WO2013117776A1 - Glucokinase enzymes with increased activity and use thereof in the treatment and/or prevention of diabetes mellitus - Google Patents

Glucokinase enzymes with increased activity and use thereof in the treatment and/or prevention of diabetes mellitus Download PDF

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Publication number
WO2013117776A1
WO2013117776A1 PCT/ES2012/070080 ES2012070080W WO2013117776A1 WO 2013117776 A1 WO2013117776 A1 WO 2013117776A1 ES 2012070080 W ES2012070080 W ES 2012070080W WO 2013117776 A1 WO2013117776 A1 WO 2013117776A1
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cell
islets
seq
amino acid
pancreatic
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PCT/ES2012/070080
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Spanish (es)
French (fr)
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Antonio Luis CUESTA MUÑOZ
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Fundación Pública Andaluza Progreso Y Salud
Instituto De Salud Carlos Iii.
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Priority to PCT/ES2012/070080 priority Critical patent/WO2013117776A1/en
Publication of WO2013117776A1 publication Critical patent/WO2013117776A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01002—Glucokinase (2.7.1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus

Definitions

  • the present invention falls within the field of gene and cell therapy, specifically it refers to nucleotide sequences coding for mutated glucokinase enzymes that exhibit greater enzymatic activity, to the amino acid sequences encoded by them, to pancreatic islets that express them and their uses. for the treatment and / or prevention of diabetes mellitus.
  • Pancreatic islet transplantation is currently the only cell therapy available for the treatment of unstable type 1 diabetes mellitus. This therapy has proven effective in achieving normalization and stability of blood glucose in these patients, in the disappearance of severe hypoglycemias and in preventing chronic and progressive complications of diabetes. However and unfortunately, the primary objective of this therapy, the long-term insulin independence, remains to be achieved.
  • pancreatic islets At the moment there are different lines of research aimed at solving the problem of limitation of available human pancreatic islets, which focus their efforts on, for example, increasing the survival and proliferation of ⁇ cells.
  • islet encapsulation O'Sullivan ES, et al., 2010, Diabetologia
  • Isle T the production of insulin-producing cells from embryonic or pancreatic progenitor cells
  • in vitro proliferation of pancreatic ⁇ cells for the expansion of human islets.
  • fibroblast growth factor or hepatocyte growth factor has allowed, although in a limited way, an expansion of human islets (Gao R, et al., 2003, Diabetes, 52 (8): 2007-2015 ).
  • overexpression of the human hepatocyte growth factor in primate islets improves both the implant and the functionality of these islets when they are transplanted in NOD-SCID diabetic mice.
  • the combination of the gastrin peptide and the epidermal growth factor also provides an expansion of the ⁇ cell mass, however the cells obtained have a short half-life (Suarez-Pinzon WL, et al., 2005, Diabetes, 54 (9): 2596-2601).
  • GLP-1 glycopeptide-1
  • GK glucokinase
  • GCK glucokinase gene
  • GK Another important aspect to consider is the important relationship between metabolism and cell apoptosis, and in which GK plays a key role.
  • GK together with BAD, PKA, PP1 and WAVE-1 forms a complex that maintains the BAD proapoptotic protein in a state of phosphorylation that prevents cell death (Danial NN., Et al., 2003, Nature, 424 (6951): 952 - 956).
  • BAD has a role in the regulation of insulin secretion by the ⁇ cell, this regulation being mediated by GK.
  • pancreatic islets that, in addition to being highly effective in terms of their ability to release glucose-stimulated insulin, eliminate the need for a large number of islets in order to achieve adequate glycemic control in the treated patient. In this way, the lack of organs that exist today can be partially replaced. Likewise, it would be desirable for the islets to have a longer survival, allowing insulin independence of the more durable and stable transplanted patients. DESCRIPTION OF THE INVENTION
  • the present invention relates to nucleotide sequences that code for variants of the glucokinase (GK) enzyme, preferably human, that exhibit an increase in their enzymatic activity, as well as to the amino acid sequences corresponding to said variants.
  • GK glucokinase
  • pancreatic islets preferably human
  • GK variants proposed herein induces a significant increase in the proliferation of ⁇ cells, which translates into a larger size of the pancreatic islets.
  • These islets also maintain a completely preserved and well defined cytoarchitecture.
  • These variants also contribute to increase the stability and survival of ⁇ cells.
  • pancreatic islets with a higher metabolic activity are obtained, since these variants of the GK have, for example, 9 times more affinity for glucose and reduce the physiological threshold of glucose-stimulated insulin secretion (UF-SIEG), going from 5 mM (established in humans with the native GK) to O, 96 mM.
  • U-SIEG glucose-stimulated insulin secretion
  • This set of characteristics gives the islet that expresses the variants described in the invention: (i) a greater resistance to survive the mechanical stress that the transplant procedure entails, (ii) a more lasting functionality over time, (iii) ability to proliferation once transplanted, and (iv) due to its larger size and higher metabolic rate, the necessary number of this type of islets to be transplanted in the patient in order to achieve adequate blood glucose is less than what is currently needed.
  • nucleotide and amino acid sequences described in the present invention are useful for the creation of pancreatic islets. of high efficacy that can be transplanted to patients, during somatic cell therapy procedures, for the treatment and / or prevention of diabetes mellitus, with the associated advantages described above.
  • a first aspect of the invention relates to an isolated nucleotide sequence, hereafter referred to as the "nucleotide sequence of the invention", which encodes an amino acid sequence of the glucokinase or GK enzyme that has at least one activating mutation of the activity of said enzyme.
  • nucleotide sequence refers to a polymeric form of nucleotides of any length that may or may not be chemical or biochemically modified. They refer, therefore, to any polyiribonucleotide or polydeoxyribonucleotide, both single-stranded and double-stranded.
  • the polynucleotide of the invention can be obtained artificially by conventional methods of cloning and selection, or by sequencing.
  • the polynucleotide in addition to the coding sequence, can carry other elements, such as, but not limited to, introns, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, or stabilizing sequences. These polynucleotides may additionally include coding sequences for amino acids. additional that may be useful, for example, but not limited to increase the stability of the peptide generated from it or allow a better purification thereof.
  • the "glucokinase enzyme” or "GK” is an isozyme hexokinase that is involved in the phosphorylation of glucose to give rise to glucose-e-phosphate.
  • amino acid sequence of the GK enzyme is preferably the amino acid sequence of the human native GK (SEQ ID NO: 4) encoded, for example, but not limited to, by the nucleotide sequence SEQ ID NO: 5 (human GCK gene), although the amino acid sequences of GK enzymes orthologous to human GK from other organisms and that fulfill the same function as human GK in the organism from which they are derived, are also within the scope of the present invention. as the nucleotide sequences that code for them.
  • activating mutations of the enzyme activity refers to those mutations present in the amino acid and / or nucleotide sequence of the GK that give rise to an enzyme that has a higher enzyme activity compared to the native enzyme that does not have these mutations.
  • Such mutations may be, but are not limited to, point mutations (changing one or several amino acids or one or several nucleotides for another / s), deletion, substitution, addition, etc. mutations.
  • the identification and selection of enzymes that have a higher enzyme activity, obtained by means of Induction of such mutations, can be carried out by enzymatic activity tests in the presence of substrate (in this case glucose) known by the person skilled in the art, which allow to select the modified enzyme variants of interest that have greater activity, less inhibition per substrate, greater enzymatic stability, greater range of pH and / or T at which the enzyme is active, etc.
  • substrate in this case glucose
  • variants of the invention is used to refer to GK enzymes that have at least one activating mutation of the activity of said enzyme.
  • the nucleotide sequence of the invention can be used to obtain high-efficiency pancreatic islets, by their introduction, by molecular biology methods known to a person skilled in the art, in a cell, preferably pancreatic, more preferably in a pancreatic ⁇ cell , even more preferably isolated from the patient, so that it expresses the amino acid sequence of any of the variants of the invention, and thus a "proliferative autologous ⁇ cell cell population is obtained" in vitro "and highly efficient in insulin release in response to glucose. Therefore, in a preferred embodiment, the nucleotide sequence of the invention is capable of generating high-efficiency pancreatic islets.
  • pancreatic islets here also referred to as “islets of the invention” as will be seen below, comprise modified pancreatic cells as explained in this paragraph, preferably pancreatic ⁇ cells, which have a greater proliferative capacity and induce a reduction in UF-SIEG, so they begin to release insulin at lower glucose concentrations than islets that do not comprise cells so modified.
  • this type of islets are larger than pancreatic islets that do not comprise cells to which the nucleotide sequence of the invention has been introduced.
  • amino acid sequence of the glucokinase enzyme having at least one activating mutation of the activity of said enzyme is selected from the list consisting of: SEQ ID NO : 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • SEQ ID NO: 1 corresponds to SEQ ID NO: 4 where the residue from position 64 (S, serine) has been replaced by a phenylalanine (F) , said variant will also be referred to as GCK-S64F.
  • SEQ ID NO: 2 corresponds to SEQ ID NO: 4 where the residue from position 91 (V, valine) has been replaced by a leucine (L), this variant will also be referred to as GCK-V91 L.
  • SEQ ID NO: 3 corresponds to SEQ ID NO: 4 where the residue of position 214 (Y, tyrosine) has been replaced by a cysteine (C), said variant will also be referred to as GCK-Y214C.
  • An example of a nucleotide sequence encoding SEQ ID NO: 1 is, but not limited to, SEQ ID NO: 6.
  • the nucleotide sequences encoding SEQ ID NO: 2 and SEQ ID NO: 3 can be obtained, but not limited to, by a method comprising introducing mutations into the human GK gene (SEQ ID NO : 5) by PCR with primers SEQ ID NO: 7 and SEQ ID NO: 8 (to obtain the nucleotide sequence encoding SEQ ID NO: 3) and SEQ ID NO: 9 and SEQ ID NO: 10 (to obtain the nucleotide sequence encoding SEQ ID NO: 2).
  • Another aspect of the invention thus relates to the isolated nucleotide sequences obtained according to the procedure described in this paragraph, which code for amino acid sequences of the GK enzyme that have at least one activating mutation of the activity of said enzyme, specifically for SEQ ID NO: 2 and SEQ ID NO: 3.
  • Another aspect of the invention relates to a genetic construct, hereafter "genetic construct of the invention", which comprises the nucleotide sequence of the invention.
  • the genetic construction of the invention can include control sequences operatively linked to the nucleotide sequence of the invention.
  • control sequence refers to nucleotide sequences that are necessary to effect the expression of the sequences to which they are linked.
  • control sequences is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous. Examples of control sequences are, but are not limited to, promoters, transcription initiation signals, transcription termination signals, polyadenylation signals or transcriptional activators.
  • operably linked refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended manner.
  • a control sequence "operably linked" to a polynucleotide is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • promoter refers to a region of DNA, generally “upstream” or “upstream” of the transcription start point, which is capable of initiating transcription in a cell.
  • This term includes, for example, but not limited to, constitutive promoters, cell or tissue specific promoters or inducible or repressible promoters. Control sequences depend on the origin of the cell in which the nucleic acid is to be expressed. Examples of promoters Prokaryotes include, for example, but not limited to, promoters of the trp, recA, lacZ, lacl, tet, gal, trc, or tac genes of E. coli, or the promoter of the B. subtilis ⁇ -amylase gene.
  • nucleic acid for the expression of a nucleic acid in a prokaryotic cell, the presence of an upstream ribosomal binding site of the coding sequence is also necessary.
  • Appropriate control sequences for the expression of a polynucleotide in eukaryotic cells are known in the state of the art.
  • the genetic construct of the invention is an expression vector, hereinafter "vector of the invention".
  • vector of the invention refers to a DNA or RNA fragment that has the ability to replicate in a given host and can serve as a vehicle for carrying out the transcription of a sequence of interest that has been inserted therein.
  • the vector can be a plasmid, a cosmid, a phagemid, artificial yeast chromosomes (YAC), artificial bacterial chromosomes (BAC), artificial human chromosomes (HAC), a bacteriophage or a viral vector such as, for example, but not limited to , adenovirus, retrovirus, adeno-associated virus, lentivirus, poxvirus or herpesvirus, without excluding other types of vectors that correspond to the definition made of vector.
  • the vector of the invention is a lentivirus, that is, a lentiviral vector.
  • Another aspect of the invention relates to an amino acid sequence of the glucokinase enzyme selected from the list consisting of: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the nucleotide sequence of the invention or the genetic construction of the invention can be used to obtain high-efficiency pancreatic islets, by introduction, by molecular biology methods known to a person skilled in the art, in a cell, preferably pancreatic, more preferably in a pancreatic ⁇ cell, even more preferably isolated from the patient, so that it expresses the amino acid sequence of any of the variants of the invention, and thus a "population in vitro" of a cell population is obtained.
  • cell of the invention which comprises, temporarily or preferably, stably, the nucleotide sequence of the invention, the genetic construction of the invention, the vector of the invention, or an amino acid sequence of any of the variants of the invention, more preferably an amino acid sequence selected from the list consisting of: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO : 3.
  • the cell of the invention can be any eukaryotic or prokaryotic cell, although preferably it is a eukaryotic cell.
  • the cell of the invention is a pancreatic cell.
  • the "pancreatic cell" of the invention may be an alpha cell, a beta cell, a delta cell, a G cell or an F cell, although preferably it is a beta cell ( ⁇ cell).
  • the cell of the invention can be both autologous, as allogeneic or xenogenic.
  • the possibility that the cell of the invention is of autologous origin allows its subsequent transplantation, after "in vitro" manipulation, for the treatment and / or prevention of diabetes mellitus can be performed without the immunosuppression of the subject being necessary. transplanted Therefore, in a more preferred embodiment, the cell of the invention is of autologous origin.
  • autologous origin any origin of the sample, taken from the tissues or cells of an individual or patient, which is the same in a donor and the recipient thereof when they are administered after treatment or transplanted after modification.
  • the cell of the invention can come from any mammal, although preferably it comes from a human. Therefore, in an even more preferred embodiment, the cell of the invention is of human origin.
  • pancreatic islet which comprises the cell of the invention.
  • the islet of the invention is of autologous origin.
  • said islet is of human origin.
  • Pantencreatic islets are clusters of cells that are responsible for producing hormones such as insulin and glucagon, with a purely endocrine function. They also secrete immunoglobulins. They form various clusters or islets scattered throughout the pancreas, with about one million such islets found in the human pancreas. They consist mainly of ⁇ cells, insulin secretors, around which larger cells are found in small groups such as a cells, glucagon secretors, ⁇ , somatostatin secretors, F, producing a pancreatic polypeptide that inhibits secretions exocrines of the pancreas, or G, secretors of gastrin.
