct comprises a promoter which is responsive to carbohydrate metabolism of said cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein, comprising administering said nucleic acid construct to said cell, whereby said gene coding for a therapeutic protein is expressed in said cell in response to carbohydrate stimulation.
In a further aspect the present invention relates to a method for producing a therapeutic protein in vivo in a mammal, said method comprising:
(a) providing an in vitro population of recombinant cells into an implantable semi-permeable device;
(b) implanting the device with the cell population into a mammalian host; and
(c) maturing the cell population in said device in vivo such that at least some cells of the cell population are cells that produce a therapeutic protein in response to carbohydrate stimulation in vivo.
In a further aspect, the invention relates to an in vitro cell culture comprising the recombinant cell, wherein said recombinant cell is expressing a protein, preferably a therapeutic protein in the presence of carbohydrates.
So that the invention may be more readily understood, certain terms are first defined. Unless otherwise defined within the specification, all technical and scientific terms used herein have their art-recognized meaning Although similar or equivalent methods and materials to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. The materials, methods, and examples are illustrative only and not intended to be limiting. The terms “comprising”. “having”, and “including” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.
As used herein, the term “beta-cell” or “β-cell” refers to a cell type found in the pancreas, in particular in the mammalian, more particular in the human pancreatic islets. Beta cells are the primary producers of insulin.
As used herein, the term “recombinant cell” refers to cells, preferably mammalian cells, more preferably human cells, which have been artificially manipulated to express genes which are introduced to the mammalian cells by e.g. transfection or transformation using nucleic acid constructs, such as e.g. expression vectors in which those genes are incorporated.
As used herein, the term “transfection” or “transfected” refers to the introduction of a nucleic acid e.g. the introduction of a nucleic acid construct as described herein into a cell. In general the nucleic acid is a DNA sequence, in particular a vector or a plasmid carrying a gene of interest like a gene coding for a therapeutic protein as described herein, operably linked to a suitable promoter as described herein. Transfection methods which can be used are e.g. those using carrier molecules like cationic lipids such as DOTAP (Roche), TransFast (Promega), and Lipofectamine (Invitrogene), or polyethylenimine (PEI), calcium phosphate and DEAE dextran. Other useful transfection techniques include electroporation, bombardment with nucleic-acid-coated carrier particles (gene gun), microinjection and using of viral vectors.
As used herein, the term “transiently transfected” of “transient transfection” refer to the transient, i.e. non-permanent expression of the gene of interest due to the episomal nature of the introduced nucleic acid. Episomal nucleic acids, including DNA (plasmids or vectors), is degraded by the cells after two to seven days, and hence the expression of the gene of interest ceases then.
As used herein, the term “stably transfected” or “stable transfection” refers to the permanent expression of a gene of interest due to the integration of the transfected DNA into the genome of the cell. Most if not all cells have the potential to incorporate episomal DNA into their genome albeit at a very low rate. However, sophisticated selection strategies are employed to expand those cells that have integrated the transfected DNA. For that a nucleic acid construct to be stably integrated, a vector carrying the DNA to be transfected normally contains at least one gene that encodes for a selection marker such as e.g. a puromycin-resistance gene.
As used herein, the term “nucleic acid construct” refers to a nucleic acid, preferably to a recombinant nucleic acid construct, i.e. a genetically engineered nucleic acid construct which includes the nucleic acid of a gene and at least one promoter for directing transcription of the nucleic acid in a host cell. Nucleic acid constructs of the present invention are preferably suitable for mammalian cell expression. The nucleic acid construct (also referred to herein as an “expression vector”) may include additional sequences that render the construct e.g. the vector, suitable for replication and integration in eukaryotes (e.g. shuttle vectors). In addition, a typical nucleic acid construct such as e.g. a cloning vector may also contain transcription and translation initiation sequences, transcription and translation terminators, and a polyadenylation signal.
As used herein, the term “promoter” refers to a regulatory DNA sequence generally located upstream of a gene that mediates the initiation of transcription by directing RNA polymerase to bind to DNA and initiating RNA synthesis. The term “Pmin” as used herein refers to a minimal promoter, preferably to the promoter as shown in SEQ ID NO: 33. A minimal promoter usually does not contain an enhancer i.e. do not comprise enhancer elements and is not a constitutive promoter. Preferably a minimal promoter shows no or only minimal transcriptional activity in the absence of transcription factors.
As used herein, the term “enhancer” as used herein refers to a nucleotide sequence that acts to potentiate the transcription of genes independent of the identity of the gene, the position of the sequence in relation to the gene, or the orientation of the sequence.
As used herein, the terms “functionally linked” and “operably linked” are used interchangeably and refer to a functional relationship between two or more DNA segments, in particular gene sequences to be expressed and those sequences controlling their expression. For example, a promoter and/or enhancer sequence, including any combination of cis-acting transcriptional control elements is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Promoter regulatory sequences that are operably linked to the transcribed gene sequence are physically contiguous to the transcribed sequence.
As used herein, the term “promoter which is responsive to carbohydrate metabolism of a cell” refers to a promoter which is activated or repressed for transcription in response to the presence or absence of a product of the carbohydrate metabolism of a cell. A product of the carbohydrate metabolism of a cell can be a metabolic product of the cellular pathway of carbohydrate transformation or carbohydrate degradation e.g. a product of glycolysis such as intracellular ATP production or can be a cellular response to the metabolism of a carbohydrate in the cell e.g. production of intracellular ATP lead to the closure of KATP, channels which causes membrane depolarization as cellular response.
As used herein, the term “carbohydrate metabolism of a cell” refers to the various biochemical processes responsible for the formation, breakdown and interconversion of carbohydrates in living organisms. Oligosaccharides and/or polysaccharides are typically cleaved into smaller monosaccharides by enzymes called glycoside hydrolases. The monosaccharide units then enter the cellular pathway of carbohydrate transformation or carbohydrate degradation, i.e. the cellular pathway of glucose transformation or degradation such as glycolysis.
As used herein, the term “cellular component for sensing extracellular carbohydrates” refers to a cellular component such as e.g. a transporter of carbohydrates like e.g. a transporter of glucose or a glucose linked transporter, a receptor involved in the regulation of carbohydrate homeostasis e.g. glucose homeostasis, or a membrane protein like potassium channels, calcium channels or sodium channels which is capable of sensing extracellular carbohydrates such as glucose by activating a gene, preferably a gene coding for a therapeutic protein whose expression level correlates with the extracellular carbohydrate levels. Usually the cellular component for sensing extracellular carbohydrates of the recombinant cell of the present invention is capable of sensing extracellular carbohydrates such as glucose by activating a gene, preferably a gene coding for a therapeutic protein whose expression level correlates with the extracellular carbohydrate levels via a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for the therapeutic protein. Preferably the cellular component for sensing extracellular carbohydrates is of mammalian, more preferably human origin.
As used herein, the term “carbohydrate” or “saccharide” are interchangeably and equivalently used within this context refer to a biological molecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen-oxygen atom ratio of 2.1 (as in water); in other words, with the empirical formula Cm(H2O)n (where m could be different from n). Carbohydrates include monosaccharides, disaccharides, oligosaccharides, and polysaccharides, preferably monosaccharides and disaccharides, more preferably monosaccharides, most preferably monosaccharides selected from the group consisting of glucose, galactose, fructose and xylose and epimeric forms thereof like mannose, in particular glucose and mannose. The term “glucose” in its broadest sense relates to glucose and its epimeric forms like mannose. Preferably the term “glucose” relates to glucose (D-Glucose, (2R,3S,4R,5R)-2,3,4,5,6-Pentahydroxyhexanal).
As used herein, the term “membrane protein” refers to a protein molecule that is attached to or associated with the membrane of a cell or organelle. The membrane protein is preferably a membrane protein of a human cell or organelle.
As used herein, the term “fragment of a membrane protein” refers to a region of a membrane protein that is shorter in length as compared with the full-length membrane protein. It is however, a requirement of the present invention that any fragment of a membrane protein used as part retain the activity of the full-length membrane protein.
As used herein, the term “subunit of a membrane protein” or “monomer of a membrane protein” are interchangeably and equivalently used within this context and refers to a separate polypeptide chain that makes a membrane protein which is made up of two or more polypeptide chains joined together. In a membrane protein molecule composed of more than one subunit, each subunit can form a stable folded structure by itself. The amino acid sequences of subunits of a protein can be identical, similar, or completely different.
As used herein, the term “physiological effect of membrane depolarization” refers to a physiological effect due to depolarization of a cell's membrane such as e.g. the variation in intra-cellular cation, e.g. intra-cellular calcium, intra-cellular sodium or intra-cellular potassium concentration, in particular intra-cellular calcium concentration within a cell which has been caused by physiological activities, in particular by the carbohydrate metabolism of said cell.
As used herein, the term “calcium-responsive promoter” refers to a promoter which is activated or repressed for transcription in response to the presence or absence of calcium in the cell. As used herein, the term “expression system” refers to a set of transgenic genetic elements within a cell as well as proteins encoded by such genetic elements.
The term “G-protein coupled receptors” as used herein refers to a family of transmembrane receptors, that sense molecules outside the cell and activate responses inside the cell by coupling with specific intracellular signaling pathways via G proteins. Preferred G-protein coupled receptors are GPR1, TAS1R2, TAS1R3, GLP1R or anyone of their orthologues. Most preferred G-protein coupled receptors are GPR1 (GenBank: CAA98593.1), TAS1R2 (UniProtKB/Swiss-Prot: Q8TE23.2), TAS1R3 (UniProtKB/Swiss-Prot: Q7RTX0.2) and GLP1R (UniProtKB/Swiss-Prot: P43220.2).
The term “glucoincretin receptor” as used herein refers to glucoincretin receptors such as gastric inhibitory polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP1R), preferably to human glucoincretin receptors such as human GIPR e.g. human GIPR (UniProtKB: P48546) and/or human GLP1R e.g. human GLP1R (UniProtKB/Swiss-Prot: P43220.2).
The term “SLC2A family glucose transporters” (also known as GLUT) as used herein refers to a family of transmembrane proteins that catalyze the entry of carbohydrates into mammalian cells. Preferred SLC2A family glucose transporters are GLUT1 (SLC2A1), GLUT2 (SLC2A2), GLUT3 (SLC2A3), GLUT4 (SLC2A4), GLUT5 (SLC2A5), GLUT6 (SLC2A6), GLUT7 (SLC2A7), GLUT8 (SLC2A8), GLUT9 (SLC2A9). GLUT10 (SLC2A10). GLUT11 (SLC2A11), GLUT12 (SLC2A12) and GLUT13 (SLC2A13) or anyone of their orthologues. Most preferred SLC2A family glucose transporters are human GLUT1, human GLUT2 and human GLUT3.
