Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/605—Glucagons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the present invention relates to gene therapy and provides stable, transformed mammalian cell lines and vectors useful as vehicles for transferring functional DNA sequences whose protein products are useful in the treatment of diabetes melli tus .
• the human hormone glucagon is a 29-amino acid hormone produced in pancreatic A-cells.
• the hormone belongs to a multi-gene family of structurally related peptides that include secretin, gastric inhibitory peptide, vasoactive intestinal peptide and glicentin. These peptides variously regulate carbohydrate metabolism, gastrointestinal mobility and secretory processing.
• the principal recognized actions of pancreatic glucagon are to promote hepatic glycogenolysis and gluconeogenesis, resulting in an elevation of blood sugar levels.
• the actions of glucagon are counter regulatory to those of insulin and may contribute to the hyperglycemia that accompanies diabetes mellitus (Lund, P.K., et al . , Proc. Natl . Acad. Sci . U. S. A . , 7£:345-349 (1982)) .
• glucagon When glucagon binds to its receptor on insulin producing cells, CAMP production increases which in turn stimulates insulin expression (Korman, L.Y., et al . , Diabetes , 11:717-722 (1985)) . Moreover, high levels of insulin down-regulate glucagon synthesis by a feedback inhibition mechanism (Ganong, W.F., .Review of Medical Physiology, Lange Publications, Los Altos, California, p. 273 (1979)) . Thus, the expression of glucagon is carefully regulated by insulin, and ultimately by serum glucose levels.
• Preproglucagon the precursor form of glucagon, is encoded by a 360 base pair gene and is processed to form proglucagon (Lund, et al . , Proc. Natl . Acad. Sci . U. S. A . 2£:345-349 (1982)) . Patzelt, et al . (Nature, 2£2.:260-266 (1979)) demonstrated that proglucagon is further processed into glucagon and a second peptide. Later experiments demonstrated that proglucagon is cleaved carboxyl to Lys-Arg or Arg-Arg residues (Lund, P.K. , et al . , Lopez L.C., et al .
• GLP-1 and GLP-2 are present in the pancreas as 37 and 34 amino acid peptides respectively.
• GLP-1 The similarity between GLP-1 and glucagon suggested to early investigators that GLP-1 might have biological activity. Although some investigators found that GLP-1 could induce rat brain cells to synthesize cAMP (Hoosein, ⁇ .M. , et al . , Febs Lett . 178:83-86 (1984)), other investigators failed to identify any physiological role for GLP-1 (Lopez, L.C., et al . supra ) . The failure to identify any physiological role for GLP-1 caused some investigators to question whether GLP-1 was in fact a hormone and whether the relatedness between glucagon and GLP-1 might be artifactual.
• GLP-K7-34) and GLP-K7-35) are disclosed in U.S. Patent No: 5,118,666, herein incorporated by reference.
• GLP-K7-37) is disclosed in U.S. Patent No: 5,120,712, herein incorporated by reference.
• GLP-1 Variants and analogs of GLP-1 are known in the art. These variants and analogs include, for example, GLP-l(7-36), Gln 9 -GLP-l(7-37) , Thr 16 -Lys 18 -GLP-1 (7-37) , and Lys 18 -GLP-1 (7- 37).
• Derivatives of GLP-1 include, for example, acid addition salts, carboxylate salts, lower alkyl esters, and amides (see, e.g., W091/11457) .
• the various disclosed forms of GLP-1 are known to stimulate insulin secretion (insulinotropic action) and cAMP formation (see, e.g., Mojsov, S., int . J. Peptide Protein Research, 40:333- 343 (1992)) .
• the fundamental defects responsible for causing hyperglycemia m mature onset diabetes include impaired secretion of endogenous insulin and resistance to the effects of insulin by muscle and liver tissue (Galloway, J.S.,
• Diabetes Care 11:1209-1239, (1990)
• the latter defect results in excess glucose production in the liver.
• a normal individual releases glucose at the rate of approximately 2 mg/kg/minute
• a patient with mature onset diabetes releases glucose at a rate exceeding 2.5 mg/kg/minute, resulting in a net excess of at least 70 grams of glucose per 24 hours.
• Retroviral-mediated gene transfer was suggested for treating human diseases involving malfunctioning bone marrow. Anderson et al . , Science 226: 401 (1984). In addition, PCT
• RNA or DNA vector which is capable of encoding the desired, therapeutic protein.