  • composition of the invention which comprises the nucleotide sequence of the invention, the genetic construction of the invention, the vector of the invention, the amino acid sequence of the variants of the invention, preferably SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, the cell of the invention or the islet of the invention.
  • the composition of the invention is a pharmaceutical composition.
  • the composition of the The invention further comprises a pharmaceutically acceptable carrier.
  • the composition of the invention further comprises another active ingredient.
  • the "pharmaceutically acceptable carrier” or carrier is preferably an inert substance.
  • the carrier is a substance that is used in the composition to dilute any of the components of said pharmaceutical composition of the present invention to a given volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the composition.
  • the composition of the invention optionally comprises another active substance or principle.
  • active substance is any matter, whatever its origin, human, animal, plant, chemical or other, to which an appropriate activity is attributed to constitute a medicine .
  • composition of the present invention can be presented in the form of solutions or any other form of clinically permitted administration and in an amount therapeutically effective.
  • the composition described in the invention can be formulated in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, gel, microsphere or aerosol, for oral, topical or parenteral administration.
  • the composition of the invention can also be formulated in the form of liposomes or nanospheres, sustained release formulations or any other conventional release system.
  • pancreatic islets of the invention are highly effective islets, in that they are larger than pancreatic islets that do not comprise cells of the invention, comprise cells, preferably pancreatic, more preferably pancreatic ⁇ cells , with a greater proliferative capacity and that induce a reduction of UF-SIEG, so they begin to release insulin at lower glucose concentrations than islets that do not comprise cells of the invention.
  • both the cells and the islets of the invention are useful in the treatment and / or prevention of diabetes mellitus, by administration as a medicine or by transplantation to the pancreas of an individual suffering from diabetes mellitus, all with the in order to increase, restore or partially or totally replace the functional activity of insulin secreting ⁇ cells present in the pancreas of the individual affected by the disease.
  • said cells and islets of the invention have a greater resistance to survive the mechanical stress involved in the transplant procedure, a more lasting functionality over time, capacity proliferation once transplanted, and due to its larger size and higher metabolic rate, the necessary number of this type of cells or islets to be transplanted in the patient in order to achieve adequate blood glucose is less than what is currently needed.
  • nucleotide sequence of the invention, the genetic construction of the invention, the vector of the invention, the composition of the The invention or the amino acid sequence of the variants of the invention can be administered as a medicine to an individual for the treatment and / or prevention of diabetes mellitus. Therefore, another aspect of the invention relates to the use of the nucleotide sequence of the invention, of the genetic construction of the invention, of the vector of the invention, of the amino acid sequence of the variants of the invention, preferably of SEQ. ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, of the cell of the invention, of the islet of the invention or of the composition of the invention, for the preparation of a medicament, hereinafter "medicament of the invention ".
  • the medicament is for the treatment and / or prevention of diabetes mellitus.
  • diabetes mellitus refers to the chronic disease that has high blood sugar levels and can be caused by low insulin production, resistance to insulin or both. This pathology can be detected by diagnostic methods known to those skilled in the art such as, for example, but not limited to, urinalysis, fasting blood glucose test, hemoglobin A1c analysis, oral glucose tolerance test or analysis. of blood glucose. This term includes, but is not limited to, gestational diabetes, metabolic syndrome, type 1 diabetes and type 2 diabetes. In an even more preferred embodiment, the medication is for the treatment and / or prevention of diabetes mellitus. type 1 or insulin dependent, which is characterized in that the pancreas does not produce or produces little insulin, which makes the daily injection of this hormone necessary.
  • treatment refers to combating the effects caused as a result of a disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:
  • prevention as understood in the present invention consists in preventing the onset of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably mammal, and more preferably a human), in particularly, when said subject has a predisposition for the pathological condition.
  • the medicament referred to in the present invention can be for human or veterinary use.
  • the "medicine for human use” is any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in humans or that can be used in humans or administered to humans in order to restore, correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action, or establishing a medical diagnosis.
  • the "veterinary medicinal product” is any substance or combination of substances that is presented as having curative or preventive properties with respect to animal diseases or that can be administered at animal in order to restore, correct or modify its physiological functions exerting a pharmacological, immunological or metabolic action, or to establish a veterinary diagnosis. "Premixes for medicated feed” prepared to be incorporated into a feed will also be considered “veterinary medicinal products”.
  • the medicament of the invention is a somatic cell therapy medication.
  • somatic cell therapy means the use of live somatic cells, both autologous (from the patient itself), and allogeneic (from another human being) or xenogeneic (from animals), whose biological characteristics have been substantially altered as a result of their manipulation, to obtain a therapeutic, diagnostic or preventive effect, by metabolic, pharmacological or immunological means.
  • somatic cell therapy drugs are, for example, but not limited to: cells manipulated to modify their immunological, metabolic or other functional properties in qualitative or quantitative aspects; classified cells, selected and manipulated, which are subsequently subjected to a manufacturing process in order to obtain the finished product; cells manipulated and combined with non-cellular components (for example, matrices or biological or inert medical devices) that exert the intended action in principle on the finished product; autologous cell derivatives expressed ex vivo ⁇ in vitro) under specific culture conditions; or cells genetically modified or subjected to another type of manipulation to express homologous or non-homologous functional properties previously not expressed.
  • non-cellular components for example, matrices or biological or inert medical devices
  • FIG. 1 Shows the result of the histological examination of islets coming from the pancreas of a patient whose ⁇ cells express the GCK-S64F variant (left panel) as well as islets coming from the pancreas of a control individual (right panel).
  • FIG. 2 Shows the result of the proliferation analysis of ⁇ cells in islets from pancreatic tissue of a patient whose ⁇ cells express the GCK-S64F variant (left panel) as well as islets from pancreatic tissue of a control individual (panel right). Cellular mapping done with Ki67.
  • FIG. 4. Shows the incorporation of BrdU into uninfected mouse islets.
  • DAPI BrdU
  • EGFP Islet control not infected.
  • B Islets infected with EGFP.
  • C Islets infected with natural GK that have a 1, 6-fold increase in BrdU incorporation compared to A and B.
  • D islets infected with GCK-V91 L that have a 6.8-fold increase in BrdU incorporation compared to A and B and a 5, 1 fold increase in BrdU incorporation compared to C.
  • Example 1 Genetic manipulation of the GCK gene to obtain both mild and severe activating forms of GCK capable of coding for glucokinase enzymes with increased activity, and their subsequent introduction into isolated human pancreatic islets.
  • the process of isolation of human pancreatic islets was carried out following the semi-automatic method of Ricordi (Ricordi et al., 1989, Diabetes, 38: Suppl 1: 140-142).
  • the islets were purified using a gradient continuity of densities with Ficoll in a COBE centrifuge. After several washes with culture medium the islets were observed under a microscope. After counting, they were distributed in culture bottles and stored at 37 ° C and 5% CO2.
  • the Adeno-X Tet-ON system developed modulates and controls the expression of GK, in order to obtain an optimal and non-deleterious expression for the functionality of the islets.
  • the human cDNAs of both the native GCK (WT), and the inactive GCK-E440G, mild GCK-E442K and severe GCK-V91 L variants were subcloned into the pTRE-Shuttle2 vector (Clontech Laboratories, Inc); subsequently, the inducible cassette was transferred to the Adeno-X vector to generate the recombinant adenoviruses: native Adv-hGCK-WT, Adv-hGCK-E440G, Adv-hGCK-E442K and Adv-hGCK-V91 L.
  • the infection protocol was optimized in the INS-1 E cell line to establish the amount of viral particles and the concentration of doxycycline necessary to achieve adequate GK expression.
  • human and rat islets co-infected well with native Adv-hGCK, with Adv-hGCK-E440G, with Adv-hGCK-E442K, or with Adv-hGCK-V91 L, in addition to the adenoviral construction with the transcriptional activator for doxycycline (Ad-X Tet-on).
  • qPCR quantitative PCR
  • immunofluorescence assays were performed.
  • rat islets human and rat glucokinase were analyzed, and in human islets, human glucokinase.
  • islet RNA was extracted by Trizol reagent (Invitrogen) and then, 2 ⁇ g of RNA was converted to cDNA by RT-PCR using oligo- (d) T primers.
  • d oligo- (d) T primers.
  • the cDNA was amplified using the Sybr green interleaver in a ABI Prism 7700 platform; the primers were designed using the Primer 3 Express software (applera Europe). Fluorescence data was extracted from the platform, and Ct values and efficiency were obtained by software miner 2.1. The quantification was determined with the qBASE software.
  • cDNAs Complementary DNA coding (cDNAs) of GCK-WT, GCK-E440G, GCK-V91 L and GCK-E442K, were cloned into the skeleton of the associated adenoviral vector subtype 8 (AAV8), under the control of the insulin gene promoter mouse (mlP), resulting in plasmids AAV8-mlP-GCKX-ires-eGFP.
  • AAV8-mlP-der vectors to be expressed exclusively in pancreatic beta cells.
  • the transduction of the islets to be transplanted was performed with AAVs replicating the optimal expression of GK obtained with the Adeno-X Tet-ON system, which was done by using different vg of AAV.
  • the possibility of constructing inducible AAVs equivalent to the inducible nonassociated adenoviruses discussed above With the doxycycline-inducible AAVs, human pancreatic islets were transduced and subsequently transplanted.
  • the virus and cell mixture was centrifuged (1000 xg, 1 h, 37 ° C), after which the cells were resuspended in RPMI 1640 medium containing 20% FCS, 10 mM HEPES, 2 mM L-glutamine, 50 ⁇ 9 / ⁇ gentamicin, and rhlL-2 (50 lU / ml), and incubated for four days.
  • the packing of the AAVs, the purification of the different AAVs by ultracentrifugation by CsCI gradient (Cesium Chloride), the titration of the genome particles of the vector (eg), as well as the confirmation of their expression were performed in cells of line I NS-1 E.
  • the transduction process was carried out in a total of 6 days, until the process efficiency was checked:
  • Day 1 8,105 cells were plated in 6-well plates with 2 ml of medium / well. The cells were allowed to incubate for about 48 hours.
  • Day 3 The culture mixture was prepared with 8 ⁇ g of polybrene / ml of medium. Polybrene is a low molecular weight, positively charged polymer that is apparently capable of binding to cells neutralizing surface charge. This allows viral glycoproteins to bind more effectively to their receptors, reducing the repulsion between the viral particle and the cell. 1.5ml of 10% DMEM medium FBS (BioWhittaker) was added with polybrene.
  • 10% DMEM medium FBS BioWhittaker
  • SN was added in the desired proportion (in infections performed, dilutions 1: 25, 1: 4, 1: 2 and 1: 1 were made) and the plate was centrifuged for 30 minutes at 37 ° C at 1000g (process called spinoculation). The cells were contacted with the viral particles and the mixture was incubated overnight at 37 ° C and 5% CO2. The mixture should not be left for more than 12 hours or the concentration of polybrene should be reduced. Day 4: The medium was changed and incubated until day 5 (approximately 62 hours after leaving the infection).
  • a "de novo" mutation was found that strongly activated the natural glucokinase (GCK) gene (SEQ ID NO: 5) in a patient with severe neonatal hypoglycemia.
  • GCK glucokinase
  • a novel nonsense mutation was found (change 191 C> T in SEQ ID NO: 5 to give rise to SEQ ID NO: 6) which results in the substitution of a residue S with an F in codon 64 of the exon 3 of the amino acid sequence of the native GK (SEQ ID NO: 4, to give rise to SEQ ID NO: 1 or GCK-S64F variant).
  • the test was heterozygous for this mutation, which was not identified in the DNA of their parents.
  • This catalytic and structural activation of GCK-S64F caused this enzyme to have an insulin release threshold in response to glucose (UF-SIEG) of 0.96 mmol / l, far removed from the value of 5 mmol / l of GK- WT and close to the values of 0.8 and 0.9 mmo / l found in other mutations that strongly activated the GK identified.
  • UF-SIEG glucose
  • the markedly high glucose affinity, and the extraordinarily low UF-SIEG relative activity index found for GCK-S64F fully explain the severe hypoglycemia observed in the patient.
  • Example 3 Analysis of the effect of the GCK-V91 L variant on the proliferation and efficiency of mouse pancreatic islets.
  • Replication of these highly effective islets can be used as a new cell therapy tool in Type 1 Diabetes Mellitus.

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Abstract

The present invention relates to coding nucleotide sequences for mutated glucokinase enzymes that have greater enzymatic activity, to genetic constructions that include same, to the amino acid sequences coded by same, to pancreatic islets that express same and to the uses thereof for the treatment and/or prevention of diabetes mellitus. The pancreatic islets that express the amino acid sequences corresponding to the glucokinase enzyme variants described in the invention are highly effective, since they include pancreatic β-cells which have greater capacity for proliferation and induce a reduction in the insulin release threshold in response to glucose. In addition, said types of islets are larger than the pancreatic islets from control individuals, and are therefore useful in cell-therapy treatment of diabetes mellitus.

Description

ENZIMAS GLUCOCINASAS CON ACTIVIDAD AUMENTADA Y SU USO EN EL TRATAMIENTO Y/O PREVENCIÓN DE LA DIABETES  GLUCOCINASE ENZYMES WITH INCREASED ACTIVITY AND ITS USE IN THE TREATMENT AND / OR PREVENTION OF DIABETES
MELLITUS La presente invención se encuadra en el campo de la terapia génica y celular, específicamente se refiere a secuencias nucleotídicas codificantes para enzimas glucocinasas mutadas que presentan mayor actividad enzimática, a las secuencias aminoacídicas codificadas por ellas, a islotes pancreáticos que las expresan y a sus usos para el tratamiento y/o prevención de la diabetes mellitus.  MELLITUS The present invention falls within the field of gene and cell therapy, specifically it refers to nucleotide sequences coding for mutated glucokinase enzymes that exhibit greater enzymatic activity, to the amino acid sequences encoded by them, to pancreatic islets that express them and their uses. for the treatment and / or prevention of diabetes mellitus.