The term “SLC5A family sodium-glucose linked transporters” (also known as SGLT) as used herein refers to a family of transmembrane proteins that mediate sodium-dependent co-transport of carbohydrates across the plasma membrane of mammalian cells Preferred SLC5A family sodium-glucose linked transporters are SGLT1 (SLC5A1), SGLT2 (SLC5A2) and SGLT3 (SLC5A3) or anyone of their orthologues. Most preferred SLC5A family sodium-glucose linked transporters are human SGLT1 and human SGLT3.
The term “potassium channels” as used herein refers to a family of pore-forming transmembrane proteins that facilitate the transport of potassium ions across the cell plasma membrane Preferred potassium channels are ATP-sensitive potassium channels (KATP), calcium-activated potassium channels (BKCa), inward rectifier potassium channels (Kir) and voltage-dependent potassium channels (Kv). Most preferred potassium channels are human ATP-sensitive potassium channels (KATP), human calcium-activated potassium channels (BKCa), human inward rectifier potassium channels (Kir) and human voltage-dependent potassium channels (Kv).
The term “calcium channels” as used herein refers to a family of pore-forming transmembrane proteins that facilitate the transport of calcium ions across the cell plasma membrane Preferred calcium channels are voltage-gated calcium channels (VGCC), N-methyl-D-aspartate type of Glutamate (NMDA) receptors, Ca2+ release-activated Ca2+ current (CRAC) channels and transient receptor potential channels (TRPCs) Most preferred calcium channels are human voltage-gated calcium channels (VGCC), human N-methyl-D-aspartate type of Glutamate (NMDA) receptors, human Ca2+ release-activated Ca2+ current (CRAC) channels and human transient receptor potential channels (TRPCs).
The term “sodium channels” as used herein refers to a family of pore-forming transmembrane proteins that facilitate the transport of sodium ions across the cell plasma membrane Preferred sodium channels are voltage gated-sodium channels, most preferably human voltage gated-sodium channels
As used herein, the term “voltage-gated calcium channel” (VDCC) refers to a group of voltage-gated ion channels, preferably human voltage-gated calcium channels, found in the membrane of excitable cells whose permeability to the calcium ion Ca2+ correlates with the membrane potential. Voltage-dependent calcium channels are formed as a complex of several different subunits. Subunits known are the pore-forming Cavα1, the intracellular Cavβ, the transmembrane Cavγ, and a disulfide-linked dimer Cavα2δ. The α1 subunit is the primary subunit necessary for channel functioning in the VDCC, and consists of the characteristic four homologous I-IV domains containing six transmembrane α-helices each forms the ion conducting pore while the associated subunits have several functions including modulation of gating. Voltage-gated calcium channels are functionalized by their al subunit, which sets the activation threshold of the entire channel. Non-limiting examples of al subunits are Cav1, Cav1.2, Cav1.3, Cav1.4, Cav2.1, Cav2.2, Cav2.3, Cav3.1, Cav3.2 and Cav3.3.
As used herein, the term “therapeutic protein” refers to a protein which is therapeutically applicable i.e. a therapeutic protein is any protein or polypeptide that can be expressed to provide a therapeutic effect, in particular a protein that can be expressed to provide a therapeutic effect with respect to metabolic diseases.
As used herein, the term “orthologues” with respect to a protein e.g. a receptor or channel refers to one of two or more homologous gene sequences found in different species.
As used herein, the term “insulin” refers to the protein hormone produced by beta cells in the pancreas which decreases blood glucose concentrations and is therefore involved in the regulation of blood sugar levels. One international unit of insulin (1 IU) is defined as the “biological equivalent” of 34.7 μg pure crystalline insulin, which corresponds to the amount required to reduce the concentration of blood glucose in a fasting rabbit to 45 mg/dl (2.5 mmol/L) Insulin is produced as a proinsulin precursor consisting of the B and A chains of insulin linked together via a connecting C-peptide. Insulin itself is comprised of only the B and A chains. Human insulin is encoded by the INS gene corresponding to GenBank Accession No: NM-000207.2 The term “insulin” or “insulin molecule” is a generic term that designates the 51 amino acid heterodimer comprising the A-chain peptide and the B-chain peptide, wherein the cysteine residues a positions 6 and 11 of the A chain are linked in a disulfide bond, the cysteine residues at position 7 of the A chain and position 7 of the B chain are linked in a disulfide bond, and the cysteine residues at position 20 of the A chain and 19 of the B chain are linked in a disulfide bond. The term “insulin” means the active principle of the pancreas that affects the metabolism of carbohydrates in the animal body and which is of value in the treatment of diabetes mellitus. The term includes synthetic and biotechnologically derived products that are the same as, or similar to, naturally occurring insulins in structure, use, and intended effect and are of value in the treatment of diabetes mellitus.
The term “insulin analogue” as used herein includes any heterodimer analogue or single-chain analogue that comprises one or more modification(s) of the native A-chain peptide and/or B-chain peptide. Modifications include but are not limited to substituting an amino acid for the native amino acid at a position selected from A4. A5, A8, A9, A10, A12, A13, A14, A15, A16, A17, A18, A19, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B15, B16, B17, B18, B20, B21, B22, B23, B26. B27, B28. B29, and B30; deleting any or all of positions B1-4 and B26-30; or conjugating directly or by a polymeric or non-polymeric linker one or more acyl, polyethylglycine (PEG), or saccharide moiety (moieties); or any combination thereof. Examples of insulin analogues include but are not limited to the heterodimer and single-chain analogues disclosed in published international application WO20100080606, WO2009099763, and WO2010080609, the disclosures of which are incorporated herein by reference. Examples of single-chain insulin analogues also include but are not limited to those disclosed in published International Applications WO9634882, WO95516708, WO2005054291, WO2006097521, WO2007104734, WO2007104736, WO2007104737, WO2007104738, WO2007096332, WO2009132129; U.S. Pat. Nos. 5,304,473 and 6,630,348; and Kristensen et al., Biochem. J. 305: 981-986 (1995), the disclosures of which are each incorporated herein by reference. The term “insulin analogues” further includes single-chain and heterodimer polypeptide molecules that have little or no detectable activity at the insulin receptor but which have been modified to include one or more amino acid modifications or substitutions to have an activity at the insulin receptor that has at least 1%, 10%, 50%, 75%, or 90% of the activity at the insulin receptor as compared to native insulin and which further includes at least one N-linked glycosylation site. In particular aspects, the insulin analogue is a partial agonist that has from 2′ to 100′ less activity at the insulin receptor as does native insulin. In other aspects, the insulin analogue has enhanced activity at the insulin receptor, for example, the IGF <B16B17> derivative peptides disclosed in published international application WO02010080607 (which is incorporated herein by reference) These insulin analogues, which have reduced activity at the insulin growth hormone receptor and enhanced activity at the insulin receptor, include both heterodimers and single-chain analogues.
As used herein, the term “autologous” or “endogenous” refers to any material that is present in a cell or an organism which is native to said recombinant cell or organism.
The term “stem cell” as used herein refers to undifferentiated biological cells that can differentiate into specialized cells and which is capable of proliferation to produce more stem cells.
As used herein, the term “somatic cell” refers to any cell forming the body of an organism, as opposed to germline cells or undifferentiated stem cells.
The terms “individual.” “subject” or “patient” are used herein interchangeably. In certain embodiments, the subject is a mammal. Mammals include, but are not limited to primates (including human and non-human primates). In a preferred embodiment, the subject is a human.
The term “about” as used herein refers to +/−10% of a given measurement.
In a first aspect, the present invention provides a recombinant cell comprising a nucleic acid construct comprising a promoter which is responsive to carbohydrate metabolism of said cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein. Preferably, the promoter which is responsive to carbohydrate metabolism is responsive to glucose metabolism.
In one embodiment the promoter which is responsive to carbohydrate metabolism is responsive to a physiological effect of membrane depolarization caused by the carbohydrate metabolism of said recombinant cell. Preferably the physiological effect of membrane depolarization caused by the carbohydrates metabolism of the cell is extracellular calcium influx Thus in a preferred embodiment the promoter which is responsive to carbohydrate metabolism is responsive to a physiological effect of membrane depolarization caused by the carbohydrate metabolism of said recombinant cell, wherein the physiological effect of membrane depolarization caused by the carbohydrates metabolism of said cell is extracellular calcium influx. Thus in a further preferred embodiment the promoter which is responsive to carbohydrate metabolism is responsive to extracellular calcium influx.
In one embodiment the promoter which is responsive to carbohydrate metabolism is a calcium-responsive promoter, preferably a calcium-responsive promoter comprising nucleic acid sequences bound by transcription factors of the NFAT family, the NFkB family, the AP-1 family, and/or the CREB family and/or cFOS, more preferably a calcium-responsive promoter comprising nucleic acid sequences bound by transcription factors of the NFAT family.
The NFAT family is a family of transcription factors shown to be important in immune response. One or more members of the NFAT family is expressed in most cells of the immune system. NFAT is also involved in the development of cardiac, skeletal muscle, and nervous systems. The NFAT family comprises the NFAT1, NFAT2, NFAT3, NFAT4, and NFAT5 proteins that specifically bind their cognate promoters. Preferred promoters that contain NFAT-binding sites are synthetic or natural promoters containing one or multiple 5′-GGAAA-3′ consensus sites, more preferred are mammalian cytokine promoters e.g. mammalian cytokine promoters selected from the group consisting of interleukin (IL)-2, IL-3, IL-4 promoter, peroxisome-proliferator-activated receptor-γ (PPARγ) promoter, orphan nuclear receptor 77 (NUR77) promoter, interferon γ promoter, GATA-binding protein 3 (GATA3) promoter and promoters of the T-box family of transcription factors (T-bex and eomesodermin), preferably the interleukin (IL)-2, L-4 promoter and PPARγ promoter Most preferred is the murine interleukin (IL)-4 promoter. Thus in one embodiment the promoter which is responsive to carbohydrate metabolism of the recombinant cell is a synthetic or natural promoter that contains NFAT-binding sites containing one or multiple 5′-GGAAA-3′ consensus sites, more preferably a mammalian cytokine promoter e.g. a mammalian cytokine promoter selected from the group consisting of interleukin (IL)-2, IL-3, IL-4 promoter, peroxisome-proliferator-activated receptor-γ (PPARγ) promoter, orphan nuclear receptor 77 (NUR77) promoter, interferon γ promoter, GATA-binding protein 3 (GATA3) promoter and promoters of the T-box family of transcription factors (T-bex and eomesodermin), preferably the interleukin (IL)-2, IL-4 promoter and PPARγ promoter and most preferrably the murine interleukin (IL)-4 promoter. The murine interleukin (IL)-4 promoter is described e.g. in Rooney J W et al., EMBO J 13, 625-633 (1994).