• Ex vivo gene therapy consists of four primary steps: (1) Primary cells (target cells) are removed from the individual m need of therapy; (2) The gene therapy nucleotide sequence is incorporated into the target cell m vitro; (3) The transformed cells expressing the incorporated nucleotide sequence encoding the protein of interest are identified, isolated, and expanded; and (4) The transformed cells are reintroduced into the individual.
• Ex vivo therapy generally results in the incorporation of the nucleotide sequence encoding the protein of interest into the chromosomal DNA of the target cell.
• the critical step of ex vivo gene therapy is the proper introduction of the nucleotide sequence encoding the desired protein into the target cell. This transfer of DNA can be accomplished by a number of well documented methods such as: calcium phosphate precipitation, electroporation, and adenoviral or retroviral vectors (Current Protocols in
• In vivo gene therapy generally describes the transfer of a desired nucleic acid sequence, located on a transport vector, directly into an individual in need of therapy. This gene therapy approach does not require that the target cells be first removed and manipulated in vi tro . Once the vector containing the desired nucleotide sequence is introduced into the individual, the vector moves into the nucleus of the target cell and the nucleotide sequence of interest integrates into the chromosomal DNA of the target cell with varying degrees of efficiency.
• a number of well documented methods exist for introducing nucleic acids into an individual requiring therapy such as direct injection of - 6 -
• the present invention provides a method of treating both Type I and Type II diabetics through a different gene therapy approach.
• a stable mammalian cell line is transformed by a nucleic acid vector such that it secretes a GLP-1 (7-37) -based protein, as defined by SEQ ID NO 1, followed by implantation into an individual needing treatment.
• the GLP-1 (7-37) -based protein in conjunction with high serum glucose levels, causes pancreatic beta cells to produce insulin in non-insulin dependent diai>etes melli tus (NIDDM) patients and delays gastric emptying in both NIDDM and insulin dependent diaj etes melli tus IDDM patients.
• NIDDM non-insulin dependent diai>etes melli tus
• one embodiment of this invention provides a method of treating Type I or Type II diabetes in a mammal in need thereof comprising implanting a cell line transformed with a vector comprising a promoter driving expression of a DNA sequence encoding a protein of SEQ ID NO 1
• Xaa 2 is Glu, Gin, Ala, Thr, Ser, and Gly;
• Xaa 3 is Lys, and Arg;
• Xaa 4 is Glu, Gin, Ala, Thr, Ser, and Gly; and, Xaa 5 is Gly-OH or is absent; into said mammal such that it is immunologically isolated from the mammal ' s immune system and secretes a protein of SEQ ID NO 1 into said patient.
• Preferred proteins of SEQ ID NO 1 are those in which Xaa 1 is Ala, Xaa 2 is Glu, Xaa 3 is Lys. Xaa 4 is Glu, and Xaa 5 is Gly-OH. More preferred are those in which Xaa 1 is
• Xaa 2 is Glu, Gin, Ala, Thr, Ser, and Gly
• Xaa 3 is Lys, and Arg
• Xaa 4 is Glu, Gin, Ala, Thr, Ser, and Gly
• Xaa 5 is Gly-OH or is absent.
• Another preferred group are those in which Xaa 1 is Val and Xaa 3 is Lys.
• a further preferred group of proteins are those wherein Xaa 1 is Ala or Val, Xaa 2 Glu, Xaa 3 is Lys or Arg, Xaa 4 is Glu, and Xaa 5 Gly-OH or is absent.
• Yet another preferred group are those in which Xaa 1 is Ala, Xaa 2 Glu, Xaa 3 is Lys, Xaa 4 is Glu, and Xaa 5 Gly-OH. Still another preferred group is when Xaa 1 is Val, Xaa 2 Glu, Xaa 3 is Lys, Xaa 4 is Glu, and Xaa 5 Gly-OH.
• Nucleotide sequences encoding any one of the polypeptides of SEQ ID NO 1 may be prepared by a variety of means readily apparent to those skilled in the art. Wholly synthetic nucleotide sequences or semi-synthetic sequences derived in part from a natural GLP-1 gene may be used. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of nucleotide sequences may be constructed which encode the proteins of SEQ ID NO 1.
• a synthetic DNA sequence encoding a GLP-1-based protein of SEQ ID NO 1 may be prepared by techniques well known in the art in substantial accordance with the teachings of Brown, e_t &1 .