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
El trasplante de islotes pancreáticos es en la actualidad la única terapia celular disponible para el tratamiento de la diabetes mellitus tipo 1 inestable. Esta terapia ha demostrado ser eficaz en conseguir la normalización y estabilidad de la glucemia en estos pacientes, en la desaparición de hipoglucemias graves y en prevenir las complicaciones crónicas y progresivas de la diabetes. No obstante y desafortunadamente, el objetivo primario de esta terapia, la independencia insulínica a largo plazo, sigue aún sin ser alcanzado. Entre las causas más importantes por las que no se ha conseguido dicho objetivo primario están el estrés, tanto isquémico como mecánico, al que son sometidos los islotes (tan sólo un 10-20% de los islotes sobreviven al procedimiento del implante) y la baja supervivencia y funcionalidad de los islotes trasplantados, dando lugar a que menos de un 15% de los pacientes trasplantados mantengan la independencia a la insulina a los 2 años pos-trasplante (Ryan EA, et al., 2005, Diabetes, 54(7):2060-2069). Otra importante limitación que presenta esta terapia es la ausencia de suficientes órganos para realizarla en el número deseado de pacientes, ya que se necesitan alrededor de 12.000 equivalentes de islotes/Kg de peso corporal para tratar con éxito a un único paciente, lo que implica la necesidad de disponer de 2 a 3 páncreas de donantes cadáveres por paciente. Es por ello que mejorar la supervivencia y función de los islotes trasplantados, junto con poder disminuir el número de islotes necesarios para conseguir un buen control glucémico, son los principales objetivos que deben ser perseguidos y alcanzados para conseguir el objetivo primario del trasplante de islotes pancreáticos. Pancreatic islet transplantation is currently the only cell therapy available for the treatment of unstable type 1 diabetes mellitus. This therapy has proven effective in achieving normalization and stability of blood glucose in these patients, in the disappearance of severe hypoglycemias and in preventing chronic and progressive complications of diabetes. However and unfortunately, the primary objective of this therapy, the long-term insulin independence, remains to be achieved. Among the most important causes for which this primary objective has not been achieved are the stress, both ischemic and mechanical, to which the islets are subjected (only 10-20% of the islets survive the implant procedure) and low survival and functionality of transplanted islets, resulting in less than 15% of transplant patients maintaining insulin independence at 2 years post-transplant (Ryan EA, et al., 2005, Diabetes, 54 (7) : 2060-2069). Another important limitation presented by this therapy is the absence of sufficient organs to perform it in the desired number of patients, since about 12,000 equivalents of islets / kg of body weight are needed to successfully treat a single patient, which implies need to have 2 to 3 pancreas of cadaver donors per patient. That is why improving the survival and function of transplanted islets, together with being able to reduce the number of islets necessary to achieve good glycemic control, are the main objectives that must be pursued and achieved to achieve the primary goal of pancreatic islet transplantation. .
En estos momentos existen diferentes líneas de investigación encaminadas a resolver el problema de limitación de islotes pancreáticos humanos disponibles, las cuales centran sus esfuerzos en, por ejemplo, incrementar la supervivencia y proliferación de células β. Entre otras, la encapsulación de islotes (O'Sullivan ES, et al., 2010, Diabetologia), la producción de células productoras de insulina a partir de células embrionarias o progenitoras pancreáticas (Aye T, et al., 2010, J. Histochem. Cytochem. ) y la proliferación "in vitro" de las células β pancreáticas para la expansión de islotes humanos (Juhl K, et al., 2010, Curr Opin Organ Transplant., 15(1 ):79-85), son líneas en las que se está trabajando intensamente. Ciertamente, el uso del factor de crecimiento de fibroblastos o el de crecimiento del hepatocito han posibilitado, aunque de forma limitada, una expansión de los islotes humanos (Gao R, et al., 2003, Diabetes, 52(8):2007-2015). En otro estudio se ha observado que la sobreexpresión del factor de crecimiento del hepatocito humano en islotes de primates, mejora tanto el implante como la funcionalidad de estos islotes cuando son trasplantados en ratones diabéticos NOD-SCID. La combinación del péptido gastrina y el factor de crecimiento epidermal también proporciona una expansión de la masa celular β, no obstante las células obtenidas presentan una vida media corta (Suarez-Pinzon WL, et al., 2005, Diabetes, 54(9):2596-2601 ). El uso de otro péptido como el GLP-1 ("glucagon-like peptide-1 ") ha dado también resultados alentadores, y es que debido a su capacidad de promover la proliferación de la célula β e inhibir la apoptosis se ha observado que en ratones diabéticos NOD-SCID, a los que se les había trasplantado islotes humanos con células exocrinas bajo su cápsula renal, el tratamiento con GLP-1 y gastrina daba lugar a una expansión de la masa celular β, especialmente a partir de células del ducto pancreático asociadas con los islotes, mejorando la hiperglucemia de estos ratones (Suarez-Pinzon WL, Lakey JR, Rabinovitch A., 2008, Cell Transplant., 17(6):631 -640). At the moment there are different lines of research aimed at solving the problem of limitation of available human pancreatic islets, which focus their efforts on, for example, increasing the survival and proliferation of β cells. Among others, islet encapsulation (O'Sullivan ES, et al., 2010, Diabetologia), the production of insulin-producing cells from embryonic or pancreatic progenitor cells (Aye T, et al., 2010, J. Histochem Cytochem.) And "in vitro" proliferation of pancreatic β cells for the expansion of human islets (Juhl K, et al., 2010, Curr Opin Organ Transplant., 15 (1): 79-85), are lines in which you are working intensely. Certainly, the use of fibroblast growth factor or hepatocyte growth factor has allowed, although in a limited way, an expansion of human islets (Gao R, et al., 2003, Diabetes, 52 (8): 2007-2015 ). In another study, it has been observed that overexpression of the human hepatocyte growth factor in primate islets improves both the implant and the functionality of these islets when they are transplanted in NOD-SCID diabetic mice. The combination of the gastrin peptide and the epidermal growth factor also provides an expansion of the β cell mass, however the cells obtained have a short half-life (Suarez-Pinzon WL, et al., 2005, Diabetes, 54 (9): 2596-2601). The use of another peptide such as GLP-1 ("glucagon-like peptide-1") has also given encouraging results, and due to its ability to promote β cell proliferation and inhibit apoptosis, it has been observed that in NOD-SCID diabetic mice, which had been transplanted islets humans with exocrine cells under their renal capsule, treatment with GLP-1 and gastrin resulted in an expansion of β cell mass, especially from pancreatic duct cells associated with islets, improving the hyperglycemia of these mice (Suarez- Pinzon WL, Lakey JR, Rabinovitch A., 2008, Cell Transplant., 17 (6): 631-640).
Por otro lado, la enzima glucocinasa (GK), codificada por el gen glucocinasa (GCK), ejerce un extraordinario control sobre la secreción de insulina y, por lo tanto, en la homeostasis de la glucosa en el humano. Así, GK es considerada como el "sensor de la glucosa sanguínea" de la célula β pancreática, gobernando el metabolismo de la secreción de insulina estimulada por glucosa (SIEG) en dicha célula y definiendo el umbral fisiológico de dicha secreción (UF-SIEG), que en el humano es de 5 mM (Barbetti F, et al., 2009, Mol. Endocrinology., 23(12): 1983-1989). On the other hand, the enzyme glucokinase (GK), encoded by the glucokinase gene (GCK), exerts an extraordinary control over insulin secretion and, therefore, in glucose homeostasis in humans. Thus, GK is considered as the "blood glucose sensor" of the pancreatic β cell, governing the metabolism of glucose stimulated insulin secretion (SGEI) in said cell and defining the physiological threshold of said secretion (UF-SIEG) , which in humans is 5 mM (Barbetti F, et al., 2009, Mol. Endocrinology., 23 (12): 1983-1989).
Otro importante aspecto a considerar es la importante relación existente entre metabolismo y apoptosis celular, y en la que GK juega un papel clave. GK junto con BAD, PKA, PP1 y WAVE-1 forma un complejo que mantiene la proteína proapoptótica BAD en un estado de fosforilación que evita la muerte celular (Danial NN., et al., 2003, Nature, 424(6951 ):952- 956). Además, BAD posee un papel en la regulación de la secreción de insulina por la célula β, estando esta regulación mediada por GK. Another important aspect to consider is the important relationship between metabolism and cell apoptosis, and in which GK plays a key role. GK together with BAD, PKA, PP1 and WAVE-1 forms a complex that maintains the BAD proapoptotic protein in a state of phosphorylation that prevents cell death (Danial NN., Et al., 2003, Nature, 424 (6951): 952 - 956). In addition, BAD has a role in the regulation of insulin secretion by the β cell, this regulation being mediated by GK.
En resumen, es deseable poder reproducir islotes pancreáticos que, además de ser altamente eficaces en lo que se refiere a su capacidad de liberación de insulina estimulada por glucosa, eliminen la necesidad de disponer de un elevado número de islotes para poder alcanzar un adecuado control glucémico en el paciente tratado. De este modo, se podría suplir en parte la carencia de órganos que existe en la actualidad. Así mismo, sería deseable que los islotes presentasen una supervivencia mayor, permitiendo una independencia a la insulina de los pacientes trasplantados más duradera y estable. DESCRIPCIÓN DE LA INVENCIÓN In summary, it is desirable to be able to reproduce pancreatic islets that, in addition to being highly effective in terms of their ability to release glucose-stimulated insulin, eliminate the need for a large number of islets in order to achieve adequate glycemic control in the treated patient. In this way, the lack of organs that exist today can be partially replaced. Likewise, it would be desirable for the islets to have a longer survival, allowing insulin independence of the more durable and stable transplanted patients. DESCRIPTION OF THE INVENTION
La presente invención se refiere a secuencias nucleotídicas que codifican para variantes de la enzima glucocinasa (GK), preferiblemente humana, que presentan un aumento en su actividad enzimática, así como a las secuencias aminoacídicas correspondientes a dichas variantes. The present invention relates to nucleotide sequences that code for variants of the glucokinase (GK) enzyme, preferably human, that exhibit an increase in their enzymatic activity, as well as to the amino acid sequences corresponding to said variants.
La invención demuestra que la activación suprafisiológica de GK en islotes pancreáticos, preferiblemente humanos, mediada por la expresión de las variantes de la GK aquí propuestas, induce un importante aumento en la proliferación de las células β, lo que se traduce en un mayor tamaño de los islotes pancreáticos. Estos islotes además mantienen una citoarquitectura completamente conservada y bien definida. Dichas variantes también contribuyen a aumentar la estabilidad y supervivencia de las células β. Además, se consiguen obtener islotes pancreáticos con una mayor actividad metabólica, ya que éstas variantes de la GK presentan, por ejemplo, 9 veces más afinidad por la glucosa y reducen el umbral fisiológico de secreción de insulina estimulada por glucosa (UF- SIEG), pasando así de 5 mM (establecido en humanos con la GK nativa) a O,96 mM. The invention demonstrates that supraphysiological activation of GK in pancreatic islets, preferably human, mediated by the expression of the GK variants proposed herein, induces a significant increase in the proliferation of β cells, which translates into a larger size of the pancreatic islets. These islets also maintain a completely preserved and well defined cytoarchitecture. These variants also contribute to increase the stability and survival of β cells. In addition, pancreatic islets with a higher metabolic activity are obtained, since these variants of the GK have, for example, 9 times more affinity for glucose and reduce the physiological threshold of glucose-stimulated insulin secretion (UF-SIEG), going from 5 mM (established in humans with the native GK) to O, 96 mM.
Este conjunto de características otorgan al islote que expresa las variantes descritas en la invención: (i) una mayor resistencia para sobrevivir al estrés mecánico que conlleva el procedimiento de trasplante, (ii) una funcionalidad más duradera en el tiempo, (iii) capacidad de proliferación una vez trasplantados, y (iv) debido a su mayor tamaño y mayor tasa metabólica, el número necesario de este tipo de islotes a trasplantar en el paciente para poder alcanzar una glucemia adecuada es menor al que actualmente se necesita. This set of characteristics gives the islet that expresses the variants described in the invention: (i) a greater resistance to survive the mechanical stress that the transplant procedure entails, (ii) a more lasting functionality over time, (iii) ability to proliferation once transplanted, and (iv) due to its larger size and higher metabolic rate, the necessary number of this type of islets to be transplanted in the patient in order to achieve adequate blood glucose is less than what is currently needed.
Por ello, las secuencias nucleotídicas y aminoacídicas descritas en la presente invención son de utilidad para la creación de islotes pancreáticos de alta eficacia que pueden ser trasplantados a pacientes, durante procedimientos de terapia celular somática, para el tratamiento y/o prevención de la diabetes mellitus, con las ventajas asociadas anteriormente descritas. Therefore, the nucleotide and amino acid sequences described in the present invention are useful for the creation of pancreatic islets. of high efficacy that can be transplanted to patients, during somatic cell therapy procedures, for the treatment and / or prevention of diabetes mellitus, with the associated advantages described above.
Por tanto, un primer aspecto de la invención se refiere a una secuencia nucleotídica aislada, de ahora en adelante "secuencia nucleotídica de la invención", que codifica para una secuencia aminoacídica de la enzima glucocinasa o GK que presenta al menos una mutación activadora de la actividad de dicha enzima. Therefore, a first aspect of the invention relates to an isolated nucleotide sequence, hereafter referred to as the "nucleotide sequence of the invention", which encodes an amino acid sequence of the glucokinase or GK enzyme that has at least one activating mutation of the activity of said enzyme.
Los términos "secuencia nucleotídica", "secuencia de nucleótidos", "ácido nucleico", "oligonucleótido" y "polinucleótido" se usan aquí de manera intercambiable y se refieren a una forma polimérica de nucleótidos de cualquier longitud que pueden estar o no, química o bioquímicamente modificados. Se refieren, por tanto, a cualquier polirribonucleótido o polidesoxirribonucleótido, tanto de cadena sencilla como de doble hebra. The terms "nucleotide sequence", "nucleotide sequence", "nucleic acid", "oligonucleotide" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length that may or may not be chemical or biochemically modified. They refer, therefore, to any polyiribonucleotide or polydeoxyribonucleotide, both single-stranded and double-stranded.
Debido a la degeneración del código genético, en el cual diversos tripletes de nucleótidos dan lugar a un mismo aminoácido, existen diversas secuencias de nucleótidos que dan lugar a una misma secuencia aminoacídica. Due to the degeneracy of the genetic code, in which several nucleotide triplets give rise to the same amino acid, there are several nucleotide sequences that give rise to the same amino acid sequence.
El polinucleótido de la invención puede obtenerse de manera artificial mediante métodos convencionales de clonación y selección, o mediante secuenciación. The polynucleotide of the invention can be obtained artificially by conventional methods of cloning and selection, or by sequencing.