The NFkB family is as family of protein complexes that act as transcription factors controlling the transcription of DNA, cytokine production and cell survival. The NFkB family comprises NF-κB1, NF-κB2, RelA, RelB and c-Rel. Preferred members of the NFkB family are NF-κB1 and NF-κB2, more preferably human NF-κB1 and NF-κB2.
The AP-1 family is as family of transcription factors that regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress and infections AP-1 is a heterodimer composed of proteins belonging to the c-Fos, c-Jun, ATF and JDP families. Preferred members of the AP-1 family are c-Fos and c-Jun cFOS is a human proto-oncogene that belongs to the FOS family of transcription factors and encodes a 62 kDa protein, which forms a heterodimer with c-jun (part of Jun family of transcription factors), resulting in the formation of AP-1 (Activator Protein-1) complex which binds DNA at AP-1 specific sites at the promoter and enhancer regions of target genes and converts extracellular signals into changes of gene expression.
The CREB (cAMP-responsive element-binding protein) family is as family of transcription factors that binds cAMP response elements (CRE) containing the highly conserved nucleotide sequence, 5′-TGACGTCA-3′, thereby modulating target gene expression from CRE-containing promoters (PCRE). Preferred members of the CREB family are CREB1 and ATF4, more preferably human CREB1 and ATF4.
In one embodiment the promoter which is responsive to carbohydrate metabolism is a calcium-responsive promoter, wherein the calcium responsive promoter is a synthetic promoter consisting of one or multiple tandem repeats of binding sites of transcription factors selected from the group consisting of the NFAT family, the NFkB family, the AP-1 family, and/or the CREB family and/or cFOS operably linked to one or multiple promoters wherein the one or multiple promoters do not comprise enhancer elements, i.e. is not a constitutive promoter.
In one embodiment the promoter which is responsive to carbohydrate metabolism is a calcium-responsive promoter wherein the calcium responsive promoter is a synthetic promoter consisting of one or multiple tandem repeats of binding sites of NFAT operably linked to one or multiple promoters wherein die one or multiple promoters do not comprise enhancer elements, wherein the sequence of the NFAT-binding sites of the calcium responsive promoter contains one or multiple tandem repeats of the binding site of a mammalian cytokine promoter selected from the group consisting of interleukin (IL)-2, IL-3, IL-4 promoter, peroxisome-proliferator-activated receptor-γ (PPARγ) promoter, orphan nuclear receptor 77 (NUR77) promoter, interferon γ promoter, GATA-binding protein 3 (GATA3) promoter and promoters of the T-box family of transcription factors (T-bex and eomesodermin), preferably the interleukin (IL)-2, IL-4 promoter and PPARγ promoter and most preferrably the murine interleukin (IL)-4 promoter in particular one or multiple tandem repeats of the binding site of the murine interleukin (IL)-4 promoter as shown in SEQ ID NO: 34, preferably 3 to 9 tandem repeats, more preferably 3, 5, 7 or 9 tandem repeats. In one embodiment the promoter which is responsive to carbohydrate metabolism is a calcium-responsive promoter, wherein the calcium responsive promoter is a CRE-containing synthetic mammalian promoter preferably the PCRE promoter as described e.g. in Auslander D et al., Mol Cell 55, 397-408 (2014), more preferably the PCRE promoter comprising SEQ ID NO: 60.
In one embodiment the promoter which is responsive to carbohydrate metabolism comprises SEQ ID NO: 4
In one embodiment the promoter which is responsive to carbohydrate metabolism comprises SEQ ID NO. 5
In one embodiment the promoter which is responsive to carbohydrate metabolism comprises SEQ ID NO: 6
In one embodiment the promoter which is responsive to carbohydrate metabolism comprises SEQ ID NO: 39
In one embodiment the recombinant cell further comprises a nucleic acid construct coding for a cellular component for sensing extracellular carbohydrates, in particular coding for a cellular component for sensing extracellular glucose. Preferably the cellular component for sensing extracellular carbohydrates e.g. extracellular glucose is a membrane protein or a fragment thereof or a subunit of a membrane protein or a fragment thereof, more preferably a membrane protein or a fragment or subunit of a membrane protein or a fragment thereof selected from the group consisting of G-protein coupled receptors, SLC2A family glucose transporters, SLC5A family sodium-glucose linked transporters, potassium channels, calcium channels and sodium channels, most preferably a membrane protein or a fragment or subunit of a membrane protein or a fragment thereof selected from the group consisting of potassium channels, calcium channels and sodium channels, in particular calcium channels, more particular voltage-gated calcium channels.
In one embodiment the recombinant cell may comprise two or more nucleic acid constructs comprising a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein. Preferably each nucleic acid construct comprises a gene coding for a different therapeutic protein. Thus in a preferred embodiment, the recombinant cell comprises two or more nucleic acid constructs, wherein the first nucleic acid construct comprises a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a first therapeutic protein and wherein the second or a further nucleic acid construct comprises a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a second or a further therapeutic protein, wherein the first therapeutic protein is different from the second or further therapeutic protein.
In one embodiment the recombinant cell comprises a nucleic acid construct comprising a first promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the first promoter is operably linked to a gene coding for a first therapeutic protein, and further comprises a nucleic acid construct comprising a second promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a second therapeutic protein, wherein the first therapeutic protein is different from the second therapeutic protein, and wherein the first promoter is different from or identical to the second promoter, preferably the first promoter is different from the second promoter.
In one embodiment the recombinant cell comprises a nucleic acid construct comprising a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein and further comprises a nucleic acid construct coding for a cellular component for sensing extracellular carbohydrates.
In one embodiment the recombinant cell comprises a nucleic acid construct comprising a first promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the first promoter is operably linked to a gene coding for a first therapeutic protein, and further comprises a nucleic acid construct comprising a second promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a second therapeutic protein, wherein the first therapeutic protein is different from the second therapeutic protein, and wherein the first promoter is different from or identical to the second promoter, preferably the first promoter is different from the second promoter, wherein the recombinant cell further comprises a nucleic acid construct coding for a cellular component for sensing extracellular carbohydrates.
In one embodiment the recombinant cell further comprises a nucleic acid construct coding for a glucoincretin receptor. The glucoincretin receptor is preferably a gastric inhibitory polypeptide receptor (GIPR) or a glucagon-like peptide-1 receptor (GLP1R), more preferably a human GIPR such as GIPR (UniProtKB: P48546) and/or a human GLP1R such as GLP1R (UniProtKB/Swiss-Prot: P43220.2), most preferably a human GLP1R, in particular GLP1R (UniProtKB/Swiss-Prot: P43220.2). In a preferred embodiment the recombinant cell further comprises a nucleic acid construct coding for a constitutively expressed glucoincretin receptor.
In one embodiment the recombinant cell comprises a nucleic acid construct comprising a first promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the first promoter is operably linked to a gene coding for a first therapeutic protein, and further comprises a nucleic acid construct comprising a second promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a second therapeutic protein, wherein the first therapeutic protein is different from the second therapeutic protein, and wherein the first promoter is different from or identical to the second promoter, preferably the first promoter is different from the second promoter, wherein the recombinant cell further comprises a nucleic acid construct coding for a glucoincretin receptor.
In one embodiment the recombinant cell comprises a nucleic acid construct comprising a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein and further comprises a nucleic acid construct coding for a cellular component for sensing extracellular carbohydrates and a nucleic acid construct coding for a glucoincretin receptor.
In one embodiment the recombinant cell comprises a nucleic acid construct comprising a first promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the first promoter is operably linked to a gene coding for a first therapeutic protein, and further comprises a nucleic acid construct comprising a second promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a second therapeutic protein, wherein the first therapeutic protein is different from the second therapeutic protein, and wherein the first promoter is different from or identical to the second promoter, preferably the first promoter is different from the second promoter, wherein the recombinant cell further comprises a nucleic acid construct coding for a cellular component for sensing extracellular carbohydrates and a nucleic acid construct coding for a glucoincretin receptor.
In one embodiment the recombinant cell further comprises a nucleic acid construct coding for a cellular component for sensing extracellular carbohydrates e.g. extracellular glucose, wherein the cellular component for sensing extracellular carbohydrates e.g. extracellular glucose is a voltage-gated calcium channel, usually a voltage-gated calcium channel selected from the group consisting of Cav1.1, Cav1.2, Cav1.3, Cav1.4, Cav2.1, Cav2.2, Cav2.3, Cav3.1, Cav3.2 and Cav3.3, more preferably Cav1.2, Cav1.3, Cav2.2, and most preferably Cav1.3. In a preferred embodiment the cellular component for sensing extracellular carbohydrates e.g. extracellular glucose is a combination of subunits of a voltage-gated calcium channel, more preferably a combination of a β subunit selected from the group consisting of Cavβ1 (CACNB1), Cavβ2 (CACNB2), Cavβ3 (CACNB3) and Cavβ4 (CACNB4), a α2δ subunit selected from the group consisting of Cavα2δ1 (CACNA2D1), Cavα2δ2 (CACNA2D2), Cavα2δ3 (CACNA2D3) and Cavα2δ4 (CACNA2D4) and a α1 subunit selected from the group consisting of Cav1.1 (CACNA1S), Cav1.2 (CACNA1C), Cav1.3 (CACNA1D), Cav1.4 (CACNA1F), Cav2.1 (CACNA1A), Cav2.2 (CACNA1B), Cav2.3 (CACNA1E), Cav3.1 (CACNA1G), Cav3.2 (CACNA1H) and Cav3.3 (CACNA1I), even more preferably a α1 subunit of Cav1.2 (CACNA1C), Cav1.3 (CACNA1D), Cav2.2 (CACNA1B) combined with Cavβ3 (CACNB3) and Cavα2δ1 (CACNA21), and most preferably a α1 subunit of Cav1.3 (CACNA1D) combined with Cavβ3 (CACNB3) and Cavα2δ1 (CACNA2D1).
