GLP-1 PROMOTER MEDIATED INSULIN EXPRESSION FOR THE TREATMENT OF DIABETES

Insulin gene therapy is one of many envisioned alternative treatments of diabetes. Diabetes gene therapy would be possible if insulin could be produced in a regulated and specifically in a sensitive manner dependent on the blood glucose level. Therefore, the present invention relates to a method for the isolation of GLP-1 expressing cells, to nucleic acids sequence construction or vectors useful for isolating GLP-1 expressing cell and to the GLP-1 expressing cells isolated therewith. Furthermore, the invention relates to a method of nucleic acids sequence construction or vectors under the control of the GLP-1 promoter expressing insulin in a recombinant GLP-1 expressing cell line. The cells of the present invention are particular useful for the treatment of diabetes and may be used in a gene therapy approach to treat diabetes and other disorders related to the nutrient metabolism.

DETAILED DESCRIPTION OF THE INVENTION

The GLP-1 promoter (glucagon) was obtained from the rat genomic sub clone Glu.BS plasmid containing the glucagon promoter (−2300 bp), the first exon and 100 bp of first intron of the rat glucagon gene in the pBS-SK+ (pBluscript phagemid vector) (FIG. 1). The Glu.BS plasmid was used as a source for the GLP-1 promoter sequence (Gosmain et al., 2007).

Previous studies demonstrated that ˜2300 bp fragment of rat proglucagon sequence is essential for the expression of GLP-1 gene in intestinal L cell (Jin et al., 1995). The sequence of rat glucagon was checked on gene bank (ref|NW—047655.1) (Appendix 1). A fragment of rat proglucagon gene (pro Glu) was amplified from Glu.BS plasmid by PCR. Table 1 shows the sequences of primers and the position of restriction sites. Spe I (−2214) and Hind III (+77) sites were included on the upstream and downstream primers, respectively to facilitate subsequent cloning.

The Insulin Gene

The human insulin gene was obtained from a human genomic DNA. The genomic DNA was extracted from human blood by manual method. The sequence of human insulin was checked on gene bank (ref|NG—007114.1) (Appendix 2). Based on previous studies, about 1800 bp of insulin gene constitutes of introns, exons and other fragments that are needed for insulin expression.

The fragment of human insulin gene was amplified by PCR from human genomic DNA. The sequences of primers and the position of restriction site are showed in the table 2. The Sal I (+18) and BamH I (+1844) restriction sites were designed upstream and downstream of primers to facilitate subsequent cloning.

Purification of PCR Products

Following amplification, PCR products (GLP-1 promoter and insulin gene) were purified from agarose gel to omit undesired bands, primer dimmers and leftover of PCR mixture by DNA Gel Extraction kit.

Ligation with pJET1.2 Cloning Vector

Pure PCR products (GLP-1 promoter and Insulin gene) were sub-cloned into the pJET1.2 cloning vector. The pJET1.2 cloning vector is an advanced positive selective system for the highest efficiency cloning of PCR products. Additionally, this system increases the effectiveness of restriction enzyme activity by creating enough space to be placed on the restriction sites. Moreover, sequencing of PCR products are more convenient in the plasmid form. This vector contains a lethal gene, which is disrupted by ligation of a DNA insert into the cloning site. As a result, only cells with recombinant plasmids are able to propagate (FIG. 2). The recombinant plasmids are named GLP-1pro/pJET (GLP-1 promoter inside the pJET1.2 cloning vector) and Ins/pJET (Insulin gene inside the pJET1.2 cloning vector) (FIG. 3).

Transformation into TOP-10

The ligation products (GLP-1pro/pJET and Ins/pJET) were transformed into the bacteria competent cells by head shock method to amplify plasmids construct (FIG. 3). TheE. colistrain TOP-10 was employed as bacterial host for propagation of plasmid in whole project. Competent bacterial cells were prepared by treating the cell with a divalent cation like calcium chloride. The pJET1.2 cloning vector includes Ampicillin selectable marker (antibiotic resistance markers) that allows only cells that receive recombinant vector, grow in the selective medium. Nevertheless, these selection steps did not absolutely guarantee that the DNA insert was present in the cells. Further investigations of the resulting colonies were performed to confirm that cloning was successful. These were accomplished by means of restriction mapping analysis and DNA sequencing.