El polinucleótido, adicionalmente a la secuencia codificante, puede llevar otros elementos, como por ejemplo aunque sin limitarse, intrones, secuencias no codificantes en los extremos 5' o 3', sitios de unión a ribosomas, o secuencias estabilizadoras. Estos polinucleótidos adicionalmente pueden incluir secuencias codificantes para aminoácidos adicionales que puedan ser útiles, por ejemplo, aunque sin limitarse, para aumentar la estabilidad del péptido generado a partir de él o permitir una mejor purificación del mismo. La "enzima glucocinasa" o "GK" es una hexoquinasa isozima que interviene en la fosforilación de la glucosa para dar lugar a glucosa-e- fosfato. En humanos, al igual que en la mayoría de los vertebrados, se expresa en células del hígado, páncreas, intestino y cerebro, órganos en los cuales juega un importante papel en la regulación del metabolismo de carbohidratos actuando como un sensor de glucosa, provocando cambios en el metabolismo o función de la célula en respuesta al aumento o disminución de los niveles de glucosa. The polynucleotide, in addition to the coding sequence, can carry other elements, such as, but not limited to, introns, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, or stabilizing sequences. These polynucleotides may additionally include coding sequences for amino acids. additional that may be useful, for example, but not limited to increase the stability of the peptide generated from it or allow a better purification thereof. The "glucokinase enzyme" or "GK" is an isozyme hexokinase that is involved in the phosphorylation of glucose to give rise to glucose-e-phosphate. In humans, as in most vertebrates, it is expressed in cells of the liver, pancreas, intestine and brain, organs in which it plays an important role in the regulation of carbohydrate metabolism acting as a glucose sensor, causing changes in the metabolism or function of the cell in response to the increase or decrease of glucose levels.
En la presente invención la "secuencia aminoacídica de la enzima GK" es, preferiblemente, la secuencia aminoacídica de la GK nativa humana (SEQ ID NO: 4) codificada, por ejemplo, aunque sin limitarnos, por la secuencia nucleotídica SEQ ID NO: 5 (gen GCK humano), aunque también se encuentran dentro del ámbito de la presente invención las secuencias aminoacídicas de enzimas GK ortólogas a la GK humana procedentes de otros organismos y que cumplen la misma función que la GK humana en el organismo del que proceden, así como las secuencias nucleotídicas que codifican para éstas. In the present invention the "amino acid sequence of the GK enzyme" is preferably the amino acid sequence of the human native GK (SEQ ID NO: 4) encoded, for example, but not limited to, by the nucleotide sequence SEQ ID NO: 5 (human GCK gene), although the amino acid sequences of GK enzymes orthologous to human GK from other organisms and that fulfill the same function as human GK in the organism from which they are derived, are also within the scope of the present invention. as the nucleotide sequences that code for them.
La expresión "mutaciones activadoras de la actividad de la enzima" se refiere a aquellas mutaciones presentes en la secuencia aminoacídica y/o nucleotídica de la GK que dan lugar a una enzima que presenta una mayor actividad enzimática en comparación con la enzima nativa que no presenta dichas mutaciones. Dichas mutaciones pueden ser, aunque sin limitarnos, mutaciones puntuales (de cambio de uno o varios aminoácidos o uno o varios nucleótidos por otro/s), mutaciones de deleción, de sustitución, de adición, etc. La identificación y selección de enzimas que presentan una mayor actividad enzimática, obtenidas mediante la inducción de tales mutaciones, se puede llevar a cabo mediante ensayos de actividad enzimática en presencia de sustrato (en este caso glucosa) conocidos por el experto en la materia, los cuales permiten seleccionar las variantes enzimáticas modificadas de interés que presentan mayor actividad, menor inhibición por sustrato, mayor estabilidad enzimática, mayor rango de pH y/o Ta a la que la enzima es activa, etc. En la presente invención se utiliza el término "variantes de la invención" para hacer referencia a las enzimas GK que presentan al menos una mutación activadora de la actividad de dicha enzima. The expression "activating mutations of the enzyme activity" refers to those mutations present in the amino acid and / or nucleotide sequence of the GK that give rise to an enzyme that has a higher enzyme activity compared to the native enzyme that does not have these mutations. Such mutations may be, but are not limited to, point mutations (changing one or several amino acids or one or several nucleotides for another / s), deletion, substitution, addition, etc. mutations. The identification and selection of enzymes that have a higher enzyme activity, obtained by means of Induction of such mutations, can be carried out by enzymatic activity tests in the presence of substrate (in this case glucose) known by the person skilled in the art, which allow to select the modified enzyme variants of interest that have greater activity, less inhibition per substrate, greater enzymatic stability, greater range of pH and / or T at which the enzyme is active, etc. In the present invention the term "variants of the invention" is used to refer to GK enzymes that have at least one activating mutation of the activity of said enzyme.
La secuencia nucleotídica de la invención puede ser usada para la obtención de islotes pancreáticos de alta eficacia, mediante su introducción, por métodos de biología molecular conocidos por un experto en la materia, en una célula, preferiblemente pancreática, más preferiblemente en una célula β pancreática, aun más preferiblemente aislada del propio paciente, para que ésta exprese la secuencia aminoacídica de alguna de las variantes de la invención, y se obtenga así "in vitro" una población celular de células β autóloga altamente proliferativa y altamente eficiente en la liberación de insulina en respuesta a glucosa. Por ello, en una realización preferida, la secuencia nucleotídica de la invención es capaz de generar islotes pancreáticos de alta eficacia. Estos "islotes pancreáticos de alta eficacia", aquí denominados también "islotes de la invención" como se verá más adelante, comprenden células pancreáticas modificadas como se ha explicado en este párrafo, preferiblemente células β pancreáticas, las cuales presentan una mayor capacidad proliferativa e inducen una reducción del UF-SIEG, por lo que comienzan a liberar insulina a concentraciones más bajas de glucosa que los islotes que no comprenden células así modificadas. Además, este tipo de islotes presentan un mayor tamaño que los islotes pancreáticos que no comprenden células a las que se les ha introducido la secuencia nucleotídica de la invención. En otra realización preferida, la secuencia aminoacídica de la enzima glucocinasa que presenta al menos una mutación activadora de la actividad de dicha enzima, o "secuencia aminoacídica de la variante de la invención", se selecciona de la lista que consiste en: SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3. La SEQ ID NO: 1 corresponde a la SEQ ID NO: 4 donde el residuo de la posición 64 (S, serina) ha sido sustituido por una fenilalanina (F), a dicha variante se hará referencia también como GCK- S64F. La SEQ ID NO: 2 corresponde a la SEQ ID NO: 4 donde el residuo de la posición 91 (V, valina) ha sido sustituido por una leucina (L), a dicha variante se hará referencia también como GCK-V91 L. La SEQ ID NO: 3 corresponde a la SEQ ID NO: 4 donde el residuo de la posición 214 (Y, tirosina) ha sido sustituido por una cisteína (C), a dicha variante se hará referencia también como GCK-Y214C. Un ejemplo de secuencia nucleotídica que codifica para la SEQ ID NO: 1 es, aunque sin limitarnos, la SEQ ID NO: 6. The nucleotide sequence of the invention can be used to obtain high-efficiency pancreatic islets, by their introduction, by molecular biology methods known to a person skilled in the art, in a cell, preferably pancreatic, more preferably in a pancreatic β cell , even more preferably isolated from the patient, so that it expresses the amino acid sequence of any of the variants of the invention, and thus a "proliferative autologous β cell cell population is obtained" in vitro "and highly efficient in insulin release in response to glucose. Therefore, in a preferred embodiment, the nucleotide sequence of the invention is capable of generating high-efficiency pancreatic islets. These "high efficiency pancreatic islets," here also referred to as "islets of the invention" as will be seen below, comprise modified pancreatic cells as explained in this paragraph, preferably pancreatic β cells, which have a greater proliferative capacity and induce a reduction in UF-SIEG, so they begin to release insulin at lower glucose concentrations than islets that do not comprise cells so modified. In addition, this type of islets are larger than pancreatic islets that do not comprise cells to which the nucleotide sequence of the invention has been introduced. In another preferred embodiment, the amino acid sequence of the glucokinase enzyme having at least one activating mutation of the activity of said enzyme, or "amino acid sequence of the variant of the invention", is selected from the list consisting of: SEQ ID NO : 1, SEQ ID NO: 2 or SEQ ID NO: 3. SEQ ID NO: 1 corresponds to SEQ ID NO: 4 where the residue from position 64 (S, serine) has been replaced by a phenylalanine (F) , said variant will also be referred to as GCK-S64F. SEQ ID NO: 2 corresponds to SEQ ID NO: 4 where the residue from position 91 (V, valine) has been replaced by a leucine (L), this variant will also be referred to as GCK-V91 L. SEQ ID NO: 3 corresponds to SEQ ID NO: 4 where the residue of position 214 (Y, tyrosine) has been replaced by a cysteine (C), said variant will also be referred to as GCK-Y214C. An example of a nucleotide sequence encoding SEQ ID NO: 1 is, but not limited to, SEQ ID NO: 6.
Por ejemplo, las secuencias nucleotídicas que codifican para la SEQ ID NO: 2 y la SEQ ID NO: 3 se pueden obtener, aunque sin limitarnos, mediante un método que comprende la introducción de mutaciones en el gen de la GK humana (SEQ ID NO: 5) mediante PCR con los cebadores SEQ ID NO: 7 y SEQ ID NO: 8 (para obtener la secuencia nucleotídica que codifica para la SEQ ID NO: 3) y SEQ ID NO: 9 y SEQ ID NO: 10 (para obtener la secuencia nucleotídica que codifica para la SEQ ID NO: 2). Otro aspecto de la invención se refiere, por tanto, a las secuencias nucleotídicas aisladas obtenidas según el procedimiento descrito en este párrafo, las cuales codifican para secuencias aminoacídicas de la enzima GK que presentan al menos una mutación activadora de la actividad de dicha enzima, concretamente para la SEQ ID NO: 2 y SEQ ID NO: 3. Otro aspecto de la invención se refiere a una construcción genética, de ahora en adelante "construcción genética de la invención", que comprende la secuencia nucleotídica de la invención. La construcción genética de la invención puede llevar incluidas secuencias de control unidas operativamente a la secuencia nucleotídica de la invención. El término "secuencia de control", tal y como se utiliza en la presente descripción, se refiere a secuencias nucleotídicas que son necesarias para efectuar la expresión de las secuencias a las que están ligadas. Se pretende que el término "secuencias de control" incluya, como mínimo, todos los componentes cuya presencia es necesaria para la expresión, y también puede incluir componentes adicionales cuya presencia sea ventajosa. Ejemplos de secuencias de control son, pero sin limitarse, promotores, señales de inicio de la transcripción, señales de terminación de la transcripción, señales de poliadenilación o activadores transcripcionales. For example, the nucleotide sequences encoding SEQ ID NO: 2 and SEQ ID NO: 3 can be obtained, but not limited to, by a method comprising introducing mutations into the human GK gene (SEQ ID NO : 5) by PCR with primers SEQ ID NO: 7 and SEQ ID NO: 8 (to obtain the nucleotide sequence encoding SEQ ID NO: 3) and SEQ ID NO: 9 and SEQ ID NO: 10 (to obtain the nucleotide sequence encoding SEQ ID NO: 2). Another aspect of the invention thus relates to the isolated nucleotide sequences obtained according to the procedure described in this paragraph, which code for amino acid sequences of the GK enzyme that have at least one activating mutation of the activity of said enzyme, specifically for SEQ ID NO: 2 and SEQ ID NO: 3. Another aspect of the invention relates to a genetic construct, hereafter "genetic construct of the invention", which comprises the nucleotide sequence of the invention. The genetic construction of the invention can include control sequences operatively linked to the nucleotide sequence of the invention. The term "control sequence", as used herein, refers to nucleotide sequences that are necessary to effect the expression of the sequences to which they are linked. The term "control sequences" is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous. Examples of control sequences are, but are not limited to, promoters, transcription initiation signals, transcription termination signals, polyadenylation signals or transcriptional activators.
La expresión "unidos operativamente", tal y como se utiliza en la presente descripción, se refiere a una yuxtaposición en la que los componentes así descritos tienen una relación que les permite funcionar en la manera intencionada. Una secuencia de control "unida de forma operativa" a un polinucleótido, está ligada de tal manera que la expresión de la secuencia codificadora se consigue en condiciones compatibles con las secuencias de control. The term "operably linked", as used in the present description, refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended manner. A control sequence "operably linked" to a polynucleotide is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.
Como se usa aquí, el término "promotor" hace referencia a una región del ADN, generalmente "aguas arriba" o "upstream" del punto de inicio de la transcripción, que es capaz de iniciar la transcripción en una célula. Este término incluye, por ejemplo, pero sin limitarse, promotores constitutivos, promotores específicos de tipo celular o de tejido o promotores inducibles o reprimibles. Las secuencias de control dependen del origen de la célula en la que se quiere expresar el ácido nucleico. Ejemplos de promotores procariotas incluyen, por ejemplo, pero sin limitarnos, los promotores de los genes trp, recA, lacZ, lacl, tet, gal, trc, o tac de E. coli, o el promotor del gen α-amilasa de B. subtilis. Para la expresión de un ácido nucleico en una célula procariota también es necesaria la presencia de un sitio de unión ribosomal situado "upstream" de la secuencia codificante. Secuencias de control apropiadas para la expresión de un polinucleótido en células eucariotas son conocidas en el estado de la técnica. As used herein, the term "promoter" refers to a region of DNA, generally "upstream" or "upstream" of the transcription start point, which is capable of initiating transcription in a cell. This term includes, for example, but not limited to, constitutive promoters, cell or tissue specific promoters or inducible or repressible promoters. Control sequences depend on the origin of the cell in which the nucleic acid is to be expressed. Examples of promoters Prokaryotes include, for example, but not limited to, promoters of the trp, recA, lacZ, lacl, tet, gal, trc, or tac genes of E. coli, or the promoter of the B. subtilis α-amylase gene. For the expression of a nucleic acid in a prokaryotic cell, the presence of an upstream ribosomal binding site of the coding sequence is also necessary. Appropriate control sequences for the expression of a polynucleotide in eukaryotic cells are known in the state of the art.