In one embodiment the recombinant cell is a cell which express an autologous cellular component (i.e. a cell which autologously express) a cellular component for sensing extracellular carbohydrates, wherein the autologous cellular component for sensing extracellular carbohydrates is a membrane protein selected from the group consisting of G-protein coupled receptors, the SLC2A family glucose transporters, the SLC5A family sodium-glucose linked transporters, potassium channels, calcium channels and sodium channels, in particular calcium channels, more particular voltage-gated calcium channels in a preferred embodiment, the recombinant cell autologously express a calcium channel, preferably a voltage-gated calcium channel, in particular a voltage-gated calcium channel selected from the group consisting of Cav1.1, Cav1.2, Cav1.3, Cav1.4, Cav2.1, Cav2.2, Cav2.3, Cav3.1, Cav3.2 and Cav3.3, more preferably Cav1.2, Cav1.3, Cav2.2, and most preferably Cav1.3. In a preferred embodiment the cellular component for sensing extracellular carbohydrates e.g. extracellular glucose is a combination of subunits of a voltage-gated calcium channel, more preferably a combination of a β subunit selected from the group consisting of Cavβ1 (CACNB1), Cavβ2 (CACNB2), Cavβ3 (CACNB3) and Cavβ4 (CACNB4), a α2δ subunit selected from the group consisting of Cavα2δ1 (CACNA2D1), Cavα2δ2 (CACNA2D2), Cavα2δ3 (CACNA2D3) and Cavα2δ4 (CACNA2D4) and a α1 subunit selected from the group consisting of Cav1.1 (CACNA1S), Cav1.2 (CACNA1C), Cav1.3 (CACNA1D), Cav1.4 (CACNA1F), Cav2.1 (CACNA1A), Cav2.2 (CACNA1B), Cav2.3 (CACNA1E), Cav3.1 (CACNA1G), Cav3.2 (CACNA1H) and Cav3.3 (CACNA1I), even more preferably a α1 subunit of Cav1.2 (CACNA1C). Cav1.3 (CACNA1D), Cav2.2 (CACNA1B) combined with Cavβ3 (CACNB3) and Cavα2δ1 (CACNA2D1), and most preferably a α1 subunit of Cav1.3 (CACNA1D) combined with Cavβ3 (CACNB3) and Cavα2δ1 (CACNA2D1).
Usually the cellular component for sensing extracellular carbohydrates of the recombinant cell of the present invention activates the promoter which is responsive to carbohydrate metabolism comprised by said recombinant cell e.g. the cellular component for sensing extracellular carbohydrates of the recombinant cell of the present invention activates the promoter which is responsive to carbohydrate metabolism comprised by said recombinant cell, wherein the gene coding for a therapeutic protein to which the promoter is operably linked is expressed, wherein the expression level of said gene correlates with the levels of extracellular carbohydrates. In a particular embodiment the cellular component for sensing extracellular carbohydrates of the recombinant cell of the present invention activates the promoter via calcium influx into the recombinant cell.
In one embodiment the recombinant cell is a non-pancreatic cell, preferably a non-pancreatic mammalian cell, more preferably a non-pancreatic human cell.
In one embodiment the recombinant cell is a mammalian cell, preferably a human cell. Recombinant mammalian cells are preferably mammalian cells selected from the group consisting of kidney cells, liver cells, stem cells, blood cells, brain cells, nerve cells, intestinal cells, fibroblasts, and adipose-derived cells, more preferably stem cells, kidney cells or liver cells, most preferably stem cells or kidney cells, in particular kidney cells. Recombinant human cells are preferably human cells selected from the group consisting of HEK-293, HeLa, mesenchymal stem cells (MSC), induced pluripotent stem cells (iPSC), pheochromocvtoma of the rat adrenal medulla (PC12), the mouse neuroblastoma cell line (N2A), liver hepatocellular carcinoma (HepG2), enteroendocrine L-cells, human epithelial colorectal adenocarcinoma (Caco2), and clinical-grade human neural stein cell (CTX), preferably HEK-293, HeLa and MSC cells, most preferably HEK-293 cells.
In one embodiment the recombinant cell is a transiently transfected recombinant cell. In one embodiment the recombinant cell is a recombinant cell transiently transfected with a nucleic acid construct comprising a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein, preferably a recombinant cell transiently co-transfected with a nucleic acid construct comprising a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein and a nucleic acid construct coding for a cellular component for sensing extracellular carbohydrates and/or a nucleic acid construct coding for a glucoincretin receptor.
In one embodiment the recombinant cell is a stably transfected recombinant cell. In one embodiment the recombinant cell is a recombinant cell stably transfected with a nucleic acid construct comprising a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein, preferably a recombinant cell stably co-transfected with a nucleic acid construct comprising a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein and a nucleic acid construct coding for a cellular component for sensing extracellular carbohydrates and/or a nucleic acid construct coding for a glucoincretin receptor.
In a specific embodiment, the recombinant cell of the present invention comprises a nucleic acid construct comprising a promoter which is responsive to glucose metabolism of said cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein and optionally the recombinant cell further comprises a nucleic acid construct coding for a cellular component for sensing extracellular glucose and/or a nucleic acid construct coding for a glucoincretin receptor.
In another specific embodiment, the recombinant cell of the present invention comprises a nucleic acid construct comprising a calcium-responsive promoter, wherein the promoter is operably linked to a gene coding for a therapeutic protein and optionally the recombinant cell further comprises a nucleic acid construct coding for a membrane protein or a fragment thereof or a subunit of a membrane protein or a fragment thereof, wherein the membrane protein is selected from the group consisting of G-protein coupled receptors, SLC2A family glucose transporters, SLC5A family sodium-glucose linked transporters, potassium channels, calcium channels and sodium channels, in particular calcium channels, and/or a nucleic acid construct coding for a glucoincretin receptor.
In another specific embodiment, the recombinant cell of the present invention comprises a nucleic acid construct comprising a calcium-responsive promoter wherein the promoter is operably linked to a gene coding for a therapeutic protein and optionally the recombinant cell further comprises a nucleic acid construct coding for a voltage-gated calcium channel and/or a nucleic acid construct coding for a glucoincretin receptor.
A particular, exemplars embodiment of the expression system of the invention is shown in
Another particular, exemplary embodiment of the expression system of the invention is shown in
In one embodiment the therapeutic protein is an insulinogenic agent selected from the group consisting of GLP1R-agonists, insulin, insulin analogues, growth hormones, insulin-like growth factors; an anorexic hormone; or a protein that activates brown fat metabolism, preferably selected from the group consisting of GLP1R-agonists, insulin and insulin analogues.
In one preferred embodiment, the recombinant cell comprises two nucleic acid constructs, wherein the first nucleic acid construct comprises a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a first therapeutic protein and wherein the second nucleic acid construct comprises a promoter which is responsive to carbohydrate metabolism of said recombinant cell, wherein the promoter is operably linked to a gene coding for a second therapeutic protein, wherein the first therapeutic protein is different from the second therapeutic protein. In a particular embodiment, the first therapeutic protein is a GLP1R-agonist, preferably shGLP1 as shown in SEQ ID NO: 35 or exedin-4 and the second therapeutic protein is an insulin analogue or insulin, preferably human insulin.
A GLP1R-agonist is any molecule that activates the GLP-1 receptor (GLP1R). GLP1R-agonists are usually selected from the group consisting of GLP-1, shGLP1, preferably shGLP1 as shown in SEQ ID NO 35, exedin-4, exenatide, liraglutide, lixisenatide, albiglutide and dulaglutide. Preferred GLP1R-agonists are selected from the group consisting of shGLP1, preferably shGLP1 as shown in SEQ ID NO 35 and exedin-4. Most preferred is shGLP1 or shGLP1 as shown in SEQ ID NO: 35.
Insulin analogues are usually selected from the group consisting of compounds derived from insulin that has been altered in its structure for the primary purpose of enhanced pharmaceutics or pharmacology. Preferred insulin analogues are selected from the group consisting of human, rodent, porcine, or bovine insulin, as well as Lispro, Aspar, Glulisine, Glargine and Detemir.
Growth hormones are usually selected from the group of hormones which stimulates growth in animal or plant cells, especially (in animals) that secreted by the pituitary gland. Preferred growth hormones are selected from the group consisting of ephinephrine, norepheniphrine and glucocorticoids.
Insulin-like growth factors are usually selected from the group consisting of hormones that are similar in molecular structure to insulin. Preferred insulin-like growth factors are selected from the group consisting of IGF1, IGF2 and IGFBP-6.
Anorexic hormones are usually selected from the group consisting of adiponectin, amylin, calcitonin, cholecystokinin (CCK), gastrin, gastric inhibitory polypeptide (GIP), ghrelin, leptin, motilin, pramlintide, secretin, somatostatin and peptide YY. Preferred anorexic hormones are selected from the group consisting of amylin, adiponectin, amylin- and adiponectin-analogues. Amylin- and adiponectin-analogues have usually an amino acid sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95%, in particular at least 97%, more particular at least 99% with the naturally occurring amylin and adiponectin, preferably with the naturally occurring human amylin and human adiponectin.
A protein that activates brown fat metabolism is usually selected from the group consisting of β2 AR activators. BMP7 irisin107, fibroblast growth factor 21 and natriuretic peptides. Preferred proteins that activates brown fat metabolism is selected from the group consisting of β2 AR activators and natriuretic peptides.
In a preferred embodiment the therapeutic protein is human insulin, human GLP-1 or a modified or truncated GLP-1, preferably the modified GLP1R-agonist shGLP1 as shown in SEQ ID NO 35.
In one embodiment the therapeutic protein is an agent against a metabolic disease, wherein the metabolic disease is selected from the group consisting of T1D (type-1 diabetes), T2D (type-2 diabetes), diabetic ketoacidosis, obesity, cardiovascular disease, the metabolic syndrome and cancer. Preferably the metabolic disease is selected from the group consisting of T1D (type-1 diabetes), T2D (type-2 diabetes), diabetic ketoacidosis, and the metabolic syndrome.
Type 1 diabetes (also known as diabetes mellitus type 1) is a form of diabetes mellitus that results from the autoimmune destruction of the insulin-producing beta cells in the pancreas. The subsequent lack of insulin leads to increased glucose in blood and urine.
Type 2 diabetes (also known as diabetes mellitus type 2) is a long term metabolic disorder that is characterized by high blood sugar, insulin resistance, and relative lack of insulin.
Cardiovascular diseases are usually selected from the group consisting of consisting of: myocardial interstitial disease, cardiac fibrosis, heart failure such as heart failure with diastolic heart failure (DHF), heart failure with preserved ejection fraction (HFpEF), congestive heart failure (CHF), asymptomatic left ventricular diastolic dysfunction (ALVDD), coronary atherosclerosis, cancer and diabetes, inflammatory bowel disease, chronic prostatitis, infections, pulmonary inflammation, osteomyelitis, renal disease, gout, arthritis and shock.
Metabolic syndrome (also referred to as syndrome X) is a cluster of risk factors that is responsible for increased cardiovascular morbidity and mortality. The National Cholesterol Education Program-Adult Treatment panel (NECP-ATP III) identified metabolic syndrome as an independent risk factor for cardiovascular disease. (National Institutes of Health: Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Executive publication no. 01-3670). As used herein, metabolic syndrome is defined according to the World Health Organization criteria (1999) which require presence of diabetes mellitus, impaired glucose tolerance, impaired fasting glucose or insulin resistance, AND two of the following: blood pressure: ≥140/90 mmHg, dyslipidaemia: triglycerides (TG): ≥1.695 mmol/L and high-density lipoprotein cholesterol (HDL-C)≤0.9 mmol/L (male), ≤1.0 mmol/L (female); central obesity: waist:hip ratio >0.90 (male); >0.85 (female), and/or body mass index >30 kg/m2; microalbuminuria:urinary albumin excretion ratio ≥20 mg/min or albumin:creatinine ratio ≥30 mg/g.