Plasmid Extraction

Some single colonies randomly chose and were cultured on the selective medium to grow overnight. Recombinant plasmids were isolated from the bacterial by plasmid miniprep kit for further analysis. The size of GLP-1pro/pJET is about 5265 bp and Ins/pJET is about 4800 bp (FIG. 3).

Restriction Mapping Analysis

Ins/pJET plasmid were digested by Sal I and BamH I restriction enzymes and GLP-1 pro/pJET plasmid were cut by Spe I and Hind III restriction enzymes to examine the correctness of the plasmid structure. Consequence of Ins/pJET plasmid digestion with Sal I and BamH I, were two fragments, insulin gene with the size of 1826 bp (insert) and linear pJET1.2 cloning vector with the size of 2974 bp (vector). In addition, consequence of GLP-1 pro/pJET plasmid digestion with Spe I and Hind III were two fragments, GLP-1 promoter with the size of 2291 bp (insert) and linear pJET1.2 cloning vector with the size of 2974 bp (vector). Only the colonies that produce these fragments during digestion analysis were selected for next experiments.

Sequencing

Random colony samples which have gone through extraction of Ins/pJET and GLP-1 pro/pJET plasmid were sent for sequencing analysis to confirm the correctness of nucleotides sequence of insulin gene and GLP-1 promoter. The results of sequencing were compared with sequence of rat GLP-1 promoter and human insulin gene in gene bank database (ref|NW—047655.1 and ref|NG—007114.1) (Appendix 1, 2).

To construct GLP-1/Ins/pbud plasmid, the pBudCE4.1 was employed as cloning vector. The pBudCE4.1 vector was designed for simultaneous expression of two genes in mammalian cell line. The vector contains the two promoters (CMV and EF-1α promoter) and two multiple cloning sites that allow independent expression of two recombinant proteins. The pBudCE4.1 includes Zeocin resistant gene for selection inE. colias well as serves to create stable mammalian cell line. MostE. colistrains are suitable for the growth of this vector including TOP-10 and DH5α (FIG. 4).

It should be noted that, CMV promoter and EF-1α promoter was eliminated in the new construct development, because the aim of the project is to study of GLP-1 promoter ability to express insulin gene, so to avoid complication and confusion with the GLP-1 promoter, promoters of the vector were deleted. Therefore, EF-1α promoter was omitted completely and CMV promoter was replaced with GLP-1 promoter.

In order to omit EF-1α promoter, the pBudCE4.1 vector was digested with Nhe I and Not I restriction enzymes. Next, pBud vector band was purified from agarose gel to omit undesired bands (EF-1α promoter) as well as any leftover mixture of digestion by DNA Gel Extraction kit. The pBud vector (“pBud pro EF less”) which now has lost its EF-1α promoter has two different sticky ends that are not able to match with each other because it was digested by two different restriction enzymes. In order to construct the circle vector, the “pBud pro EF less” fragment was treated by Klenow Fragment enzyme to make blunt ends. The blunt ends facilitate subsequence ligation in order to recircle the vector (FIG. 5).

The treated fragment was ligated by T4 DNA ligase enzyme to attach the two blunt ends with each other and make circle “pBud pro EF less” vector (FIG. 5). This new vector was employed in producing GLP-1/Ins/pbud plasmid.

The insulin gene and GLP-1 promoter were inserted into the “pBud pro EF less” vector in two steps. At first, the Ins/pJET plasmid (containing Human Insulin gene) and “pBud pro EF less” vector were digested by suitable restriction enzymes (Sal I and BamH I) to create insulin gene (insert) and linear pBud vector with same sticky ends. These digested fragments were purified from gel electrophoresis by Gel DNA Recovery Kit to omit undesired fragments. Insert (insulin) and vector (pBud pro EF less) were ligated to construct Ins/pbud plasmid include insulin gene in the Sal I and BamH I site (FIG. 6A). The ligation product was transformed into theE. colistrain TOP-10 as host bacterial for propagation of plasmid.

Single colonies obtained from Ins/pbud plasmid transformation process were extracted to check the correctness of plasmid content. In this order, some single colonies were randomly selected to extract their plasmid. The plasmids were digested by Sal I and BamH I restriction enzymes. The plasmids that contain insulin gene had two fragments on the gel that were the same size in compare with the insert (insulin gene 1826 bp) and vector (pBud pro EF less vector 3400 bp).