• the DNA sequence may be generated using conventional DNA synthesizing apparatus such as an Applied Biosystems Model 380A or 380B DNA synthesizer (commercially available from Applied Biosystems, Foster City, California) . Commercial services are also available for the construction of such nucleotide sequences based on the amino acid sequence.
• the coding sequence for a protein of SEQ ID NO 1 is the following:
• SEQ ID NO 2 This sequence encodes the following protein of SEQ ID NO 1:
• the coding sequence for a protein of SEQ ID NO 1 is the following:
• Coding regions for SEQ ID NO 1 may be fused to a leader sequence which signals the cell to secrete a precursor peptide of SEQ ID NO 1 that is subsequently processed by the cell during the secretion process to a protein of SEQ ID NO 1.
• leader sequences are know in the art.
• One well known and preferred leader sequence is the hybrid tissue plasminogen activator/protein C prepropeptide described in Berg et al . Bi ochem . Biophys . Res . Commun . 179: 1289-1296 (1991) .
• nucleotide sequences encoding precursor peptides of SEQ ID NO 1 are flanked by linker DNA to facilitate enzymatic ligation into expression vectors as is later exemplified herein.
• a suitable coding sequence of SEQ ID NO 1 is constructed and optionally fused and flanked by an appropriate leader sequence and linker DNA, the construct is ligated into an expression vector which is then introduced into an appropriate cell line.
• Construction of suitable vectors containing the desired coding and control sequences may be constructed by standard ligation techniques. Isolated plasmids or nucleotide fragments are cleaved, tailored, and religated in the manner necessary to achieve the desired plasmids.
• nucleotide sequence encoding the polypeptide into an appropriate recombinant nucleotide expression vector through the use of appropriate enzymes.
• the nucleotide sequence encoding a polypeptide of SEQ ID NO 1 is designed to possess restriction endonuclease cleavage sites at either end of the DNA to facilitate isolation from and integration into these amplification and expression plasmids.
• the coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of the coding sequence into the desired cloning vectors by techniques well known in the art.
• restriction endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed.
• the choice of restriction sites are chosen so as to properly orient the coding sequence such that it is properly associated with the promoter and ribosome binding site of the expression vector, both of which are functional in the host cell in which a compound of SEQ ID NO 1 is to be expressed.
• the expression vector may contain several other functional elements.
• One such element is the promoter and upstream regulatory sequences which control the level of expression of the protein of interest.
• Some expression vectors contain promoters and regulatory sequences which normally regulate transcription of cellular genes.
• One such promoter is the mouse metallothionein-I promoter which has been shown to function both in-vi tro and in-vivo (Palmiter et al., Nature 300: 611 - 615, 1982) .
• promoters and regulatory sequences from viruses are frequently used in expression vectors
• the expression vector also carries an origin of replication as well as marker sequences which are capable of providing phenotypic selection in transformed cells.
• the proteins useful in the present invention do not require post-translational processing mechanisms other than enzymatic removal of the propeptide leader sequence, many stable human cell lines are consistent with the practice of the invention.
• One preferred cell line is the human embryonal kidney cell line 293, available from the permanent collection of the American Type Culture Collection.
• a number of well known methods exist for introducing the genetic material into target cells such as chemical (calcium phosphate precipitation), physical (electorporation and microinjection) , and viral methods (adenovirus, retrovirus, and adeno-associated virus) (Methods for Gene Transfer. Gene Theranv. Mary Ann Liebert, Inc.,
• the plasmid pLP53-tLB was isolated from E. coli K12 AGl (on deposit under terms of the Budapest Treaty and made part of the permanent stock culture collection of the Northern Regional Research Laboratories (NRRL) , agricultural Research Service, U.S. Dept. of Agriculture, Peoria, IL 61604 under accession number NRRL B-18714) using the Plasmid Purification Midi Kit. (Qiagen, Inc., 9600 DeSoto Avenue, Chatsworth, CA 91311) .
• NRRL Northern Regional Research Laboratories
• the reaction was loaded into the preparative well of a 1.5% NuSieve GTG agarose (FMC Bioproducts, 191 Thomaston Street, Rockland, ME 04841) /TAE buffer gel and then electrophoresed. The gel was stained with ethidium bromide and the DNA was visualized by ultraviolet light. The digested DNA band was excised with a scalpel and placed into two micro-tubes. The DNA was purified from the low melting point agarose using the Wizard PCR Preps DNA Purification System (Promega, 2800 Woods Hollow Road, Madison, WI 53711-5399) .