In one specific embodiment the therapeutic protein is an agent against T1D and/or T2D.
In a further aspect the present invention provides an encapsulated cell, comprising the recombinant cell as described above and a semi-permeable membrane.
Encapsulated cell biodelivery is based on the concept of isolating cells from the recipient host's immune system by surrounding the cells with a semipermeable biocompatible membrane before implantation within the host. Cells are immunoisolated from the host by enclosing them within implantable polymeric capsules formed by a semi-permeable membrane. This approach prevents the cell-to-cell contact between host and implanted tissues, eliminating antigen recognition through direct presentation.
The encapsulated cell of the present invention has a semi-permeable membrane which is tailored to control diffusion of molecules, such as growth factor hormones, neurotransmitters, peptides, antibodies and complements, based on their molecular weight. Using encapsulation techniques, cells can be transplanted into a host without immune rejection, either with or without use of immunosuppressive drugs. Useful biocompatible polymer capsules usually contain a core that contains cells, either suspended in a liquid medium or immobilised within an immobilising matrix, and a surrounding or peripheral region of permselective matrix or membrane (“jacket”) that does not contain isolated cells, that is biocompatible, and that is sufficient to protect cells in the core from detrimental immunological attack. Encapsulation hinders elements of the immune system from entering the capsule, thereby protecting the encapsulated cells from immune destruction. The semipermeable nature of the membrane also permits the biologically active molecule of interest to easily diffuse from the capsule into the surrounding host tissue and allows nutrients to diffuse easily into the capsule and support the encapsulated cells. The capsule can be made from a biocompatible material. A “biocompatible material” is a material that, after implantation in a host, does not elicit a detrimental host response sufficient to result in the rejection of the capsule or to render it inoperable, for example through degradation. The biocompatible material is relatively impermeable to large molecules, such as components of the host's immune system, but is permeable to small molecules, such as insulin, growth factors, and nutrients, while allowing metabolic waste to be removed. A variety of biocompatible materials are suitable for delivery of growth factors by the composition of the invention Numerous biocompatible materials are known, having various outer surface morphologies and other mechanical and structural characteristics as described e.g. by WO 92/19195 or WO 95/05452. Components of the biocompatible material may include a surrounding semipermeable membrane and the internal cell-supporting scaffolding. Preferably, the recombinant cells are seeded onto the scaffolding, which is encapsulated by the permselective membrane. The filamentous cell-supporting scaffold may be made from any biocompatible material selected from the group consisting of acrylic, polyester, polyethylene, polypropylene polyacetonitrile, polyethylene teraphthalate, nylon, polyamides, polyurethanes, polybutester, silk, cotton, chitin, carbon, or biocompatible metals. Also, bonded fibre structures can be used for cell implantation (U.S. Pat. No. 5,512,600, incorporated by reference). Biodegradable polymers include those comprised of poly(lactic acid) PLA, poly(lactic-coglycolic acid) PLGA, and poly(glycolic acid) PGA and their equivalents. Foam scaffolds have been used to provide surfaces onto which transplanted cells may adhere (WO 98/05304, incorporated by reference). Woven mesh tubes have been used as vascular grafts (WO 99/52573, incorporated by reference). Additionally, the core can be composed of an immobilizing matrix formed from a hydrogel, which stabilizes the position of the cells. A hydrogel is a 3-dimensional network of cross-linked hydrophilic polymers in the form of a gel, substantially composed of water.
Various polymers and polymer blends can be used to manufacture the semipermeable membrane, including alginate, alginate-poly-(L-lysine)-alginate, polycarbonate, polyethylene, polyethylene-terephthalate, collagen, gelatin, agarose, cellulose acetate and cellulose sulfate, polyacrylates (including acrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers, polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulose nitrates, polysulfones (including polyether sulfones), polyphosphazenes, polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well as derivatives, copolymers and mixtures thereof. Such membranes, and methods of making them are disclosed by e.g. U.S. Pat. Nos. 5,284,761 and 5,158,881.
The capsule can be any configuration appropriate for maintaining biological activity and providing access for delivery of the product or function, including for example, cylindrical, rectangular, disk-shaped, patch-shaped, ovoid, stellate, or spherical. Moreover, the capsule can be coiled or wrapped into a mesh-like or nested structure. If the capsule is to be retrieved after it is implanted, configurations, which tend to lead to migration of the capsules from the site of implantation, such as spherical capsules small enough to travel in the recipient host's blood vessels, are not preferred. Certain shapes, such as rectangles, patches, disks, cylinders, and flat sheets offer greater structural integrity and are preferable where retrieval is desired.
The encapsulated cell devices are implanted according to known techniques. Many implantation sites are contemplated for the devices and methods of this invention. These implantation sites include, but are not limited to the intraperitoneal cavity, into the kidney capsules, subcutaneous tissues, the portal vein, the liver and the cerebral cortex.
In one embodiment the encapsulated cell comprises a biocompatible material selected from the group consisting of alginate, alginate-poly-(L-lysine)-alginate, polycarbonate, polyethylene, polyethylene-terephthalate, collagen, gelatin, agarose, cellulose acetate and cellulose sulfate, preferably alginate, more preferably alginate-poly-(L-lysine)-alginate.
In one embodiment the semi-permeable membrane comprises a biocompatible material selected from the group consisting of alginate, alginate-poly-(L-lysine)-alginate, polycarbonate, polyethylene, polyethylene-terephthalate, collagen, gelatin, agarose, cellulose acetate and cellulose sulfate, preferably alginate, more preferably alginate-poly-(L-lysine)-alginate
In a further aspect the present invention provides thus a method for producing a therapeutic protein in vivo in a mammal, said method comprising:
(a) providing an in vitro population of the recombinant cells as described herein into an implantable semi-permeable device;
(b) implanting the device with the cell population into a mammalian host: and
(c) maturing the cell population in said device in vivo such that at least some cells of the cell population are cells that produce a therapeutic protein in response to carbohydrate stimulation in vivo.
In one embodiment the implantable semi-permeable device are capsules or beads consisting of a biocompatible material selected from the group consisting of alginate, alginate-poly-(L-lysine)-alginate, polycarbonate, polyethylene, polyethylene-terephthalate, collagen, gelatin, agarose, cellulose acetate and cellulose sulfate, preferably alginate, more preferably alginate-poly-(L-lysine)-alginate.
In a further aspect the present invention provides an in vitro cell culture comprising the recombinant cell as described herein, wherein said recombinant cell is expressing a therapeutic protein in the presence of carbohydrates, preferably in the presence of glucose. Cells can be grown and maintained in vitro as generally known. The nutrient medium and the cells generally are contained in a suitable vessel to which an adequate supply of oxygen and carbon dioxide is furnished in order to support cell growth and maintenance. Cell cultures may be batch systems in which nutrients are not replenished during cultivation but oxygen is added as required, fed-batch systems in which both nutrient and oxygen concentrations are monitored and replenished as necessary, and perfusion systems in which nutrient and waste product concentrations are monitored and controlled. Cells may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), Minimal Essential Medium (MEM; Sigma-Aldrich Chemie GmbH), RPMI-1640 (Sigma-Aldrich Chemie GmbH, Basel, Switzerland), and Dulbecco's Modified Eagle's Medium ((DMEM; Sigma-Aldrich Chemie GmbH) are suitable for culturing the host cells.
In a further aspect the present invention provides a method, preferably an in vitro method, of producing a recombinant cell expressing a therapeutic protein, said method comprising the steps of:
(a) obtaining a population of cells;
(b) transfecting said population of cells with a nucleic acid construct comprising a promoter which is responsive to a product of the carbohydrate metabolism of said cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein;
(c) incubating the population of transfected cell in the presence of carbohydrates for a sufficient time to permit the transfected cells to express a therapeutic protein.
In one embodiment the population of cells is further transfected in step (b) with a nucleic acid construct encoding a cellular component for sensing extracellular carbohydrates. The cells, the promoter which is responsive to a product of the carbohydrates metabolism, and the cellular component for sensing extracellular carbohydrates used in the method are as described above. The carbohydrate used is preferably glucose. Incubation time in the presence of carbohydrates is usually between 0.5 to 96 hours, preferably between 1 to 12 hours.
In a further aspect the present invention provides the recombinant cell or the encapsulated cell as described herein for use as a medicament.
In a further aspect the present invention provides the recombinant cell or the encapsulated cell as described herein for use in a method for the prevention, delay of progression or treatment of a metabolic disease in a subject.
Also provided is the use of the recombinant cell or the encapsulated cell as described herein for the manufacture of a medicament for the prevention, delay of progression or treatment of a metabolic disease in a subject.
Also provided is the use of the recombinant cell or the encapsulated cell as described herein for the prevention, delay of progression or treatment of a metabolic disease in a subject.
Also provided is a method for the prevention, delay of progression or treatment of a metabolic disease in a subject, comprising administering to said subject the recombinant cell or the encapsulated cell as described herein.
In one embodiment the metabolic disease is selected from the group consisting of T1D (type-1 diabetes), T2D (type-2 diabetes), diabetic ketoacidosis, obesity, cardiovascular disease, the metabolic syndrome and cancer. Preferably the metabolic disease is selected from the group consisting of T1D (type-1 diabetes), T2D (type-2 diabetes), diabetic ketoacidosis, and the metabolic syndrome.
In a further aspect the present invention provides a method to deliver a nucleic acid construct to a cell, wherein the nucleic acid construct comprises a promoter which is responsive to carbohydrate metabolism of said cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein, comprising administering said nucleic acid construct to said cell, whereby said gene coding for a therapeutic protein is expressed in said cell in response to carbohydrate stimulation. In one embodiment the cell is further transfected with a nucleic acid construct encoding a cellular component for sensing extracellular carbohydrates. The promoter which is responsive to carbohydrate metabolism, and the cellular component for sensing extracellular carbohydrates used in the method are as described above. The carbohydrate used is preferably glucose.
In one embodiment the nucleic acid construct is delivered to a cell in a subject e.g. by gene therapy Thus in a specific embodiment the present invention provides a method to deliver a nucleic acid construct to a cell of a subject, wherein the nucleic acid construct comprises a promoter which is responsive to carbohydrate metabolism of said cell, wherein the promoter is operably linked to a gene coding for a therapeutic protein, comprising administering said nucleic acid construct to said subject, whereby said gene coding for a therapeutic protein is expressed in said cell of said subject in response to carbohydrate stimulation.