En una realización preferida, la construcción genética de la invención es un vector de expresión, de ahora en adelante "vector de la invención". El término "vector de expresión" se refiere a un fragmento de ADN o ARN que tiene la capacidad de replicarse en un determinado huésped y puede servir de vehículo para llevar a cabo la transcripción de una secuencia de interés que haya sido insertada en el mismo. El vector puede ser un plásmido, un cósmido, un fagémido, cromosomas artificiales de levaduras (YAC), cromosomas artificiales de bacterias (BAC), cromosomas humanos artificiales (HAC), un bacteriófago o un vector viral tal como por ejemplo, aunque sin limitarnos, adenovirus, retrovirus, virus adenoasociados, lentivirus, poxvirus o herpesvirus, sin excluir otro tipo de vectores que se correspondan con la definición realizada de vector. Los "lentivirus" son retrovirus no oncogénicos caracterizados por presentar largos tiempos de incubación e infección persistente, son capaces de infectar a todo tipo de células, tanto quiescentes como en división, permiten insertos de tamaño medio y se integran en el genoma de la célula huésped dando lugar a una expresión a largo plazo, sin ser inmunogénicos. Por ello, en una realización más preferida, el vector de la invención es un lentivirus, es decir, un vector lentiviral. In a preferred embodiment, the genetic construct of the invention is an expression vector, hereinafter "vector of the invention". The term "expression vector" refers to a DNA or RNA fragment that has the ability to replicate in a given host and can serve as a vehicle for carrying out the transcription of a sequence of interest that has been inserted therein. The vector can be a plasmid, a cosmid, a phagemid, artificial yeast chromosomes (YAC), artificial bacterial chromosomes (BAC), artificial human chromosomes (HAC), a bacteriophage or a viral vector such as, for example, but not limited to , adenovirus, retrovirus, adeno-associated virus, lentivirus, poxvirus or herpesvirus, without excluding other types of vectors that correspond to the definition made of vector. The "lentiviruses" are non-oncogenic retroviruses characterized by having long incubation times and persistent infection, they are capable of infecting all types of cells, both quiescent and in division, allow medium-sized inserts and integrate into the genome of the host cell leading to long-term expression, without being immunogenic. Therefore, in a more preferred embodiment, the vector of the invention is a lentivirus, that is, a lentiviral vector.
Otro aspecto de la invención se refiere a una secuencia aminoacídica de la enzima glucocinasa seleccionada de la lista que consiste en: SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3. Como ya se ha mencionado, la secuencia nucleotídica de la invención o la construcción genética de la invención pueden ser usadas para la obtención de islotes pancreáticos de alta eficacia, mediante su introducción, por métodos de biología molecular conocidos por un experto en la materia, en una célula, preferiblemente pancreática, más preferiblemente en una célula β pancreática, aun más preferiblemente aislada del propio paciente, para que ésta exprese la secuencia aminoacídica de alguna de las variantes de la invención, y se obtenga así "in vitro" una población celular de células β autóloga altamente proliferativa y altamente eficiente en la liberación de insulina en respuesta a glucosa. Por ello, otro aspecto de la invención se refiere a una célula, de ahora en adelante "célula de la invención", que comprende, de forma transitoria o, preferiblemente, de forma estable, la secuencia nucleotídica de la invención, la construcción genética de la invención, el vector de la invención, o una secuencia aminoacídica de alguna de las variantes de la invención, más preferiblemente una secuencia aminoacídica seleccionada de la lista que consiste en: SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3. Another aspect of the invention relates to an amino acid sequence of the glucokinase enzyme selected from the list consisting of: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. As already mentioned, the nucleotide sequence of the invention or the genetic construction of the invention can be used to obtain high-efficiency pancreatic islets, by introduction, by molecular biology methods known to a person skilled in the art, in a cell, preferably pancreatic, more preferably in a pancreatic β cell, even more preferably isolated from the patient, so that it expresses the amino acid sequence of any of the variants of the invention, and thus a "population in vitro" of a cell population is obtained. Highly proliferative and highly efficient autologous β cells in insulin release in response to glucose. Therefore, another aspect of the invention relates to a cell, hereafter referred to as "cell of the invention", which comprises, temporarily or preferably, stably, the nucleotide sequence of the invention, the genetic construction of the invention, the vector of the invention, or an amino acid sequence of any of the variants of the invention, more preferably an amino acid sequence selected from the list consisting of: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO : 3.
La célula de la invención puede ser cualquier célula eucariota o procariota, aunque preferiblemente es una célula eucariota. En otra realización preferida, la célula de la invención es una célula pancreática. La "célula pancreática" de la invención puede ser una célula alfa, una célula beta, una célula delta, una célula G o una célula F, aunque preferiblemente es una célula beta (célula β). The cell of the invention can be any eukaryotic or prokaryotic cell, although preferably it is a eukaryotic cell. In another preferred embodiment, the cell of the invention is a pancreatic cell. The "pancreatic cell" of the invention may be an alpha cell, a beta cell, a delta cell, a G cell or an F cell, although preferably it is a beta cell (β cell).
Además, la célula de la invención puede ser tanto de origen autólogo, como alogénico o xenogénico. La posibilidad de que la célula de la invención sea de origen autólogo permite que el posterior trasplante de ésta, tras su manipulación "in vitro", para el tratamiento y/o prevención de la diabetes mellitus pueda realizarse sin que sea necesaria la inmunosupresión del sujeto trasplantado. Por ello, en una realización más preferida, la célula de la invención es de origen autólogo. Se entiende por "origen autólogo" cualquier procedencia de la muestra, tomada de los tejidos o células de un individuo o paciente, que es la misma en un donante y en el receptor de los mismos cuando le son administrados tras su tratamiento o trasplantados tras su modificación. La célula de la invención puede proceder de cualquier mamífero, aunque preferiblemente procede de un humano. Por ello, en una realización aun más preferida, la célula de la invención es de origen humano. In addition, the cell of the invention can be both autologous, as allogeneic or xenogenic. The possibility that the cell of the invention is of autologous origin allows its subsequent transplantation, after "in vitro" manipulation, for the treatment and / or prevention of diabetes mellitus can be performed without the immunosuppression of the subject being necessary. transplanted Therefore, in a more preferred embodiment, the cell of the invention is of autologous origin. It is understood by "autologous origin" any origin of the sample, taken from the tissues or cells of an individual or patient, which is the same in a donor and the recipient thereof when they are administered after treatment or transplanted after modification. The cell of the invention can come from any mammal, although preferably it comes from a human. Therefore, in an even more preferred embodiment, the cell of the invention is of human origin.
Otro aspecto de la invención se refiere a un islote pancreático, "islote de la invención", que comprende la célula de la invención. En una realización preferida, el islote de la invención es de origen autólogo. En una realización más preferida, dicho islote es de origen humano. Another aspect of the invention relates to a pancreatic islet, "islet of the invention", which comprises the cell of the invention. In a preferred embodiment, the islet of the invention is of autologous origin. In a more preferred embodiment, said islet is of human origin.
Los "islotes pancreáticos" son acúmulos de células que se encargan de producir hormonas como la insulina y el glucagón, con función netamente endocrina. También secretan inmunoglobulinas. Forman diversos racimos o islotes dispersos por todo el páncreas, encontrándose alrededor de un millón de tales islotes en el páncreas humano. Están formados principalmente por células β, secretoras de insulina, alrededor de las cuales se encuentran en grupos pequeños células de mayor tamaño como las células a, secretoras de glucagón, δ, secretoras de somatostatina, F, productoras de un polipéptido pancreático que inhibe las secreciones exocrinas del páncreas, o G, secretoras de gastrina. Otro aspecto de la invención se refiere a una composición, de ahora en adelante "composición de la invención", que comprende la secuencia nucleotídica de la invención, la construcción genética de la invención, el vector de la invención, la secuencia aminoacídica de las variantes de la invención, preferiblemente la SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3, la célula de la invención o el islote de la invención. En otra realización preferida, la composición de la invención es una composición farmacéutica. En una realización más preferida, la composición de la invención además comprende un vehículo farmacéuticamente aceptable. En una realización aun más preferida, la composición de la invención además comprende otro principio activo. El "vehículo farmacéuticamente aceptable" o portador, es preferiblemente una sustancia inerte. La función del vehículo es facilitar la incorporación de otros compuestos, permitir una mejor dosificación y administración o dar consistencia y forma a la composición farmacéutica. Por tanto, el vehículo es una sustancia que se emplea en la composición para diluir cualquiera de los componentes de dicha composición farmacéutica de la presente invención hasta un volumen o peso determinado; o bien que aún sin diluir dichos componentes es capaz de permitir una mejor dosificación y administración o dar consistencia y forma a la composición. Por otra parte, la composición de la invención comprende opcionalmente otra sustancia o principio activo. Además del requerimiento de la eficacia terapéutica, donde dicha composición farmacéutica puede necesitar el uso de otros agentes terapéuticos, pueden existir razones fundamentales adicionales que obligan o recomiendan en gran medida el uso de una combinación de un compuesto de la invención y otro agente terapéutico. El término "principio activo", "sustancia activa" o "ingrediente activo" es toda materia, cualquiera que sea su origen, humano, animal, vegetal, químico o de otro tipo, a la que se atribuye una actividad apropiada para constituir un medicamento. "Pancreatic islets" are clusters of cells that are responsible for producing hormones such as insulin and glucagon, with a purely endocrine function. They also secrete immunoglobulins. They form various clusters or islets scattered throughout the pancreas, with about one million such islets found in the human pancreas. They consist mainly of β cells, insulin secretors, around which larger cells are found in small groups such as a cells, glucagon secretors, δ, somatostatin secretors, F, producing a pancreatic polypeptide that inhibits secretions exocrines of the pancreas, or G, secretors of gastrin. Another aspect of the invention relates to a composition, hereinafter "composition of the invention", which comprises the nucleotide sequence of the invention, the genetic construction of the invention, the vector of the invention, the amino acid sequence of the variants of the invention, preferably SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, the cell of the invention or the islet of the invention. In another preferred embodiment, the composition of the invention is a pharmaceutical composition. In a more preferred embodiment, the composition of the The invention further comprises a pharmaceutically acceptable carrier. In an even more preferred embodiment, the composition of the invention further comprises another active ingredient. The "pharmaceutically acceptable carrier" or carrier is preferably an inert substance. The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition. Therefore, the carrier is a substance that is used in the composition to dilute any of the components of said pharmaceutical composition of the present invention to a given volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the composition. On the other hand, the composition of the invention optionally comprises another active substance or principle. In addition to the requirement of therapeutic efficacy, where said pharmaceutical composition may require the use of other therapeutic agents, there may be additional fundamental reasons that compel or strongly recommend the use of a combination of a compound of the invention and another therapeutic agent. The term "active substance", "active substance" or "active ingredient" is any matter, whatever its origin, human, animal, plant, chemical or other, to which an appropriate activity is attributed to constitute a medicine .
En cada caso la forma de presentación de la composición de la invención se adaptará al tipo de administración utilizada, por ello, la composición de la presente invención se puede presentar bajo la forma de soluciones o cualquier otra forma de administración clínicamente permitida y en una cantidad terapéuticamente efectiva. La composición descrita en la invención se puede formular en formas sólidas, semisólidas, líquidas o gaseosas, tales como comprimido, cápsula, polvo, gránulo, ungüento, solución, supositorio, inyección, inhalante, gel, microesfera o aerosol, para su administración oral, tópica o parenteral. Por otra parte, la composición de la invención también puede ser formulada en forma de liposomas o nanosferas, de formulaciones de liberación sostenida o de cualquier otro sistema convencional de liberación. In each case the form of presentation of the composition of the invention will be adapted to the type of administration used, therefore, the composition of the present invention can be presented in the form of solutions or any other form of clinically permitted administration and in an amount therapeutically effective. The composition described in the invention can be formulated in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, gel, microsphere or aerosol, for oral, topical or parenteral administration. On the other hand, the composition of the invention can also be formulated in the form of liposomes or nanospheres, sustained release formulations or any other conventional release system.
Como se ha explicado anteriormente, los islotes pancreáticos de la invención son islotes de alta eficacia, en el sentido de que presentan un mayor tamaño que los islotes pancreáticos que no comprenden células de la invención, comprenden células, preferiblemente pancreáticas, más preferiblemente células β pancreáticas, con una mayor capacidad proliferativa y que inducen una reducción del UF-SIEG, por lo que comienzan a liberar insulina a concentraciones más bajas de glucosa que los islotes que no comprenden células de la invención. Así, tanto las células como los islotes de la invención son de utilidad en el tratamiento y/o prevención de la diabetes mellitus, mediante su administración como medicamento o bien mediante su trasplante al páncreas de un individuo que padezca diabetes mellitus, todo ello con el fin de incrementar, restaurar o sustituir parcial o totalmente la actividad funcional de las células β secretoras de insulina presentes en el páncreas del individuo afectado por la enfermedad. Las ventajas asociadas a dicha aplicación clínica son las que se han descrito anteriormente, a saber, dichas células e islotes de la invención presentan una mayor resistencia para sobrevivir al estrés mecánico que conlleva el procedimiento de trasplante, una funcionalidad más duradera en el tiempo, capacidad de proliferación una vez trasplantados, y debido a su mayor tamaño y mayor tasa metabólica, el número necesario de este tipo de células o islotes a trasplantar en el paciente para poder alcanzar una glucemia adecuada es menor al que actualmente se necesita. As explained above, the pancreatic islets of the invention are highly effective islets, in that they are larger than pancreatic islets that do not comprise cells of the invention, comprise cells, preferably pancreatic, more preferably pancreatic β cells , with a greater proliferative capacity and that induce a reduction of UF-SIEG, so they begin to release insulin at lower glucose concentrations than islets that do not comprise cells of the invention. Thus, both the cells and the islets of the invention are useful in the treatment and / or prevention of diabetes mellitus, by administration as a medicine or by transplantation to the pancreas of an individual suffering from diabetes mellitus, all with the in order to increase, restore or partially or totally replace the functional activity of insulin secreting β cells present in the pancreas of the individual affected by the disease. The advantages associated with said clinical application are those described above, namely, said cells and islets of the invention have a greater resistance to survive the mechanical stress involved in the transplant procedure, a more lasting functionality over time, capacity proliferation once transplanted, and due to its larger size and higher metabolic rate, the necessary number of this type of cells or islets to be transplanted in the patient in order to achieve adequate blood glucose is less than what is currently needed.