In the second step, GLP-1 promoter was inserted to the Ins/pbud plasmid in such a manner that it was placed upstream of the insulin gene (FIG. 6B). In this case, the GLP-1pro/pJET plasmid (containing rat GLP-1 promoter,FIG. 3) and Ins/pbud were digested with Spe I and Hind III restriction enzymes to generate GLP-1 promoter fragment (as insert) and linear Ins/pbud fragment (as vector) with sticky ends. These digested fragments were purified from gel electrophoresis by Gel DNA Recovery Kit to omit undesired fragments. Insert (GLP-1 promoter) and vector (Ins/pbud plasmid) were ligated to construct GLP-1/Ins/pbud plasmid include GLP-1 promoter in the Spe I and Hind III sites and insulin gene in the Sal I and BamH I sites (FIG. 6B). The ligation product was transformed into theE. colistrain TOP-10 as host bacteria for propagation of plasmid.

The accomplishment of GLP-1/Ins/pbud plasmid transformation was examined by analyzing several single colonies. In this order, some colonies randomly were selected to extract their plasmid. The plasmids were digested by Spe I and Hind III restriction enzymes. The correct plasmids have two fragments on the gel that were the same size in compare with the insert (GLP-1 promoter 2291 bp) and vector (Ins/pbud plasmid 4790 bp).

One sample from extraction of GLP-1/Ins/pbud plasmid was sent for sequencing analysis to confirm the correctness of nucleotides sequence of insulin gene and GLP-1 promoter. The positions of primers that used for sequencing of GLP-1/Ins/pbud are showed inFIG. 7and the sequences of primers are listed in table 3. The results of sequencing were compared with sequence of rat GLP-1 promoter and human insulin gene in gene bank database (ref|NW—047655.1 and ref|NG—007114.1) (Appendix 1, 2).

TABLE 3The sequence of primers that used for sequencingof GLP-1/Ins/pbud plasmidPrimers for sequencing of GLP-1/Ins/pbudProM-R5′ AC AAC ACT AGT GCT TCC AGT CAA ACC 3′LP-V5′ G ACG TCA AAA TTC ACT TCA GAG AGC 3′LPC-F5′ G CTA AAT CTG GGT GTC CAA GTG 3′LPC-R5′ A AGC TCC ATG TCC ACC AGT TAG 3′InsCo-R5′ A TAG GAT CCA CAG GGA CTC CAT CAG 3′INC-F5′ CT CAC GGC AGC TCC ATA GTC 3′INC-R5′ TGT TCC ACA ATG CCA CGC TTC 3′

Construct Plasmid for L Cell Selection

Neomycin Gene

Suitable selected marker for mammalian cell line is needed to be expressed under GLP-1 promoter to extract L cells from heterogeneous population of STC-1 cell line. In this order, neomycin resistant gene causing resistance against geneticin antibiotic in mammalian cell line was placed downstream of the GLP-1 promoter in the new constructs. After transfection of the STC-1 cell line with plasmid containing neomycin resistant GLP-1 promoter, the cells only could determine GLP-1 promoter (L cell respectively) and express neomycin resistant protein were able to survive under geneticin antibiotic treatment condition.

The neomycin resistant gene was amplified from pcDNA3 plasmid by PCR with two specific primers that include restriction enzyme sites (EcoR I and Xba I respectively) to facilitate subsequent cloning (Table 4) (Appendix 3). The PCR product with 1202 bp fragment was purified from agarose gel to omit undesired bands, primer dimmers and leftover PCR mixture. Pure PCR product was sub-cloned into the pJET1.2 cloning vector to construct Neo/pJET plasmid (FIG. 8A). The ligation product was transformed into theE. colistrain TOP-10 competent cells to amplify new construct.

The Neo/pJET plasmid was digested by EcoR I and Xba I restriction enzymes. Consequence of Neo/pJET plasmid digestion was neomycin resistant gene with size of 1202 bp (insert) and pJET1.2 cloning vector with size of 2974 bp (vector). For confirmation, one sample from extraction of Neo/pJET plasmid was sent for sequencing analysis to check the correctness of nucleotides sequence of neomycin resistant gene. The result of sequencing was compared with sequence of neomycin resistant gene in pcDNA3 plasmid sequence (ACCESSION EF550208). The single colony that had correct structure and sequence was selected for next experiment.