For delivery of a nucleic acid construct to a cell in a subject it may be valuable in some instances to utilize or design vectors to deliver the nucleic acid construct to a particular cell type. Certain vectors exhibit a natural tropism for certain tissue types. Cell type specificity or cell type targeting may be achieved in vectors derived from viruses having characteristically broad infectivities by the modification of the viral envelope proteins. For example, cell targeting has been achieved with adenovirus vectors by selective modification of the viral genome knob and fiber coding sequences to achieve expression of modified knob and fiber domains having specific interaction with unique cell surface receptors. Other methods of cell specific targeting have been achieved by the conjugation of antibodies or antibody fragments to the envelope proteins. Alternatively, particularly moieties may be conjugated to the viral surface to achieve targeting. Additionally, the virally encoded nucleic acid construct may also be under control of a tissue specific promoter region allowing expression of the gene coding for a therapeutic protein preferentially in particular cell types.
It will be apparent to one skilled in the art that nucleic acid constructs according to the invention may be introduced into an animal subject in a variety of ways including enterally (orally, rectally or sublingually) or parenterally (intravenously, subcutaneously, or by inhalation). The nucleic acid constructs may be provided to the mammal by e.g. implanted catheters. The nucleic acid constructs can be instilled into a body cavity to facilitate transduction of the surrounding tissues. Examples of such body cavities into which the solutions may be provided for the delivery of nucleic acids include the peritoneal cavity, pleural cavity, and the abdominal cavity. Additionally the nucleic acid constructs may be provided in other fluid containing spaces.
The nucleic acid constructs to be delivered to a cell of a subject may further comprise additional carriers, excipients or diluants. The compositions comprising the nucleic acid constructs may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The concentration of the nucleic acid constructs in the compositions can vary widely, i.e., from less than about 0.1%0, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
General Experimental Procedures
Vector Design.
Comprehensive design and construction details for all expression vectors are provided in Table 1. Some expression vectors were constructed by Gibson assembly using the GeneArt® Seamless Assembly Cloning Kit (Obio Technology, Shanghai, China; cat. no. BACR(C)20144001). Plasmids encoding KAT-subunits (pCMV Human SUR1 and pCMV6c hKir6.2(BIR)) were kindly provided by Susumu Seino (Kobe University, Kobe, Japan). Plasmids encoding Cav2.2-, Cav1.2- and Cav1.3-subunits (CaV1.3e[8a,11,31b,Δ32,42a], CaV1.2, Cav2.2e[Δa10, Δ18a, Δ24a, 31a, 37a, 46], Cavb3 and CaVα2δ1) were kindly provided by Diane Lipscombe (Brown University, RI, USA).
[ctagctacattggaaaattttatacacgtt]3AGACTCTAGAGGGTATAT
Oligonucleotides: restriction endonuclease-specific sites are underlined in oligonucleotide sequences. Annealing base pairs contained in oligonucleotide sequences are shown in capital letters.
attR1/2, Gateway®-compatible recombination sites; BFP, blue fluorescent protein; BlastR, gene conferring blasticidin resistance; Cav1.2, member 2 of the Cav1 family of L-type voltage-gated Ca2+ channels; Cav1.3, member 3 of the Cav1 family of L-type voltage-gated Ca2+ channels; Cav2.2, N-type voltage-gated Ca2+ channel; c-fos, human proto-oncogene from the Fos family of transcription factors; Cacna1b, α1-subunit of rat Cav2.2 (NCBI Gene ID: 257648); Cacna1c, α1-subunit of mouse Cav1.2 (NCBI Gene ID: 12288); Cacna1d, α1-subunit of rat Cav1.3 (NCBI Gene ID: 29716); Cacnb3, β3-subunit of rat Cav1.3 (NCBI Gene ID: 25297); Cacna2d1, α2δ-subunit of rat Cav1.3 (NCBI Gene ID: 25399); ccdB, DNA gyrase toxin for positive selection; Cmr, chloramphenicol-resistance gene for negative selection; CRE, cAMP-response element; dTomato, destabilized red fluorescent protein variant; EGFP, enhanced green fluorescent protein (Genbank: U55762); GLP-1, glucagon-like peptide 1; GLP1R, human GLP-1 receptor; GLuc, Gaussia princeps luciferase; hGCK, human pancreas glucokinase (also known as hexokinase 4; NCBI Gene ID: 2645); hGlut2, human facilitated glucose transporter member 2 (also known as SLC2A2; NCBI Gene ID: 6514); hGNAT3, human guanine nucleotide binding protein alpha transducing 3 (NCBI Gene ID: 346562); hKir6.2, human inwardly rectifying KATP-channel subunit (also known as KCNJ11; NCBI Gene ID: 3767); hSUR1, regulatory sulphonylurea receptor subunit of human KATP-channels (also known as ABCC8; NCBI Gene ID: 6833); hT1R2, member 2 of the human taste receptor type 1 family (NCBI Gene ID: 80834); hT1R3, member 3 of the human taste receptor type 1 family (NCBI Gene ID: 83756); IL2/4, interleukin 2/4; ITR, inverted terminal repeats of SB100X; KATP, adenosine triphosphate-sensitive potassium channel; Luc, Firefly Luciferase; MCS, multiple cloning site; mINS, modified rat insulin variant as shown in SEQ ID NO: 36 for optimal expression in HEK-293 cells; NFAT, nuclear factor of activated T-cells; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; P2A, picornavirus-derived ribosome skipping sequence optimized for bicistronic expression in mammalian cells; pA, polyadenylation signal; PcFOS, synthetic mammalian promoter containing a tetrameric c-fos response element ((c-fos)4-Pmin; Sheng M et al., Mol Cell Biol 8, 2787-96 (1988)); PCR, polymerase chain reaction; PCRE, CRE-containing synthetic mammalian promoter; PCREm, modified PCRE variant; PhEF1α, human elongation factor 1α promoter; PEST, peptide sequence rich in proline, glutamic acid, serine and threonine; PhCMV, human cytomegalovirus immediate early promoter; PhCMV*-1, tetracycline-responsive promoter (tetO7-PhCMVmin); PhCMVmin, minimal version of PhCMV; Pmin, minimal eukaryotic TATA-box promoter (5′-TAGAGGGTATATAATGGAAGCTCGACTTCCAG-3′) as shown in SEQ ID NO: 33; PNFAT-IL2, synthetic mammalian promoter containing three tandem repeats of a murine IL2 NFAT-binding site ((NFATIL2)3-Pmin; Rooney J W et al., EMBO J 13, 625-633 (1994)); PNFAT-IL4, synthetic mammalian promoter containing three tandem repeats of a murine IL4 NFAT-binding site ((NFATIL4)3-Pmin; Rooney J W et al., EMBO J 13, 625-633 (1994)); PNFAT-IL4×5, synthetic mammalian promoter containing five tandem repeats of a human IL4 NFAT-binding site ((NFATIL4)5-Pmin); PNFAT-IL4×7, synthetic mammalian promoter containing seven tandem repeats of a human IL4 NFAT-binding site ((NFATIL4)7-Pmin); PNFAT-IL4×9, synthetic mammalian promoter containing nine tandem repeats of a human IL4 NFATbinding site ((NFATIL4)9-Pmin); PNFkB, synthetic mammalian promoter containing a NF-κB-response element (Genbank: EU581860.1); PRPBSA, constitutive synthetic mammalian promoter; PSV40, simian virus 40 promoter; PuroR, gene conferring puromycin resistance; rtTA, reverse TetR-dependent mammalian transactivator; SB100X, optimized Sleeping Beauty transposase; SEAP, human placental secreted alkaline phosphatase; shGLP1, short human glucagon-like peptide 1; tetO, TetR-specific operator; TetR, Escherichia coli Tn10-derived tetracycline-dependent repressor of the tetracycline resistance gene; TurboGFP:dest1, PEST-tagged TurboGFP variant (Evrogen); ZeoR, gene conferring zeocin resistance. Oligonucleotides: Restriction endonuclease-specific sites are underlined and annealing base pairs are indicated in capital letters.
Cell Culture and Transfection.
Human embryonic kidney cells (HEK-293T, ATCC: CRL-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, Buchs, Switzerland; cat. no. F7524, lot no. 022M3395) and 1% (v/v) penicillin/streptomycin solution (PenStrep; Biowest, Nuaillé, France; cat. no. L0022-100) at 37° C. in a humidified atmosphere containing 5% CO2. The human 1.1E7 β-cell line (Sigma-Aldrich, cat. no. EC10070101) was cultured in RPMI 1640 medium (ThermoFisher Scientific, cat. no. 11875085) supplemented with 10% FBS and 1% PenStrep. Human islets (HIR; Prodo Laboratories, Irvine, Calif.; lot. no. HP-16161-01) were transferred from Prodo Transport medium (PIM(T)®; Prodo Laboratories; cat. no. IMT001GMP) into CMRL medium (ThermoFisher Scientific; cat. no. 11530037) supplemented with 10% FBS, 1% PenStrep, 1% ITS, 5 mM D-Glucose, 2 mM GlutaMAX, 1 mM pyruvate, 10 mM nicotinamide and 2.5 mM HEPES, and cultivated for seven days prior to encapsulation while changing fresh CMRL medium every third day. For passaging, cells of pre-confluent HEK-293 and 1.1E7 cultures were detached by incubation in 0.05% Trypsin-EDTA (Life Technologies, CA, USA; cat. no. 25300-054) for 3 min at 37° C., collected in 10 ml cell culture medium, centrifuged for 3 min at 290 g and resuspended in fresh culture medium at standard cell densities (1.5×105 cells/mL), before seeding into new tissue culture plates. Cell number and viability were quantified using an electric field multichannel cell counting device (Casy Cell Counter and Analyzer Model TT, Roche Diagnostics GmbH). For transfection, a solution containing 2-3 μg plasmid DNA and 6-9 μg polyethyleneimine (PEI; Polysciences, Eppelheim, Germany; cat. no. 24765-2) was incubated in 300 μl serum- and antibiotics-free DMEM for 30 min at 22° C. and subsequently added dropwise to 3×105 cells seeded per well of a 6-well plate. 12 h after addition of PEI, transfected HEK-293 cells were detached by incubation in Trypsin-EDTA, centrifuged (3 min at 290 g) and resuspended in low/no-glucose medium (glucose-free DMEM [Life Technologies, CA, USA; cat. no. 11966-025] supplemented with 10% FBS, 1% PenStrep, 0-2 mM D-glucose and 0.7 mM CaCl2) and reseeded at a cell density of 2×105/mL. Unless stated otherwise, D-glucose or other control compounds were added to transfected cells after cultivation under low/no-glucose conditions for another 12 h.
Quantification of Target Gene Expression.