Así mismo, la secuencia nucleotídica de la invención, la construcción genética de la invención, el vector de la invención, la composición de la invención o la secuencia aminoacídica de las variantes de la invención, pueden ser administrados como medicamento a un individuo para el tratamiento y/o prevención de la diabetes mellitus. Por todo ello, otro aspecto de la invención se refiere al uso de la secuencia nucleotídica de la invención, de la construcción genética de la invención, del vector de la invención, de la secuencia aminoacídica de las variantes de la invención, preferiblemente de la SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3, de la célula de la invención, del islote de la invención o de la composición de la invención, para la elaboración de un medicamento, de ahora en adelante "medicamento de la invención". O alternativamente, a la secuencia nucleotídica de la invención, a la construcción genética de la invención, al vector de la invención, a la secuencia aminoacídica de las variantes de la invención, preferiblemente la SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3, a la célula de la invención, al islote de la invención o a la composición de la invención, para su uso como medicamento. En una realización más preferida, el medicamento es para el tratamiento y/o prevención de la diabetes mellitus. Likewise, the nucleotide sequence of the invention, the genetic construction of the invention, the vector of the invention, the composition of the The invention or the amino acid sequence of the variants of the invention can be administered as a medicine to an individual for the treatment and / or prevention of diabetes mellitus. Therefore, another aspect of the invention relates to the use of the nucleotide sequence of the invention, of the genetic construction of the invention, of the vector of the invention, of the amino acid sequence of the variants of the invention, preferably of SEQ. ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, of the cell of the invention, of the islet of the invention or of the composition of the invention, for the preparation of a medicament, hereinafter "medicament of the invention ". Or alternatively, to the nucleotide sequence of the invention, to the genetic construction of the invention, to the vector of the invention, to the amino acid sequence of the variants of the invention, preferably SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, to the cell of the invention, to the islet of the invention or to the composition of the invention, for use as a medicament. In a more preferred embodiment, the medicament is for the treatment and / or prevention of diabetes mellitus.
El término "diabetes mellitus" se refiere a la enfermedad crónica que cursa con altos niveles de azúcar en sangre y que puede ser causada por una escasa producción de insulina, resistencia a ésta o ambas. Esta patología se puede detectar por métodos diagnósticos conocidos por los expertos en la materia tales como, por ejemplo, aunque sin limitarnos, análisis de orina, test de glucemia en ayunas, análisis de la hemoglobina A1c, prueba de tolerancia a la glucosa oral o análisis de glucosa en sangre. Se engloban dentro de este término, aunque sin limitarnos, la diabetes gestacional, el síndrome metabólico, la diabetes tipo 1 y la diabetes tipo 2. En una realización aun más preferida, el medicamento es para el tratamiento y/o prevención de la diabetes mellitus de tipo 1 o insulinodependiente, la cual se caracteriza porque el páncreas no produce o produce poca insulina, lo que hace necesaria la inyección diaria de esta hormona. The term "diabetes mellitus" refers to the chronic disease that has high blood sugar levels and can be caused by low insulin production, resistance to insulin or both. This pathology can be detected by diagnostic methods known to those skilled in the art such as, for example, but not limited to, urinalysis, fasting blood glucose test, hemoglobin A1c analysis, oral glucose tolerance test or analysis. of blood glucose. This term includes, but is not limited to, gestational diabetes, metabolic syndrome, type 1 diabetes and type 2 diabetes. In an even more preferred embodiment, the medication is for the treatment and / or prevention of diabetes mellitus. type 1 or insulin dependent, which is characterized in that the pancreas does not produce or produces little insulin, which makes the daily injection of this hormone necessary.
El término "tratamiento" tal como se entiende en la presente invención se refiere a combatir los efectos causados como consecuencia de una enfermedad o condición patológica de interés en un sujeto (preferiblemente mamífero, y más preferiblemente un humano) que incluye: The term "treatment" as understood in the present invention refers to combating the effects caused as a result of a disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:
(i) inhibir la enfermedad o condición patológica, es decir, detener su desarrollo;  (i) inhibit the disease or pathological condition, that is, stop its development;
(ii) aliviar la enfermedad o la condición patológica, es decir, causar la regresión de la enfermedad o la condición patológica o su sintomatología; (ii) alleviate the disease or the pathological condition, that is, cause the regression of the disease or the pathological condition or its symptomatology;
(iii) estabilizar la enfermedad o la condición patológica. El término "prevención" tal como se entiende en la presente invención consiste en evitar la aparición de la enfermedad, es decir, evitar que se produzca la enfermedad o la condición patológica en un sujeto (preferiblemente mamífero, y más preferiblemente un humano), en particular, cuando dicho sujeto tiene predisposición por la condición patológica. (iii) stabilize the disease or pathological condition. The term "prevention" as understood in the present invention consists in preventing the onset of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably mammal, and more preferably a human), in particularly, when said subject has a predisposition for the pathological condition.
El medicamento al que se refiere la presente invención puede ser de uso humano o veterinario. El "medicamento de uso humano" es toda sustancia o combinación de sustancias que se presente como poseedora de propiedades para el tratamiento o prevención de enfermedades en seres humanos o que pueda usarse en seres humanos o administrarse a seres humanos con el fin de restaurar, corregir o modificar las funciones fisiológicas ejerciendo una acción farmacológica, inmunológica o metabólica, o de establecer un diagnóstico médico. El "medicamento de uso veterinario" es toda sustancia o combinación de sustancias que se presente como poseedora de propiedades curativas o preventivas con respecto a las enfermedades animales o que pueda administrarse al animal con el fin de restablecer, corregir o modificar sus funciones fisiológicas ejerciendo una acción farmacológica, inmunológica o metabólica, o de establecer un diagnóstico veterinario. También se considerarán "medicamentos veterinarios" las "premezclas para piensos medicamentosos" elaboradas para ser incorporadas a un pienso. The medicament referred to in the present invention can be for human or veterinary use. The "medicine for human use" is any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in humans or that can be used in humans or administered to humans in order to restore, correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action, or establishing a medical diagnosis. The "veterinary medicinal product" is any substance or combination of substances that is presented as having curative or preventive properties with respect to animal diseases or that can be administered at animal in order to restore, correct or modify its physiological functions exerting a pharmacological, immunological or metabolic action, or to establish a veterinary diagnosis. "Premixes for medicated feed" prepared to be incorporated into a feed will also be considered "veterinary medicinal products".
En una realización aun más preferida, el medicamento de la invención es un medicamento de terapia celular somática. Se entiende por "terapia celular somática" la utilización de células somáticas vivas, tanto autólogas (procedentes del propio paciente), como alogénicas (de otro ser humano) o xenogénicas (de animales), cuyas características biológicas han sido alteradas sustancialmente como resultado de su manipulación, para obtener un efecto terapéutico, de diagnóstico o preventivo, por medios metabólicos, farmacológicos o inmunológicos. Entre los medicamentos de terapia celular somática se encuentran, por ejemplo, pero sin limitarse: células manipuladas para modificar sus propiedades inmunológicas, metabólicas o funcionales de otro tipo en aspectos cualitativos o cuantitativos; células clasificadas, seleccionadas y manipuladas, que se someten posteriormente a un proceso de fabricación con el fin de obtener el producto terminado; células manipuladas y combinadas con componentes no celulares (por ejemplo, matrices o productos sanitarios biológicos o inertes) que ejercen la acción pretendida en principio en el producto acabado; derivados de células autólogas expresadas ex vivo {in vitro) en condiciones específicas de cultivo; o células modificadas genéticamente o sometidas a otro tipo de manipulación para expresar propiedades funcionales homologas o no homologas anteriormente no expresadas. In an even more preferred embodiment, the medicament of the invention is a somatic cell therapy medication. "Somatic cell therapy" means the use of live somatic cells, both autologous (from the patient itself), and allogeneic (from another human being) or xenogeneic (from animals), whose biological characteristics have been substantially altered as a result of their manipulation, to obtain a therapeutic, diagnostic or preventive effect, by metabolic, pharmacological or immunological means. Among somatic cell therapy drugs are, for example, but not limited to: cells manipulated to modify their immunological, metabolic or other functional properties in qualitative or quantitative aspects; classified cells, selected and manipulated, which are subsequently subjected to a manufacturing process in order to obtain the finished product; cells manipulated and combined with non-cellular components (for example, matrices or biological or inert medical devices) that exert the intended action in principle on the finished product; autologous cell derivatives expressed ex vivo {in vitro) under specific culture conditions; or cells genetically modified or subjected to another type of manipulation to express homologous or non-homologous functional properties previously not expressed.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y figuras se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. BREVE DESCRIPCIÓN DE LAS FIGURAS Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be apparent in part of the description and part of the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Muestra el resultado del examen histológico de islotes procedentes del páncreas de un paciente cuyas células β expresan la variante GCK-S64F (panel de la izquierda) así como de islotes procedentes del páncreas de un individuo control (panel de la derecha). FIG. 1. Shows the result of the histological examination of islets coming from the pancreas of a patient whose β cells express the GCK-S64F variant (left panel) as well as islets coming from the pancreas of a control individual (right panel).
FIG. 2. Muestra el resultado del análisis de proliferación de células β en islotes procedentes de tejido pancreático de un paciente cuyas células β expresan la variante GCK-S64F (panel de la izquierda) así como de islotes procedentes de tejido pancreático de un individuo control (panel de la derecha). Mareaje celular realizado con Ki67. FIG. 2. Shows the result of the proliferation analysis of β cells in islets from pancreatic tissue of a patient whose β cells express the GCK-S64F variant (left panel) as well as islets from pancreatic tissue of a control individual (panel right). Cellular mapping done with Ki67.
FIG. 3. Muestra islotes de ratón no infectados (control) e islotes de ratón infectados con el vector de expresión vacío (pRRL.cmv.ires.eGFP), el vector que contenía GCK-WT (pRRL.cmv.GCK-WT.ires.eGFP) y la mutación GCK-V91 L activadora de GCK encontrada en un ser humano (pRRL.cmv.GCK-V91 L.ires.eGFP). M.O.I de 2 y n= 8. FIG. 3. Shows uninfected mouse islets (control) and mouse islets infected with the empty expression vector (pRRL.cmv.ires.eGFP), the vector containing GCK-WT (pRRL.cmv.GCK-WT.ires. eGFP) and the GCK-V91 L activating GCK mutation found in a human being (pRRL.cmv.GCK-V91 L.ires.eGFP). M.O.I of 2 and n = 8.
FIG. 4. Muestra la incorporación de BrdU en islotes de ratón no infectados. DAPI, BrdU, EGFP. (A) Islote control no infectado. (B) Islotes infectados con EGFP. (C) Islotes infectados con GK natural que presentan un aumento de 1 ,6 veces en la incorporación de BrdU en comparación con A y B. Y (D) islotes infectados con GCK-V91 L que presentan un aumento de 6,8 veces en la incorporación de BrdU en comparación con A y B y un aumento de 5, 1 veces en la incorporación de BrdU en comparación con C. EJEMPLOS FIG. 4. Shows the incorporation of BrdU into uninfected mouse islets. DAPI, BrdU, EGFP. (A) Islet control not infected. (B) Islets infected with EGFP. (C) Islets infected with natural GK that have a 1, 6-fold increase in BrdU incorporation compared to A and B. And (D) islets infected with GCK-V91 L that have a 6.8-fold increase in BrdU incorporation compared to A and B and a 5, 1 fold increase in BrdU incorporation compared to C. EXAMPLES
A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores, que ponen de manifiesto la efectividad de las secuencias nucleotídicas y aminoacídicas de las variantes de GK descritas en la invención para la obtención de islotes pancreáticos de alta eficacia. Estos ejemplos específicos que se proporcionan sirven para ilustrar la naturaleza de la presente invención y se incluyen solamente con fines ilustrativos, por lo que no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ilustran la invención sin limitar el campo de aplicación de la misma. The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the nucleotide and amino acid sequences of the GK variants described in the invention for obtaining high-efficiency pancreatic islets. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.
Ejemplo 1 : Manipulación genética del gen GCK para la obtención de formas activadoras, tanto leves como severas, de GCK capaces de codificar para enzimas glucocinasas con actividad aumentada, y su posterior introducción en islotes pancreáticos humanos aislados. Example 1: Genetic manipulation of the GCK gene to obtain both mild and severe activating forms of GCK capable of coding for glucokinase enzymes with increased activity, and their subsequent introduction into isolated human pancreatic islets.
1.1. Aislamiento de islotes pancreáticos humanos. 1.1. Isolation of human pancreatic islets.
Los páncreas de donde procedían los islotes pertenecían a personas donantes de órganos, y eran páncreas que no cumplieron con los criterios necesarios para ser transplantados como órgano sólido. Así mismo existía un consentimiento informado y firmado en el que se permitía el uso en investigación de los islotes pancreáticos aislados en caso de que éstos no pudieran ser transplantados. El proceso de aislamiento de islotes pancreáticos humanos se llevó a cabo siguiendo el método semiautomático de Ricordi (Ricordi et al., 1989, Diabetes, 38:Suppl 1 : 140- 142). El páncreas, una vez limpiado de grasa y tejido adyacente, se perfundió a través del conducto pancreático con una solución de Liberasa (Roche). Posteriormente, se introdujo en la cámara de Ricordi para su digestión. Finalmente, los islotes se purificaron utilizando un gradiente continuo de densidades con Ficoll en una centrífuga COBE. Después de varios lavados con medio de cultivo los islotes se observaron al microscopio. Tras un contaje se distribuyeron en frascos de cultivo y se almacenaron a 37°C y 5% de CO2. The pancreas where the islets came from belonged to organ donors, and were pancreas that did not meet the criteria necessary to be transplanted as a solid organ. There was also an informed and signed consent that allowed the use in research of isolated pancreatic islets if they could not be transplanted. The process of isolation of human pancreatic islets was carried out following the semi-automatic method of Ricordi (Ricordi et al., 1989, Diabetes, 38: Suppl 1: 140-142). The pancreas, once cleared of fat and adjacent tissue, was perfused through the pancreatic duct with a solution of Liberase (Roche). Subsequently, it was introduced into Ricordi's chamber for digestion. Finally, the islets were purified using a gradient continuity of densities with Ficoll in a COBE centrifuge. After several washes with culture medium the islets were observed under a microscope. After counting, they were distributed in culture bottles and stored at 37 ° C and 5% CO2.
1.2. Desarrollo del sistema adenoviral inducible conteniendo ADNc de GCK-WT, y de los mutantes GCK-E440G, GCK-E442K y GCK-V91 L, para los estudios funcionales "in vitro" de los islotes humanos aislados. 1.2. Development of the inducible adenoviral system containing GCK-WT cDNA, and GCK-E440G, GCK-E442K and GCK-V91 L mutants, for "in vitro" functional studies of isolated human islets.