• the Bglll-Mung Bean-Ayrll digested pLP53-tLB DNA was dephosphorylated (removal of 5' phosphate groups) by the addition of 2 ⁇ l (2 units) of calf intestinal alkaline phosphatase to the reaction.
• the sample was incubated at 37°C for an additional 30 minutes. Twenty-four ⁇ l of 5x stop mix was added.
• the sample was heated at 70°C for 15-20 minutes to inactivate the enzymes and then spin dialyzed using G-50 Sephadex Quick Spin columns (Boehringer Mannheim Corporation, P.O. Box 50414, 9115 Hague Road, Indianapolis, IN 46250-0414) in order to remove the AyrII produced small DNA fragments.
• the DNA was precipitated by addition of 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol. This mixture was mixed thoroughly and then chilled to -20°C. The precipitate was collected by centrifugation for 30 minutes. The supernatant was discarded and the pellet was washed with 700 ⁇ l of cold 70% ethanol. The sample was centrifuged for 15 minutes. The supernatant was discarded, the DNA pellet was dried and then resuspended in 25 ⁇ l of water.
• GLP-1.1 and GLP-1.2 are complementary DNA molecules.
• the synthetic DNA molecules were dissolved in water and stored at less than 0°C.
• GLP-1 double stranded DNA linker
• the linker was stored at -20°C.
• the GLP-1 linker In order to be able to ligate into the dephosphorylated Bglll-Mung Bean-Ayrll digested pLP53-tLB DNA segment, the GLP-1 linker must have 5' phosphate groups .
• the phosphate groups were added by the use of the enzyme T4 polynucleotide kinase.
• the kinase reaction contained 80 ⁇ l of the GLP-1 linker, 0.33 ⁇ M ATP, 70mM Tris- HCl (pH 7.6), lOmM MgCl2, lOOmM KCl, ImM ⁇ -mercaptoethanol and 37.2 ⁇ l (372 units) of T4 polynucleotide kinase.
• the reaction was incubated at 37°C for 30 minutes. Sixteen ⁇ l of 500mM EDTA was added to stop the reaction.
• the reaction was extracted once with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1) followed by an extraction with chloroform:isoamyl alcohol (24:1) .
• One hundred ⁇ l of the aqueous layer was spin dialyzed using G-50 Sephadex Quick Spin columns (Boehringer Mannheim) in order to remove the reaction components.
• the DNA was precipitated by addition of 0.1 volume of 3M sodium acetate (pH 5.2), 0.1 volume of lOOmM MgCl2 and 2.5 volumes of absolute ethanol. This mixture was mixed thoroughly and then chilled at -20°C. The precipitate was collected by centrifugation for 30 minutes.
• the supernatant was discarded and the pellet was washed with 700 ⁇ l of cold 70% ethanol. The sample was centrifuged for 15 minutes. The supernatant was discarded, the DNA pellet was dried and then resuspended in 10 ⁇ l of water.
• the DNA prepared in Example IA was ligated with linker GLP-1.
• Two ⁇ l of DNA prepared in Example IA and 4 ⁇ l of GLP-1 linker were ligated in a reaction that contained 2 ⁇ l (2 units) of T4 DNA ligase, 50mM Tris-HCl (pH 7.6), lOmM MgCl2, ImM ATP, ImM DTT, and 50% (w/v) polyethylene glycol- 8000 in a total volume of 10 ⁇ l.
• the mixture was incubated at 16°C for 16 hours.
• the ligation was used to transform E. coli K12 INV ⁇ F' cells as generally described below.
• Frozen competent E. coli K12 INV ⁇ F' cells were obtained from Invitrogen (3985 B Sorrento Valley Boulevard, San Diego, CA 92121) . Two ⁇ l of 0.5M ⁇ -mercaptoethanol were added to 50 ⁇ l of thawed competent cells. About 1-2 ⁇ l of the ligation reaction was mixed with the cells. The cell-DNA mixture was incubated on ice for 30 minutes, heat-shocked at 42°C for exactly 30 seconds and then chilled on ice for 2 minutes. The cell-DNA mixture was diluted into 450 ⁇ l of SOC media (2% tryptone, 0.05% yeast extract, lOmM NaCl, 2.5mM
• KCl lOmM MgCl2, 10mM MgS04 , 20mM glucose in distilled water
• a rotary shaker set at about 225 rpm.