Expression levels of human placental secreted alkaline phosphatase (SEAP) in culture supernatants were quantified according to a p-nitrophenylphosphate-based light absorbance time course (Wang H et al., Nucleic Acids Res 43(14):e91 (2015)). SEAP levels in mouse serum were profiled using a chemiluminescence-based assay (Roche Diagnostics GmbH, Mannheim, Germany; cat. no. 11 779 842 001). Human insulin levels secreted by 1.1E7 cells and human islets were quantified using an Ultrasensitive C-peptide ELISA kit (Mercordia, Uppsala, Sweden; cat. no. 10-1141-01). Murine insulin levels (mINS) in culture supernatants and mouse serum were quantified with a Mouse Insulin ELISA kit (Mercordia, Uppsala, Sweden; cat. no. 10-1247-01). Short human glucagon-like peptide 1 (shGLP1) levels in culture supernatants were quantified with a Mouse IgG ELISA Kit (Immunology Consultants Laboratory Inc., Portland, Oreg.; cat. no. E-90G). Bioactive GLP-1 levels in mouse serum were quantified with a High Sensitivity GLP-1 Active ELISA Kit (Merck Millipore, Schaffhausen, Switzerland; cat. no. EZGLPHS-35K). TurboGFP was visualized by fluorescence microscopy using a Nikon Ti-E base Wide Field microscope (Nikon AG, Egg, Switzerland) equipped with a Hammamatsu Orca Flash 4 digital camera, a 20× objective, a 488 nm/509 nm excitation and emission filter set and NIS Elements AR software (version 4.3.0).
Generation of Stable Cell Lines.
The monoclonal HEK-293NFAT-SEAP cell line, transgenic for depolarization-stimulated SEAP expression, was constructed by co-transfecting HEK-293 cells with a 20:1 (w/w) mixture of pMX57 (PNFAT3-SEAP-pA) and pZeoSV2(+) (PSV40-zeo-pA), followed by selection in culture medium containing 1 mg/mL zeocin (Life Technologies, CA, USA; cat. no. R250-05) and FACS-mediated single-cell cloning. Sixteen cell clones were picked and the best-in-class HEK-293NFAT-SEAP was used for all follow-up studies.
The polyclonal HEKGLP1R population, transgenic for high-level GLP1R expression, was constructed by cotransfecting 3×106 HEK-293 cells with 9500 ng pMX250 (ITR-PhEF1
The polyclonal HEKMX252 s population, transgenic for stable expression of the α1D subunit of Cav1.3 (Cacna1d), was constructed by co-transfecting 3×106 HEK-293 cells with 9500 ng pMX252 (ITR-PhEF1
The polyclonal HEKCav1.3 population, transgenic for stable expression of the full Cav1.3 channel componentry, was constructed by co-transfecting 3×106 HEKMX252 cells with 9500 ng pMX251 (ITR-PhEF1
The monoclonal HEK-β cell line, transgenic for glucose-stimulated SEAP- and insulin-expression, was constructed by cotransfecting 3×106 HEKCav1.3 cells with 9500 ng pMX256 (ITR-PNFAT5-SEAP-P2A-mINS-pA:PRPBSA-EGFP-P2A-ZeoR-pA-ITR) and 500 ng pCMV-T7-SB100. After selection with 100 μg/mL zeocin for three passages, 5% of the surviving population with highest EGFP expression levels were subjected to FACS-mediated single-cell cloning. 50 cell clones were selected and clone no. 4 showing optimal glucoseinducible insulin expression was used for all follow-up studies.
FACS-Mediated Cell Sorting.
HEK-293 cells expressing EGFP (488 nm laser, 505 nm long-pass filter, 530/30 emission filter) or dTomato (561 nm laser, 570 nm long-pass filter, 586/15 emission filter) were sorted using a Becton Dickinson LSRII Fortessa flow cytometer (Becton Dickinson, Allschwil, Switzerland) while excluding dead cells and cell doublets. Untreated HEK-293 cells or parental polyclonal populations were used as negative controls.
RT-PCR.
Total RNA of untreated HEK-293 cells was isolated using the ZR RNA MiniPrep™ kit (Zymo Research, CA, USA; cat. no. R1064), treated with DNaseI (Thermo Scientific, cat. no. EN0521) and cDNA was synthesized using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit (Life Technologies, CA, USA; cat. no. 4368814). Amplicons of target components were generated by PCR reactions of 30-45 cycles of denaturation (95° C., 20 s), annealing (58° C., 30 s) and extension (68° C., 30 s) with primers listed in Table 2. PCR products were separated on 1.5% agarose gels supplemented with 1× RedSafe™ (iNtRON Biotechnology, Gyeonggi-do, Republic of Korea; cat. no. 21141) and visualized under UV light. For quantitative analysis, PCR reaction (2 min at 50° C., 20 s at 95° C. and 60 cycles of is at 95° C. followed by Imin at 60° C.) was performed on the Eppendorf Realplex2 Mastercycler (Eppendorf GmbH, Hamburg, Germany) using the SYBR® Green PCR Master Mix (Life Technologies, CA, USA; cat. no. 4309155) and the primers listed in Table 2. The relative cycle threshold (CT) was determined and normalized against the endogenous human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.
Glucose-Stimulated Insulin Secretion (GSIS).
Encapsulated human islets and 1.1E7 cells were washed (incubation for 30 min) in 0.25 mL Krebs-Ringer Bicarbonate Buffer (Sigma-Aldrich, cat. No. K4002; 129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 10 mM HEPES, 0.1% BSA, pH7.4) and incubated for 30 min in low-glucose (2.8 mM) Krebs-Ringer Bicarbonate Buffer. The culture was then switched to high-glucose (30 mM) Krebs-Ringer Bicarbonate Buffer for another 30 min. The secreted isoform of the connecting peptide (C-peptide) produced during proinsulin processing was quantified using the Ultrasensitive Human C-peptide ELISA and the capsules were then transferred to fresh culture medium and cultivated until the next GSIS assay.
Chemicals and Soft Drinks.
Ethanol (EtOH; cat. no. 02860), acetic acid (cat. no. A6283), calcium chloride dihydrate (stock solution 0.5M in ddH2O; cat. no. C7902), D-glucose (stock solution 1M in ddH2O; cat. no. G-7021), D-mannitol (stock solution 0.1M in ddH2O; cat. no. M4125), D-mannose (stock solution 1M in ddH2O; cat. no. M6020), D-galactose (stock solution 0.1M in ddH2O; cat. no. 48263), magnesium sulfate (MgSO4; cat. no. M2643), nicotinamide (stock solution 0.5M in ddH2O; cat. no. N0636), potassium phosphate monobasic (K2HPO4; cat. no. P5655), sodium bicarbonate (NaHCO3; cat. no. S5761), sucrose (stock solution 0.1M in ddH2O; cat. no. S0389), D-maltose monohydrate (stock solution 0.1M in ddH2O; cat. no. M9171), D-xylose (stock solution 0.1M in ddH2O; cat. no. X1500), L-glutamine (stock solution 0.15M in ddH2O; cat. no. G3126), 3-(N-Morpholino)propanesulfonic acid (MOPS; stock solution 0.1M in ddH2O; cat. no. M1254), palmitic acid (stock solution 0.02M in EtOH; cat. no. P0500) and alloxan monohydrate (cat. no. A7413) were purchased from Sigma-Aldrich (Buchs, Switzerland). Blasticidin S HCl (cat. no. A1113903), GlutaMAX™ Supplement (cat. no. 35050061), Insulin Transferrin Selenium liquid media supplement (ITS; cat. no. 41400045), N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES; stock solution 1M; cat no. 15630080), puromycin dihydrochloride (cat. no. A1113803), sodium pyruvate (stock solution 100 mM; cat no. 11360070) and Zeocin™ selection reagent (cat. no. R25005) were purchased from ThermoFisher Scientific (Reinach, Switzerland). Recombinant human GLP-1 (stock solution 1 mM in ddH2O; cat. no. 130-08; lot no. 0108358), IL-2 (stock solution 10 μM in 10 mM aqueous acetic acid; cat. no. 200-02; lot no. 051512-1), IL-12 p70 (stock solution 1 μM in DMEM; cat. no. 200-12; lot no. 0909S96) and IL-15 (stock solution 1 μM in ddH2O; cat. no. 200-15; lot no. 061024) were purchased from PeproTech EC Ltd (London, UK). D-fructose (stock solution 0.1M in ddH2O; cat. no. 161350010), L-leucine (stock solution 0.01M in glucose-free DMEM; cat. no. 125121000) and linoleic acid (stock solution 0.02M in EtOH; cat. no. 215040050) were purchased from Acros Organics (Geel, Belgium). L-glucose anhydrous (stock solution 1M in ddH2O; cat. no. AB116919) was purchased from abcr GmbH (Karlsruhe, Germany). Potassium chloride (KCl; stock solution 4M in ddH2O; cat. no. A3582) and sodium chloride (NaCl; stock solution 5M in ddH2O; cat. no. A2942) were purchased from AppliChem (Darmstadt, Germany). Citric acid anhydrous (stock solution 0.1M in ddH2O; cat. no. sc-211113) was purchased from Santa Cruz Biotechnology (Dallas, USA). Poly(L-lysine) hydrobromide (cat. no. PLKB50) was purchased from Alamanda Polymers (Alabama, USA). Trisodium citrate 2-hydrate (stock solution 0.1M in ddH2O; cat. no. 6448) was purchased from Merck Millipore (Schaffhausen, Switzerland). Bovine serum albumin (BSA; stock solution 10 g/L; cat. no. B9000S) was purchased from NEB Biolabs (Ipswich, Mass.). Streptozotocin (cat. no. 1621) was purchased from Tocris Bioscience (Bristol, UK). Coke was purchased at local supermarkets, degassed by extensive shaking and directly administered to mice (4×200 μl).
Animal Experiments.