El sistema Adeno-X Tet-ON desarrollado modula y controla la expresión de GK, con el fin de obtener una expresión óptima y no deletérea para la funcionalidad de los islotes. Los ADNc humanos tanto de la GCK nativa (WT), como de las variantes inactivante GCK-E440G, activante leve GCK- E442K y activante severa GCK-V91 L, fueron subclonados en el vector pTRE-Shuttle2 (Clontech laboratorios, Inc); posteriormente, el cassette inducible se transfirió al vector Adeno-X para generar los adenovirus recombinantes: Adv-hGCK-WT nativo, Adv-hGCK-E440G, Adv-hGCK- E442K y Adv-hGCK-V91 L. El protocolo de infección se optimizó en la línea celular INS-1 E para establecer la cantidad de partículas virales y la concentración de doxiciclina necesaria para alcanzar una expresión adecuada de GK. Posteriormente, islotes humanos y de rata se coinfectaron bien con Adv-hGCK nativo, con Adv-hGCK-E440G, con Adv- hGCK-E442K, o con Adv-hGCK-V91 L, además de con la construcción adenoviral con el activador transcripcional para doxiciclina (Ad-X Tet-on). Para determinar los niveles de expresión de la glucocinasa se realizaron ensayos de PCR cuantitativa (qPCR) y de inmunofluorescencia. En islotes de rata se analizaron las glucocinasa humana y de rata, y en islotes humanos la glucocinasa humana. Para ello, se extrajo el RNA de islotes mediante el reactivo Trizol (Invitrogen) y entonces, 2μg de RNA se convirtieron en ADNc por RT-PCR usando primers oligo-(d)T. Para la qPCR, el ADNc se amplificó usando el intercalante Sybr green en una plataforma ABI Prism 7700; los cebadores se diseñaron usando el software Primer 3 Express (applera Europe). Los datos de fluorescencia se extrajeron de la plataforma, y se obtuvieron los valores Ct y la eficiencia mediante el software miner 2.1 . La cuantificación se determinó con el software qBASE. Para los experimentos de fluorescencia, se solubilizaron los islotes humanos y de rata, transducidos y no transducidos, con el tampón RIPA (1 ,25% (peso / vol) NP-40, 1 ,25% (peso / vol) deoxicolato sódico 0,0125; fosfato sódico, pH 7,2 mM EDTA), complementado con inhibidores de proteasa y de fosfatasa (Roche). Posteriormente, 80μ9 de proteína total fueron fraccionados para realizar un Western Blot usando anticuerpos contra glucocinasa humana/rata (c- 20, Santa Cruz de Biotecnología). The Adeno-X Tet-ON system developed modulates and controls the expression of GK, in order to obtain an optimal and non-deleterious expression for the functionality of the islets. The human cDNAs of both the native GCK (WT), and the inactive GCK-E440G, mild GCK-E442K and severe GCK-V91 L variants were subcloned into the pTRE-Shuttle2 vector (Clontech Laboratories, Inc); subsequently, the inducible cassette was transferred to the Adeno-X vector to generate the recombinant adenoviruses: native Adv-hGCK-WT, Adv-hGCK-E440G, Adv-hGCK-E442K and Adv-hGCK-V91 L. The infection protocol was optimized in the INS-1 E cell line to establish the amount of viral particles and the concentration of doxycycline necessary to achieve adequate GK expression. Subsequently, human and rat islets co-infected well with native Adv-hGCK, with Adv-hGCK-E440G, with Adv-hGCK-E442K, or with Adv-hGCK-V91 L, in addition to the adenoviral construction with the transcriptional activator for doxycycline (Ad-X Tet-on). To determine the levels of glucokinase expression, quantitative PCR (qPCR) and immunofluorescence assays were performed. In rat islets, human and rat glucokinase were analyzed, and in human islets, human glucokinase. For this, islet RNA was extracted by Trizol reagent (Invitrogen) and then, 2μg of RNA was converted to cDNA by RT-PCR using oligo- (d) T primers. For qPCR, the cDNA was amplified using the Sybr green interleaver in a ABI Prism 7700 platform; the primers were designed using the Primer 3 Express software (applera Europe). Fluorescence data was extracted from the platform, and Ct values and efficiency were obtained by software miner 2.1. The quantification was determined with the qBASE software. For fluorescence experiments, human and rat islets, transduced and non-transduced, were solubilized with the RIPA buffer (1.25% (weight / vol) NP-40, 1.25% (weight / vol) sodium deoxycholate 0 , 0125; sodium phosphate, pH 7.2 mM EDTA), supplemented with protease and phosphatase inhibitors (Roche). Subsequently, 80μ9 of total protein were fractionated to perform a Western Blot using antibodies against human glucokinase / rat (c-20, Santa Cruz de Biotecnología).
1.3. Construcción de los vectores virales adeno-asociados. 1.3. Construction of adeno-associated viral vectors.
Los DNA complementarios codificantes (cDNAs) de GCK-WT, GCK- E440G, GCK-V91 L y GCK-E442K, fueron clonados en el esqueleto del vector adenoviral asociado subtipo 8 (AAV8), bajo el control del promotor del gen de la insulina de ratón (mlP), resultando los plásmidos AAV8-mlP- GCKX-ires-eGFP. El uso del promotor mlP hace que los vectores AAV8- mlP-der se expresen exclusivamente en las células beta pancreáticas. Complementary DNA coding (cDNAs) of GCK-WT, GCK-E440G, GCK-V91 L and GCK-E442K, were cloned into the skeleton of the associated adenoviral vector subtype 8 (AAV8), under the control of the insulin gene promoter mouse (mlP), resulting in plasmids AAV8-mlP-GCKX-ires-eGFP. The use of the mlP promoter causes AAV8-mlP-der vectors to be expressed exclusively in pancreatic beta cells.
1.4. Procedimiento de mutagénesis dirigida. Para la construcción de las diferentes mutaciones de la glucocinasa se utilizó el kit QuikChange I I XL Site-Directed Mutagénesis (Strategene). Las mutaciones se incorporaron por PCR empleando la polimerasa PfuUltra High Fidelity; y mediante la digestión con la endonucleasa Dpnl se digirieron específicamente los ADNs metilados/hemimetilados y no mutados. Los oligonucleótidos diseñados para la introducción de las mutaciones en el gen de la glucocinasa (GCK, SEQ ID NO: 5) se detallan a continuación: 1.4. Directed mutagenesis procedure. The QuikChange II XL Site-Directed Mutagenesis (Strategene) kit was used to construct the different glucokinase mutations. Mutations were incorporated by PCR using PfuUltra High Fidelity polymerase; and by digestion with the Dpnl endonuclease, the methylated / hemimethylated and non-mutated DNAs were specifically digested. Oligonucleotides designed for the introduction of mutations in the glucokinase gene (GCK, SEQ ID NO: 5) are detailed below:
Figure imgf000023_0001
1.5. Transducción celular de islotes pancreáticos humanos con vectores virales anedo-asociados.
Figure imgf000023_0001
1.5. Cellular transduction of human pancreatic islets with anedo-associated viral vectors.
Debido a que el sistema adenoviral inducible comentado anteriormente tiene una vida media muy limitada (el control de expresión proteica empieza a disminuir aproximadamente a las 3 semanas de la infección), la transducción de los islotes a trasplantar se realizó con AAVs replicando la expresión óptima de GK obtenida con el sistema Adeno-X Tet-ON, lo cual se realizó mediante el uso de diferentes v.g. de AAV. En paralelo, se valoró la posibilidad de construir AAVs inducibles equivalentes a los adenovirus no-asosciados inducibles comentados anteriormente. Con los AAVs inducibles con doxiciclina se transdujeron los islotes pancreáticos humanos que fueron posteriormente trasplantados. La mezcla de virus y células se centrifugó (1000 x g, 1 h, 37°C), tras lo cual las células fueron resuspendidas en medio RPMI 1640 que contiene 20% FCS, 10 mM HEPES, 2 mM L-glutamina, 50 μ9/ιηΙ gentamicina, y rhlL-2 (50 lU/ml), y se incubaron durante cuatro días. El empaquetamiento de los AAVs, la purificación de los diferentes AAVs mediante ultracentrifugación por gradiente CsCI (Cesium Chloride), la titulación de las partículas de genoma del vector (v.g.), así como la confirmación de su expresión se realizaron en células de la línea I NS-1 E. El proceso de transducción se llevó a cabo en un total de 6 días, hasta la comprobación de la eficiencia del proceso: Because the inducible adenoviral system discussed above has a very limited half-life (protein expression control begins to decrease approximately 3 weeks after infection), the transduction of the islets to be transplanted was performed with AAVs replicating the optimal expression of GK obtained with the Adeno-X Tet-ON system, which was done by using different vg of AAV. In parallel, the possibility of constructing inducible AAVs equivalent to the inducible nonassociated adenoviruses discussed above. With the doxycycline-inducible AAVs, human pancreatic islets were transduced and subsequently transplanted. The virus and cell mixture was centrifuged (1000 xg, 1 h, 37 ° C), after which the cells were resuspended in RPMI 1640 medium containing 20% FCS, 10 mM HEPES, 2 mM L-glutamine, 50 μ9 / ιηΙ gentamicin, and rhlL-2 (50 lU / ml), and incubated for four days. The packing of the AAVs, the purification of the different AAVs by ultracentrifugation by CsCI gradient (Cesium Chloride), the titration of the genome particles of the vector (eg), as well as the confirmation of their expression were performed in cells of line I NS-1 E. The transduction process was carried out in a total of 6 days, until the process efficiency was checked:
Día 1 : se plaquearon 8.105 células en placas de 6 pocilios con 2 mi de medio/pocilio. Las células se dejaron incubar cerca de 48 horas. Día 3: se preparó la mezcla de cultivo con 8μg de polibreno/ml de medio. El polibreno es un polímero se bajo peso molecular, positivamente cargado, que aparentemente es capaz de unirse a las células neutralizando la carga superficial. Esto permite que las glicoproteínas virales se unan más eficazmente a sus receptores, reduciendo la repulsión entre la partícula viral y la célula. Se añadieron 1 ,5ml de medio DMEM al 10% de FBS (BioWhittaker) con polibreno. Se añadió el SN en la proporción deseada (en las infecciones realizadas, se hicieron diluciones 1 :25, 1 :4, 1 :2 y 1 : 1 ) y se centrifugó la placa durante 30 minutos a 37°C a 1000g (proceso denominado espinoculación). Se dejaron en contacto las células con las partículas virales y la mezcla se incubó toda la noche a 37°C y 5% CO2. No se debe dejar la mezcla más de 12 horas o bien se debe reducir la concentración de polibreno. Día 4: se cambió el medio y se incubó hasta el día 5 (aproximadamente unas 62 horas tras dejar puesta la infección). Day 1: 8,105 cells were plated in 6-well plates with 2 ml of medium / well. The cells were allowed to incubate for about 48 hours. Day 3: The culture mixture was prepared with 8μg of polybrene / ml of medium. Polybrene is a low molecular weight, positively charged polymer that is apparently capable of binding to cells neutralizing surface charge. This allows viral glycoproteins to bind more effectively to their receptors, reducing the repulsion between the viral particle and the cell. 1.5ml of 10% DMEM medium FBS (BioWhittaker) was added with polybrene. SN was added in the desired proportion (in infections performed, dilutions 1: 25, 1: 4, 1: 2 and 1: 1 were made) and the plate was centrifuged for 30 minutes at 37 ° C at 1000g (process called spinoculation). The cells were contacted with the viral particles and the mixture was incubated overnight at 37 ° C and 5% CO2. The mixture should not be left for more than 12 hours or the concentration of polybrene should be reduced. Day 4: The medium was changed and incubated until day 5 (approximately 62 hours after leaving the infection).
Día 5: se levantaron las células mediante tripsinización, del modo habitual. Se pasaron las células a tubos eppendorf. Se centrifugaron a 1200 rpm durante 5minutos, se eliminó el sobrenadante y se resuspendieron las células en 500μΙ de PBS. Se analizaron las células por citometría de flujo. Una vez analizadas se estimó el tiempo de cultivo necesario durante el cual se deben cultivar las células que han sufrido el proceso de infección, en base al tiempo de duplicación de la línea celular, el porcentaje de células positivas para eGFP, y el número mínimo de células cultivables, para su purificación mediante FACS (Fluorescent Activated Cell Sorting; Beckman Coulter). Ejemplo 2: Análisis del efecto de la variante GCK-S64F en la proliferación y eficiencia de islotes pancreáticos humanos. Day 5: the cells were lifted by trypsinization, in the usual way. The cells were transferred to eppendorf tubes. They were centrifuged at 1200 rpm for 5 minutes, the supernatant was removed and the cells were resuspended in 500μΙ of PBS. Cells were analyzed by flow cytometry. Once analyzed, the necessary culture time during which the cells that have undergone the infection process should be cultivated was estimated, based on the doubling time of the cell line, the percentage of cells positive for eGFP, and the minimum number of cultivable cells, for purification by FACS (Fluorescent Activated Cell Sorting; Beckman Coulter). Example 2: Analysis of the effect of the GCK-S64F variant on the proliferation and efficiency of human pancreatic islets.
Se encontró una mutación "de novo" que activaba intensamente el gen de la glucocinasa (GCK) natural (SEQ ID NO: 5) en un paciente con hipoglucemia neonatal grave. Se encontró una mutación novedosa sin sentido, (cambio 191 C>T en la SEQ ID NO: 5 para dar lugar a la SEQ ID NO: 6) que da como resultado la sustitución de un residuo S por uno F en el codón 64 del exón 3 de la secuencia aminoacídica de la GK nativa (SEQ ID NO: 4, para dar lugar a la SEQ ID NO: 1 o variante GCK-S64F). El probando era heterocigótico para esta mutación, que no se identificó en el ADN de sus padres. Para determinar si esta mutación era un caso de novo, se llevó a cabo un análisis de microsatélites sobre varios cromosomas, y se excluyó una falsa paternidad. Los estudios funcionales realizados sobre la variante GCK-S64F mostraron una afinidad 9 veces superior por la glucosa (So.s), una eficacia 8 veces superior (Kcat/So,s) y un índice de actividad aumentado de manera importante en comparación con la GK natural (WT-GK) (Tabla 1 ). Esta activación catalítica y estructural del GCK-S64F originó que esta enzima tuviera un umbral de liberación de insulina en respuesta a glucosa (UF-SIEG) de 0,96 mmol/l, muy alejado del valor de 5 mmol/l de la GK-WT y cercano a los valores de 0,8 y 0,9 mmo/l encontrados en otras mutaciones que activaban fuertemente la GK identificadas. La afinidad por la glucosa, notablemente elevada, y el índice de actividad relativa junto al UF-SIEG extraordinariamente bajo encontrado para GCK-S64F explican en su totalidad la grave hipoglicemia observada en el paciente. A "de novo" mutation was found that strongly activated the natural glucokinase (GCK) gene (SEQ ID NO: 5) in a patient with severe neonatal hypoglycemia. A novel nonsense mutation was found (change 191 C> T in SEQ ID NO: 5 to give rise to SEQ ID NO: 6) which results in the substitution of a residue S with an F in codon 64 of the exon 3 of the amino acid sequence of the native GK (SEQ ID NO: 4, to give rise to SEQ ID NO: 1 or GCK-S64F variant). The test was heterozygous for this mutation, which was not identified in the DNA of their parents. To determine if this mutation was a de novo case, a microsatellite analysis was carried out on several chromosomes, and a false paternity was excluded. Functional studies performed on the GCK-S64F variant showed a 9-fold higher affinity for glucose (So.s), an 8-fold higher efficiency (Kcat / So, s) and an increased activity index of important way compared to natural GK (WT-GK) (Table 1). This catalytic and structural activation of GCK-S64F caused this enzyme to have an insulin release threshold in response to glucose (UF-SIEG) of 0.96 mmol / l, far removed from the value of 5 mmol / l of GK- WT and close to the values of 0.8 and 0.9 mmo / l found in other mutations that strongly activated the GK identified. The markedly high glucose affinity, and the extraordinarily low UF-SIEG relative activity index found for GCK-S64F fully explain the severe hypoglycemia observed in the patient.