• Aliquots of up to 200 ⁇ l were plated on TY- agar plates (1% tryptone, 0.5% yeast extract, 1% NaCl, and 1.5% agar, pH 7.4) containing lOO ⁇ g/ml ampicillin and then incubated at 37°c until colonies appear.
• ampicillin resistant cells were picked and inoculated into 3 ml of TY broth (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) containing lOO ⁇ g/ml ampicillin. These cultures were grown for about 16 hours at 37°C with aeration. Plasmid DNA was isolated from cultures using Wizard Minipreps obtained from Promega (2800 Woods Hollow Road, Madison, WI 53711-5399) . Recombinant plasmids were identified by digestion with restriction endonucleases followed by gel electrophoresis analysis.
• the plasmid pGT-h was isolated from E. coli K12 GM4£ (on deposit under terms of the Budepest Treaty and made part of the permanent stock culture collection of the NRRL under accession number B-18592 using the Plasmid Purification Midi Kit (Qiagen, Inc. ) .
• Calf intestinal alkaline phosphatase was used to remove the 5' phosphate groups from the DNA segment in order to prevent recircularization of the pGT-h.
• Ten ⁇ l of the Bell digested pGT-h DNA was treated with 1 ⁇ l (1 unit) of calf intestinal alkaline phosphatase in a 15 ⁇ l reaction containing 50mM Tris-HCl (pH 8.5 at 20°C) and 0.ImM EDTA. The reaction was allowed to proceed for 45 minutes at 37°C.
• the phosphatase was inactivated by the addition of l ⁇ l of 500mM EDTA and then heating at 65°C for 10 minutes.
• the reaction volume was increased to lOO ⁇ l with water and then extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) followed by extraction with chloroform: isoamyl alcohol (24:1) .
• the DNA was recovered from the aqueous layer by the addition of 0.1 volumes of 3M sodium acetate (pH 5.2) and 2.5 volumes of absolute ethanol. The mixture was mixed thoroughly and then chilled at -20°C. The precipitate was collected by centrifugation for 30 minutes. The supernatant was discarded and the pellet was washed with 700 ⁇ l of cold 70% ethanol. The sample was centrifuged for 15 minutes. The supernatant was discarded, the DNA pellet was dried and then resuspended in 25 ⁇ l of water.
• the gel was stained with ethidium bromide and then the DNA was visualized by ultraviolet light.
• the desired 213 base pair DNA band was excised using a scalpel.
• the DNA was purified from the low melting point agarose using Wizard PCR preps (Promega) .
• Example 2A The DNA prepared in Example 2A was ligated with DNA prepared in Example 2B.
• One ⁇ l of DNA from Example 2A and 10.5 ⁇ l of DNA from Example 2B were ligated in a reaction that contained 2 ⁇ l (2 units) of T4 DNA ligase, 50mM Tris-HCl (pH 7.6), lOmM MgCl2, ImM ATP, ImM DTT, and 50% (w/v) polyethylene glycol-8000 in a total volume of 20 ⁇ l .
• the mixture was incubated at 16°C for 16 hours.
• the ligation reaction was used to transform E. coli K12 iNV ⁇ F' as described in Example ID. Plasmid DNA was isolated from ampicillin resistant cultures as described in Example IE.
• the plasmid pGT-h+tLB+Val8GLP-l was constructed substantially in accordance with Examples 1 and 2. To accommodate the change in the amino acid sequence (Ala to Val at Xaa 1 in SEQ ID NO 1) the following coding sequences were substituted for those described in Example IB:
• the plasmid pMIOO (pOKl2) was isolated from E. coli K12 RR1 ⁇ M15 using Magic Minipreps (Promega) . See Vieira and
• the plasmid pBK-neo 1 (described in U S Patent No: 5,550,036, herein incorporated by reference, and available from the American Type Culture Collection under terms of the Budapest Treaty via Accession No. Atcc 37224) was isolated from E. coli K12 HB-1 using the large scale alkaline lysis method. Three ⁇ l (26.3 ⁇ g) of pBK-neo DNA was digested to completion with 7 ⁇ l (15 units) of EcoRI in a reaction volume of 200 ⁇ l containing 50mM Tris-HCl (pH 8.0), lOmM MgCl2, and lOOmM NaCl. The sample was incubated at 37°C.