The type 1 diabetes mouse model (T1D) was generated as described previously (Auslander D et al., Mol Cell 55, 397-408 (2014)). In brief, fasted mice (2×18 h/day) were injected with a single dose of freshly diluted alloxan monohydrate (ALX; 200 mg/kg in 300 μl phosphate buffered saline) and persistent fasting hyperglycemia (>20 mM) developed after 48 h. The type 2 diabetes mouse model (T2D) was generated as described in (Arora S et al., Global J Pharmacol 3, 81-84 (2009)). In brief, fasted mice (20 h/day) were injected with daily doses of freshly diluted streptozotocin (STZ; 40 mg/kg in 250 μl ice-cold sodium citrate buffer [pH 4.5, 0.01M, 0.11 g/L NaCl]) for five consecutive days and chronic fasting hyperglycemia (>10 mM) developed after 3 weeks. Glycemia of mice were measured with a commercial glucometer (Contour® Next; Bayer HealthCare, Leverkusen, Germany; detection range: 0.5-35 mM) purchased at local pharmacies. Intraperitoneal implants were produced by encapsulating transgenic HEK-293 cells, 1.1E7 cells or human islets into coherent alginate-poly-(L-lysine)-alginate beads (400 μm; 500 cells or 1-10 IEQs/capsule) using an Inotech Encapsulator Research Unit IE-50R (EncapBioSystems Inc., Greifensee, Switzerland) set to the following parameters: a 200-μm nozzle with a vibration frequency of 1025 Hz, a 25-mL syringe operated at a flow rate of 410 units and 1.12-kV voltage for bead dispersion (Ye H et al., PNAS 110, 141-146 (2013)). 5-9-weeks old female wild-type or ALX/STZ-pretreated CD-1 Swiss albino mice (Janvier Labs, Le Genest-Saint-Isle, France) were intraperitoneally injected with 1 mL of glucose-free DMEM containing 1×104 microcapsules. Blood serum was isolated using microtainer serum separating tubes (SST) according to the manufacturer's instructions (centrifugation for 5 min at 10 000×g; Becton Dickinson, Plymouth, UK; cat. no. 365967). Most experiments involving animals were performed according to the directive of the European Community Council (2010/63/EU), approved by the French Republic and carried out by Ghislaine Charpin-El Hamri (No. 69266309; project No. DR2013-01 (v2)) and Marie Daoud-El Baba (No. 69266310; project No. DR2013-01 (v2)) at the Institut Universitaire de Technologie, UCB Lyon 1, F-69622 Villeurbanne Cedex, France. Animal experiments related to
Glucose sensing was achieved by coupling the 3-cell-mimetic cascade of glycolysis-mediated calcium entry to a synthetic excitation-transcription coupling system (D'Arco M and Dolphin A C, Sci Signal 5, pe34 (2012)) in human embryonic kidney cells. This human cell line is widely used in studies of ion channel activities (Thomas P and Smart T G, J Pharmacol Toxicol Methods 51, 187-200 (2005)) and shows optimal production capacities for antidiabetic proteins (Auslander D et al., Mol Cell 55, 397-408 (2014)). A cell-based assay was constructed in human embryonic kidney cells (HEK-293) to evaluate the stimulus strength of membrane depolarization with a quantitative reporter protein (
To experimentally evaluate the contributing effects of each 3-cell-derived component for sensing glucose (GLUT2, GCK, KATP, Cav1.3) with the pMX57-based depolarization-induced transcriptional system, a combinatorial screening approach (
To quantitatively analyse the system, ensure consistency in the design steps, and eventually predict circuit operation in vivo, we developed a dynamic mathematical model for the β-cell-derived glucose-sensing cascade. Briefly, this ordinary differential equation (ODE) model covers the components shown in
To test the substrate specificity of the Cav1.3/PNFAT-IL4-constituted glucose-sensing system, Cav1.3/pMX57-transgenic HEK-293 cells were cultured in cell culture medium containing different sugar compounds such as osmotic controls (
To test the application potential of the glucose-induced excitation-transcription coupling system for diabetes treatment, Cav1.3-transgenic HEK-293NFAT-SEAP1 cells were microencapsulated into coherent, semi-permeable and immunoprotective alginate-poly-(L-lysine)-alginate beads and implanted them into the peritoneum of mice, where they become vascularized and connected to the animal's bloodstream with appropriate oxygen supply (Jacobs-Tulleneers-Thevissen D et al., Diabetologia 56, 1605-1614 (2013)). Also in vivo, the transcriptional regulation system operated in a dose- and Cav1.3-dependent manner, as recapitulated by the same in vitro dynamic model coupled to a mathematical representation of mouse physiology (
State-of-the-art treatment options for diabetes mellitus are either long-acting drugs, such as stabilized GLP-1 variants, in which the frequency of drug injection can be reduced to weekly periods (T2D) (Trujillo J et al., Ther Adv Endocrinol Metab 6, 19-28 (2015)), or portable, external pump systems that self-sufficiently inject fast-acting insulin analogues according to the patient's instantaneous glycaemia (T1D) (Pickup J C, N Engl J Med 366, 1616-1624 (2012)). To test whether the expression levels achieved with the glucose-inducible excitation-transcription coupling system were compatible with antidiabetic therapeutic activities, HEK-293 cells were co-transfected with Cav1.3 and the previously reported short human GLP-1 (shGLP1) construct (Ye H et al., Science 332, 1565-1568 (2011)) (pMX115; (NFATIL4)9-Pmin-shGLP1-pA) to engineer therapeutic mammalian cells that express GLP-1 exclusively under hyperglycaemic conditions (
β-cells modulate the insulin release not only in response to glucose but also by the action of glucoincretins such as GLP-1 (Lee Y S, Metabolism 63, 9-19 (2014)). We therefore engineered HEK-293 for HEK-β componentry as well as for constitutive expression of the GLP-1 receptor (GLP1R) and PCRE-driven insulin expression (
Implantation of microencapsulated HEK-β cells (
In a comparative analysis of reversible glucose-stimulated insulin secretion by the β-cell-mimetic HEK-β, the pancreatic β-cell line 1.1E7 (McCluskey J T, The Journal of biological chemistry 286, 21982-21992 (2011)) and human islets over three weeks, HEK-β showed higher insulin secretion capacity than 1.1E7 and human islets in vitro (
Coupling of CaV1.3-based glucose sensing to insulin production and secretion resulted in the β-cell-mimetic HEK-β that provided increased 3-week insulin secretion profiles compared to the pancreatic cell line 1.1E7 and human islets in vitro. Control of postprandial glucose metabolism was similar between HEK-β and 1.1E7 but only HEK-β reached the blood glucose levels of healthy mice. Interestingly, since the different insulin release dynamics of HEK-β, 1.1E7 and human islets in vitro had apparently no significant impact on postprandial glucose metabolism, the differences in the secretion modality—constitutive for HEK-β, vesicular for 1.1E7 and human islets—may not be as relevant in response to meals as generally thought. This is supported by our model simulations and by the latest generation of basal insulin analogs such as insulin degludec (Tresiba®, Novo Nordisk), which provides autonomous glucose control for up to 42 hours without the need to synchronize its administration with meals.
Implantation of microencapsulated mammalian cells into patients does not necessitate immunosuppression, as host and graft communicate via secretory metabolites that diffuse across a semi-permeable biocompatible membrane (Jacobs-Tulleneers-Thevissen D et al., Diabetologia 56, 1605-1614 (2013)). Since the first implantable alginate-poly-(L-lysine) capsules harbouring rat islet cells were presented almost 35 years ago, techniques for microencapsulated pancreatic β-cells have been continuously optimized for diabetes treatment, with several clinical trials already approved by governments. However, although current advances in stem cell research (Pagliuca F W et al., Cell 159, 428-439 (2014)) have successfully solved the previous issues of poor source availability and differentiation efficiency to generate adequate numbers of functional 3-cells that elicit observable antidiabetic functions (Kobayashi N, Cell Transplant 15, 849-854 (2006)), regenerated pancreatic n-cells are generally restricted to the treatment of insulin-deficient type-1 diabetes (Bruin J E et al., Stem Cell Reports 4, 605-620 (2015)). Type-2 diabetes, however, is much more common, accounting for more than 95% of all diabetes cases and with a pathogenesis that often involves an impaired sensitivity of body cells to excessive levels of circulating insulin (Johnson A M and Olefsky J M, Cell 152, 673-684 (2013)). Glucagon-like peptide 1 (GLP-1) is an incretin hormone naturally released from the intestine after meal ingestion that not only acts on n-cells to stimulate their postprandial release of insulin (Drucker D J et al., Lancet 368, 1696-1705 (2006)) but also circulates to other somatic cells to (i) promote satiety, (ii) improve hepatic insulin sensitivity, (iii) slow gastric emptying and (iv) inhibit glucagon secretion (Ye H et al., PNAS 110, 141-146 (2013)). Currently, subcutaneous injection of long-acting GLP-1 analogues is the state-of-the-art treatment option for most type-2 diabetes cases (Trujillo J et al., Ther Adv Endocrinol Metab 6, 19-28 (2015)).
The quest for a cell-based glucose sensor has always been in great demand for biomedical research (Auslander D et al., Mol Cell 55, 397-408 (2014)). Although a variety of putative glucose-sensing components, including GPCRs (Jang H et al., PNAS 104, 15069-15074 (2007)), bacterial transcriptional repressors (Gaigalat L et al., BMC Mol Biol 8, 104 (2007)) and human nuclear receptors (Mitro N et al., Nature 445, 219-223 (2007)) have been characterized, to the best of our knowledge, a successful translation of the glucose input into a transcriptional message that includes antidiabetic activities in vivo has not yet been achieved. In this work, we used the low threshold voltage-gated calcium channel Cav1.3, which permits long-lasting Ca2+ influxes during weak membrane depolarizations (Lipscombe D et al., J Neurophysiol 92, 2633-2641 (2004)), to sense glycaemia-relevant extracellular glucose levels but to remain insensitive to other potential trigger compounds such as metabolites and salts within their physiologically relevant concentration range (
β-cell-mimetic designer cells such as HEK-13 could combine the best of all strategies: (i) they use glucose-sensor components evolved in native β-cells, (ii) they take advantage of parental cell lines with a track record in biopharmaceutical manufacturing that are known for their robustness and reliability and (iii) they show glucose-induced insulin release performance comparable to β-cell lines and human islets. Additionally, rational programming of designer cells enables (iv) straightforward fine-tuning of performance parameters and provides (v) flexibility to couple glucose-sensing to the production of other therapeutic proteins such as GLP-1 required for the treatment of type-2 diabetes.
Although an ideal implant genotype for T1D therapy might also include postprandial insulin release to attenuate immediate perturbations of blood glucose levels following dietary sugar intake, our experimental and computational analyses showed that the glucose-induced insulin expression system achieved rapid attenuation of life-threatening hyperglycaemia in a T1D animal model by restoring near-homeostatic blood insulin and glucose levels in a self-sufficient and (predicted) robust manner. In particular, glycaemic control in T1D mice was restored in less than one week following implantation—a response time that compares favourably with experimental cell-based therapies in animals using pancreatic progenitor cells (30 weeks; (Rezania A et al., Diabetes 61, 2016-2029 (2012)) or β-cell mimetics produced from human pluripotent stem cells by a seven-stage in vitro differentiation protocol (40 days; (Rezania A et al., Nat Biotechnol 32, 1121-1133 (2014)). Furthermore, the animal experiments demonstrated an absolutely increased survival rate already with the current implant version and within only one week of treatment, which should be the foremost criterion to judge the system's efficiency. This work is therefore considered as a proof-of-principle study that introduces an attractive alternative concept for diabetes treatment with therapeutic features that have already been achieved at a standard of practical relevance.
| Number | Date | Country | Kind |
|---|---|---|---|
| 16180000.8 | Jul 2016 | EP | regional |
| 16200258.8 | Nov 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/067981 | 7/17/2017 | WO | 00 |