Tabla 1. Resultados de los estudios funcionales realizados sobre la variante GCK-S64F (SEQ ID NO: 1 ) en comparación con la GK nativa humana (SEQ ID  Table 1. Results of the functional studies performed on the GCK-S64F variant (SEQ ID NO: 1) compared to the native human GK (SEQ ID
NO: 4). En el examen histológico, el páncreas del paciente mostró islotes anormalmente grandes en comparación con un control de la misma edad (Fig. 1 ). También se encontró una proliferación de células beta en los islotes del tejido pancreático obtenidos en las pancreatectomías parciales realizadas en este paciente (Fig. 2). Tras la primera pancreatectomía parcial, el porcentaje de células beta marcadas con Ki67 fue 1 ,56, 2,59 y 4,09%, y tras el segundo procedimiento quirúrgico, el índice Ki67 fue de aproximadamente el 10%. Esto es interesante, ya que sugiere que tras la pancreatectomía se consiguió una "regeneración" de las células beta. Dos de los controles de la misma edad tuvieron 0,2 y 0,98%. Igualmente, el tamaño promedio del islote fue de 4.064, 5.023 y 6.058 micrómetros cuadrados tras el primer procedimiento quirúrgico, y de 9.865 tras el segundo. Esto sugiere que los islotes intentaron compensar la pancreatectomía parcial. Ejemplo 3: Análisis del efecto de la variante GCK-V91 L en la proliferación y eficiencia de islotes pancreáticos de ratón. NO: 4). On histological examination, the patient's pancreas showed abnormally large islets compared to a control of the same age (Fig. 1). A proliferation of beta cells was also found in the islets of the pancreatic tissue obtained in the partial pancreatectomies performed in this patient (Fig. 2). After the first partial pancreatectomy, the percentage of beta cells labeled with Ki67 was 1, 56, 2.59 and 4.09%, and after the second surgical procedure, the Ki67 index was approximately 10%. This is interesting, since it suggests that after the pancreatectomy a "regeneration" of the beta cells was achieved. Two of the same age controls had 0.2 and 0.98%. Similarly, the average islet size was 4,064, 5,023 and 6,058 square micrometers after the first surgical procedure, and 9,865 after the second. This suggests that the islets tried to compensate for partial pancreatectomy. Example 3: Analysis of the effect of the GCK-V91 L variant on the proliferation and efficiency of mouse pancreatic islets.
La eficacia de infección en todos los casos fue de aproximadamente el 93%, (Figura 3). La liberación de insulina en respuesta a glucosa estuvo significativamente por encima de concentraciones de glucosa de 4 mM en los islotes infectados con GCK-V91 L (p< 0,01 ) en comparación con los islotes de control no infectados y con los islotes infectados con eGFP y GCK-WT. Además, los islotes infectados con GCK-V91 L comenzaron a liberar insulina en respuesta a glucosa a concentraciones de glucosa inferiores (4 mM) (Tabla 2). De manera fascinante, la incorporación de BrdU fue 6,8 veces mayor en los islotes infectados con GCK-V91 L que en los islotes de control no infectados y en los islotes infectados con eGFP; y 5,1 veces mayor en comparación con los islotes infectados con GCK-WT (Figura 4). The efficacy of infection in all cases was approximately 93%, (Figure 3). Insulin release in response to glucose was significantly above 4 mM glucose concentrations in islets infected with GCK-V91 L (p <0.01) compared to uninfected control islets and islets infected with eGFP and GCK-WT. In addition, islets infected with GCK-V91 L began releasing insulin in response to glucose at lower glucose concentrations (4 mM) (Table 2). Fascinatingly, the incorporation of BrdU was 6.8 times greater in islets infected with GCK-V91 L than in uninfected control islets and in islets infected with eGFP; and 5.1 times higher compared to islets infected with GCK-WT (Figure 4).
Figure imgf000027_0001
Figure imgf000027_0001
Tabla 2. UF-SIEG en islotes de ratón. La liberación de insulina se expresa como pg de insulina^g proteína/60 min *p< 0,01 en comparación con el resto de condiciones. 7 ratones estudiados; n= 15 y 3 muestras ELISA se analizaron por triplicado. Table 2. UF-SIEG in mouse islets. Insulin release is expressed as pg of insulin ^ g protein / 60 min * p <0.01 compared to other conditions. 7 mice studied; n = 15 and 3 ELISA samples were analyzed in triplicate.
Considerando que la "desdiferenciación" de células beta y la carencia de una respuesta correcta a la glucosa son los problemas más importantes para el aumento de la proliferación de células β durante un periodo de tiempo prolongado, de forma sorprendente, la proliferación en los islotes infectados con GCK-V91 L conservaba en su totalidad una sensibilidad correcta a la glucosa y un UF-SIEG correcto tras 15 días en cultivo. Whereas the "dedifferentiation" of beta cells and the lack of a correct response to glucose are the most important problems for the increase in the proliferation of β cells over a prolonged period of time, surprisingly, the proliferation in the islets Infected with GCK-V91 L, it conserved in its entirety a correct glucose sensitivity and a correct UF-SIEG after 15 days in culture.
Los resultados confirmaron que la mutación activadora GCK-V91 L aumentaba la liberación de insulina en respuesta a glucosa a la vez que disminuía el UF-SIEG. The results confirmed that the GCK-V91 L activating mutation increased insulin release in response to glucose while decreasing UF-SIEG.
La glucosa desempeña un papel crítico en la proliferación de células beta. Por tanto, la disminución en el UF-SIEG lleva a su vez a un aumento en el flujo de glucosa intracelular, que activara directamente rutas posteriores alternativas o de amplificación, algo importante en la regulación de la proliferación de las células beta. Glucose plays a critical role in the proliferation of beta cells. Therefore, the decrease in UF-SIEG in turn leads to an increase in the flow of intracellular glucose, which will directly activate alternative or amplification posterior routes, something important in the regulation of beta cell proliferation.
La replicación de estos islotes de alta eficacia puede usarse como una nueva herramienta de terapia celular en la Diabetes Mellitus de Tipo 1 . Replication of these highly effective islets can be used as a new cell therapy tool in Type 1 Diabetes Mellitus.

Claims

REIVINDICACIONES
1 . Una secuencia nucleotídica aislada que codifica para una secuencia aminoacídica de la enzima glucocinasa que presenta al menos una mutación activadora de la actividad de dicha enzima. one . An isolated nucleotide sequence encoding an amino acid sequence of the glucokinase enzyme that has at least one activating mutation of the activity of said enzyme.
2. La secuencia nucleotídica según la reivindicación 1 capaz de generar islotes pancreáticos de alta eficacia. 2. The nucleotide sequence according to claim 1 capable of generating high-efficiency pancreatic islets.
3. La secuencia nucleotídica según cualquiera de las reivindicaciones 1 ó 2 donde la secuencia aminoacídica de la enzima glucocinasa que presenta al menos una mutación activadora de la actividad de dicha enzima se selecciona de la lista que consiste en: SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3. 3. The nucleotide sequence according to any of claims 1 or 2 wherein the amino acid sequence of the glucokinase enzyme having at least one activating mutation of the activity of said enzyme is selected from the list consisting of: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
4. Una construcción genética que comprende la secuencia nucleotídica según cualquiera de las reivindicaciones 1 a 3. 4. A genetic construct comprising the nucleotide sequence according to any one of claims 1 to 3.
5. La construcción genética según la reivindicación 4 que es un vector de expresión. 5. The genetic construct according to claim 4 which is an expression vector.
6. El vector de expresión según la reivindicación 5, que es un lentivirus. 6. The expression vector according to claim 5, which is a lentivirus.
7. Una secuencia aminoacídica de la enzima glucocinasa seleccionada de la lista que consiste en: SEQ ID NO: 1 , SEQ ID NO: 2 o SEQ ID NO: 3. 7. An amino acid sequence of the glucokinase enzyme selected from the list consisting of: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
8. Una célula que comprende la secuencia nucleotídica según cualquiera de las reivindicaciones 1 a 3, la construcción genética según cualquiera de las reivindicaciones 4 ó 5, el vector de expresión según la reivindicación 6, o la secuencia aminoacídica según la reivindicación 7. 8. A cell comprising the nucleotide sequence according to any of claims 1 to 3, the genetic construct according to any of claims 4 or 5, the expression vector according to the claim 6, or the amino acid sequence according to claim 7.
9. La célula según la reivindicación 8, que es una célula pancreática. 9. The cell according to claim 8, which is a pancreatic cell.
10. La célula según cualquiera de las reivindicaciones 8 ó 9, que es de origen humano. 10. The cell according to any of claims 8 or 9, which is of human origin.
1 1 . Un islote pancreático que comprende al menos una célula según cualquiera de las reivindicaciones 8 a 10. eleven . A pancreatic islet comprising at least one cell according to any of claims 8 to 10.
12. El islote pancreático según la reivindicación 1 1 , que es de origen humano. 12. The pancreatic islet according to claim 1, which is of human origin.
13. Una composición que comprende la secuencia nucleotídica según cualquiera de las reivindicaciones 1 a 3, la construcción genética según cualquiera de las reivindicaciones 4 ó 5, el vector de expresión según la reivindicación 6, la secuencia aminoacídica según la reivindicación 7, la célula según cualquiera de las reivindicaciones 8 a 10, o un islote pancreático según cualquiera de las reivindicaciones 1 1 ó 12. 13. A composition comprising the nucleotide sequence according to any one of claims 1 to 3, the genetic construct according to any of claims 4 or 5, the expression vector according to claim 6, the amino acid sequence according to claim 7, the cell according to any of claims 8 to 10, or a pancreatic islet according to any of claims 1 1 or 12.
14. La composición según la reivindicación 13, que es una composición farmacéutica. 14. The composition according to claim 13, which is a pharmaceutical composition.
15. La composición según cualquiera de las reivindicaciones 13 ó 14, que además comprende un vehículo farmacéuticamente aceptable. 15. The composition according to any of claims 13 or 14, further comprising a pharmaceutically acceptable carrier.
16. La composición según cualquiera de las reivindicaciones 13 a 15, que además comprende otro principio activo. 16. The composition according to any of claims 13 to 15, further comprising another active ingredient.
17. El uso de la secuencia nucleotídica según cualquiera de las reivindicaciones 1 a 3, de la construcción genética según cualquiera de las reivindicaciones 4 ó 5, del vector de expresión según la reivindicación 6, de la secuencia aminoacídica según la reivindicación 7, de la célula según cualquiera de las reivindicaciones 8 a 10, de un islote pancreático según cualquiera de las reivindicaciones 1 1 ó 12, o de la composición según cualquiera de las reivindicaciones 13 a 16, en la elaboración de un medicamento. 17. The use of the nucleotide sequence according to any of claims 1 to 3, of the genetic construct according to any of claims 4 or 5, of the expression vector according to the claim 6, of the amino acid sequence according to claim 7, of the cell according to any of claims 8 to 10, of a pancreatic islet according to any of claims 1 1 or 12, or of the composition according to any of claims 13 to 16 , in the preparation of a medicine.
18. El uso de la secuencia nucleotídica según cualquiera de las reivindicaciones 1 a 3, de la construcción genética según cualquiera de las reivindicaciones 4 ó 5, del vector de expresión según la reivindicación 6, de la secuencia aminoacídica según la reivindicación 7, de la célula según cualquiera de las reivindicaciones 8 a 10, de un islote pancreático según cualquiera de las reivindicaciones 1 1 ó 12, o de la composición según cualquiera de las reivindicaciones 13 a 16, en la elaboración de un medicamento para el tratamiento y/o prevención de la diabetes mellitus. 18. The use of the nucleotide sequence according to any one of claims 1 to 3, of the genetic construct according to any of claims 4 or 5, of the expression vector according to claim 6, of the amino acid sequence according to claim 7, of the cell according to any of claims 8 to 10, of a pancreatic islet according to any of claims 1 or 12, or of the composition according to any of claims 13 to 16, in the preparation of a medicament for the treatment and / or prevention of diabetes mellitus.
19. El uso según la reivindicación 18 donde la diabetes mellitus es de tipo 1 . 19. The use according to claim 18 wherein the diabetes mellitus is type 1.
PCT/ES2012/070080 2012-02-08 2012-02-08 Glucokinase enzymes with increased activity and use thereof in the treatment and/or prevention of diabetes mellitus WO2013117776A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017994A1 (en) * 1995-11-16 1997-05-22 Universidad Autonoma De Barcelona Treatment of diabetes with a glucokinase gene
EP1360898A1 (en) * 2000-12-20 2003-11-12 Universitat Autonoma De Barcelona Joint utilization of the insulin gene and the glucokinase gene in the development of therapeutic approaches for diabetes mellitus
WO2006043036A2 (en) * 2004-10-19 2006-04-27 The University Of Newcastle Treatment of diabetes
EP2388317A1 (en) * 2009-01-07 2011-11-23 Haidong Huang Gene encoding human glucokinase mutant, enzyme encoded by the same, recombinant vectors and hosts, pharmaceutical compositions and uses thereof, methods for treating and preventing diseases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017994A1 (en) * 1995-11-16 1997-05-22 Universidad Autonoma De Barcelona Treatment of diabetes with a glucokinase gene
EP1360898A1 (en) * 2000-12-20 2003-11-12 Universitat Autonoma De Barcelona Joint utilization of the insulin gene and the glucokinase gene in the development of therapeutic approaches for diabetes mellitus
WO2006043036A2 (en) * 2004-10-19 2006-04-27 The University Of Newcastle Treatment of diabetes
EP2388317A1 (en) * 2009-01-07 2011-11-23 Haidong Huang Gene encoding human glucokinase mutant, enzyme encoded by the same, recombinant vectors and hosts, pharmaceutical compositions and uses thereof, methods for treating and preventing diseases

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