WO2013117776A1 - Enzymes glucokinases à activité accrue, et leur utilisation pour le traitement et/ou la prévention du diabète sucré - Google Patents

Enzymes glucokinases à activité accrue, et leur utilisation pour le traitement et/ou la prévention du diabète sucré Download PDF

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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)
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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/fr
Publication of WO2013117776A1 publication Critical patent/WO2013117776A1/fr

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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

La présente invention concerne des séquences nucléotidiques codant pour des enzymes glucokinases mutées, qui présentent une activité enzymatique accrue, des constructions génétiques qui les contiennent, des séquences d'acides aminés codant pour elles, des îlots pancréatiques qui les expriment, ainsi que leurs utilisations pour le traitement et/ou la prévention du diabète sucré. Les îlots pancréatiques qui expriment les séquences d'acides aminés correspondant aux variants de glucokinases décrits dans l'invention ont une efficacité élevée, du fait qu'ils contiennent des cellules β pancréatiques qui présentent une capacité de prolifération accrue et induisent une baisse du seuil de libération d'insuline en réponse au glucose. Les îlots du type décrit présentent en outre une taille supérieure à celle des îlots pancréatiques provenant de sujets témoins, ce qui les rend utiles pour le traitement du diabète sucré par thérapie cellulaire.
PCT/ES2012/070080 2012-02-08 2012-02-08 Enzymes glucokinases à activité accrue, et leur utilisation pour le traitement et/ou la prévention du diabète sucré WO2013117776A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017994A1 (fr) * 1995-11-16 1997-05-22 Universidad Autonoma De Barcelona Traitement du diabete a l'aide d'un gene de glucokinase
EP1360898A1 (fr) * 2000-12-20 2003-11-12 Universitat Autonoma De Barcelona Utilisation conjointe du gene de l'insuline et du gene de la glucokinase dans la mise au point d'approches therapeutiques pour le diabete sucre
WO2006043036A2 (fr) * 2004-10-19 2006-04-27 The University Of Newcastle Traitement du diabete
EP2388317A1 (fr) * 2009-01-07 2011-11-23 Haidong Huang Gène codant pour un mutant de glucokinase humaine, enzyme codée par celui-ci, vecteurs recombinants et hôtes recombinants, compositions pharmaceutiques et leurs utilisations, procédés de traitement et de prévention de maladies

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017994A1 (fr) * 1995-11-16 1997-05-22 Universidad Autonoma De Barcelona Traitement du diabete a l'aide d'un gene de glucokinase
EP1360898A1 (fr) * 2000-12-20 2003-11-12 Universitat Autonoma De Barcelona Utilisation conjointe du gene de l'insuline et du gene de la glucokinase dans la mise au point d'approches therapeutiques pour le diabete sucre
WO2006043036A2 (fr) * 2004-10-19 2006-04-27 The University Of Newcastle Traitement du diabete
EP2388317A1 (fr) * 2009-01-07 2011-11-23 Haidong Huang Gène codant pour un mutant de glucokinase humaine, enzyme codée par celui-ci, vecteurs recombinants et hôtes recombinants, compositions pharmaceutiques et leurs utilisations, procédés de traitement et de prévention de maladies

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