• Example 4A The DNA prepared in Example 4A was ligated with DNA prepared in Example 4B.
• Two ⁇ l of DNA from Example 4A and 2 ⁇ l of DNA from Example 4B were ligated in a reaction that contained 1 ⁇ l (1 unit) of T4 DNA ligase, 50mM Tris-HCl (pH 7.6), lOmM MgCl2, ImM ATP, ImM DTT, and 50% (w/v) polyethylene glycol-8000 in a total volume of 16 ⁇ l.
• the mixture was incubated at 16°C for 16 hours.
• Ten ⁇ l of the ligation reaction was used to transform E. coli K12 RR1 ⁇ M15 as described in Example 5D.
• kanamycin resistant cells were picked and inoculated into 5 ml of TY broth (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) containing kanamycin. These cultures were grown for about 8 hours at 37°C with aeration. Plasmid DNA was isolated from cultures using Magic Minipreps obtained from (Promega) . Recombinant plasmids were identified by digestion with restriction endonucleases followed by gel electrophoresis analysis. To obtain larger amounts of pMlOO-neo plasmid DNA, large scale isolation was performed using the Plasmid Purification Kit (Qiagen, Inc.) .
• the plasmid pGT-h was isolated as described in substantial accordance with Example 2A. Three ⁇ g (4.6 ⁇ l) of pGT-h DNA was digested to completion with 1 ⁇ l (8 units) of Bstll07l in a 10 ⁇ l reaction volume containing lOmM Tris-HCl, lOmM MgCl 2 , lOOmM KCl (pH 8.5 at 37°C) . The sample was incubated at 37°C for 1 hour. The Bstll07I digested pGT-h DNA was purified from the reaction components using the Wizard DNA Clean-Up System (Promega) . The Bstll07l digested pGT-h DNA was concentrated in a 6.7 ⁇ l volume using a microcon 50 (Amicon, Inc. 72 Cherry Hill Drive, Beverly, MA
• the 6.7 ⁇ l of Bstll07l digested pGT-h DNA was further digested with 1 ⁇ l (10 units) of Bell in a reaction volume of 10 ⁇ l containing 50mM Tris-HCl (pH 8.0), lOmM MgCl2 / 50mM NaCl. The sample was incubated at 50°C for 1 hour. To prevent any possible recircularization, the Bstll07l-Bcll digested pGT-h was dephosphorylated (removal of 5' phosphate groups) by the addition of 1 ⁇ l (1 unit) of calf intestinal phosphatase to the sample. The sample was incubated at 37°C for 30 minutes.
• Gel loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in water) was added to the reaction.
• the reaction was loaded into the preparative well of a 1% SeaPlaque GTG agarose/TAE buffer gel and then electrophoresed. The gel was stained with ethidium bromide and then the DNA was visualized by ultraviolet light.
• the desired 1.8 Kb DNA band was excised with a scalpel and placed into a micro-tube.
• the DNA was purified from the low melting point agarose using the Wizard PCR Preps DNA Purification System (Promega) .
• the DNA prepared in Example 5A was ligated with the DNA prepared in Example 5B and the DNA prepared in Example 3 (pLP53-tLB+Val8GLP-l BamHI fragment) .
• Four ⁇ l of DNA from Example 5A, 3 ⁇ l of DNA from Example 5B and 12 ⁇ l of DNA prepared in Example 3 (pLP53-tLB+Val8GLP-l BamHI fragment) were ligated in a reaction that contained 1 ⁇ l (1 unit) of T4 DNA ligase, 50mM Tris-HCl (pH 7.6) , lOmM MgCl2, ImM ATP, ImM DTT, and 50% (w/v) polyethylene glycol-8000 in a total volume of 25 ⁇ l .
• Example 6 Construction of Stable Cell 3 ines Plasmid DNA, either pGT-h+tLB+GLP-1, pGT- h+tLB+val8GLP-l, or pMT-h+tLB+val8GLP-l was transfected into human embryonic kidney 293 cells using the stable CaP0 4 method contained in the Mammalian Transfection Kit available from Stratagene (11011 North Torrey Pines Road, LaJolla, CA 92037) .

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