Insertion of Neomycin Gene into the pBluescript Plasmid:

The pBluescript II phagemid (plasmid with a phage origin) is cloning vector designed to simplify commonly used cloning procedure. This vector has an extensive polylinker with unique restriction enzymes to facilitate insertion of new fragments (FIG. 8).

The neomycin resistant gene was inserted to the pBluescript plasmid in such a manner that it was placed between EcoR I and Xba I resistant sites (FIG. 8B). In this case, Neo/pJET plasmid and pBluescript vectors were digested with the same restriction enzymes, EcoR I and Xba I, to generate linear neomycin resistant gene fragment (as insert) and linear pBluescript vector with sticky ends. These digested fragments were purified from gel electrophoresis by Gel DNA Recovery Kit to omit undesired fragments. Insert (neomycin resistant gene) and vector (pBluescript plasmid) were ligated to construct Neo/pblu plasmid include neomycin resistant gene in the EcoR I and Xba I sites (FIG. 8B). The ligation product was transformed into theE. colistrain TOP-10 as host bacteria for propagation of plasmid.

The accomplishment of Neo/pblu plasmid transformation was again examined by analysing several single colonies. In this order, some colonies randomly were selected to extract plasmid. The plasmids were digested by EcoR I and Xba I restriction enzymes. The correct plasmids had two fragments on the gel that were the same size in compare with the insert (Neomycin resistant gene 12002 bp) and vector (pBluescript plasmid 3000 bp).

One sample from extraction of Neo/pblu plasmid was sent for sequencing analysis to confirm the correctness of nucleotides sequence of neomycin resistant gene. The results of sequencing were compared with neomycin resistant gene in gene bank database (ACCESSION EF550208).

PCR GLP-1 with New Primers

To construct the GLP-1/Neo/pblu plasmid, GLP-1 promoter was placed upstream of neomycin gene in the EcoR I and Xho I restriction sites. In this order, GLP-1 was amplified with other primers include EcoR I and Xho I restriction enzyme sites. Sequences of forward and reverse primers to amplify GLP-1 promoter are showed in the table 5. Primers include EcoR I and Xho I restriction enzyme sites.

The GLP-1 fragment was purified from the agarose gel by the Gel DNA Recovery Kit, and then was sub-cloned into the pJET1.2 cloning vector to construct GLP-1-Ex/pJET plasmid (FIG. 10A).

The GLP-1 promoter was inserted to the Neo/pblu plasmid to produce GLP-1/Neo/pBlu plasmid. At first, the GLP-1 EX/pJET plasmid (FIG. 10A) and Neo/pblu plasmid (FIG. 8B) were digested by suitable restriction enzymes (Xho I and EcoR I) to create GLP-1 promoter fragment (insert) and linear Neo/pBlu vector with same sticky ends. These digested fragments were purified from gel electrophoresis by Gel DNA Recovery Kit to omit undesired fragments.

Next, Insert (GLP-1 promoter) and vector (Neo/pBlu plasmid) were ligated to construct GLP-1/Neo/pBlu plasmid include GLP-1 promoter in the Xho I and Ecor I sites and neomycin gene in the downstream of GLP-1 promoter in the position of EcoR I and Xba I sites (FIG. 10B). The ligation product was transformed into theE. colistrain TOP-10 as host bacterial for propagation of plasmid. The correctness of plasmid structure was considered by restriction enzyme mapping and sequencing. The positions of primers that used for sequencing of GLP-1/Neo/pbud are showed inFIG. 11and the sequences of primers are listed in table 6 (Appendix 3).

In Vitro Study

STC-1 cell line was derived from an endocrine tumor of the intestine (Rindi et al., 1990). It has been demonstrated that ˜7% and 5% of this heterogeneous population of cells produce immunoreactive glucose dependent insulinotropic polypeptide (GIP) and glucagon like polypeptide I (GLP-1), respectively. In addition, there was no immunoreactivity detected for insulin antibodies in STC-1 cell line (Rindi et al., 1990). Since, STC-1 cell line is suitable source of L cells; it was applied for in vitro studies.

The concentration of 5×104 cells/ml is proper for primary culture. Based on previous studies, STC-1 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum under an atmosphere 5% CO2 and 37° C. (Rindi et al., 1990). The media of culture was changed in regular interval. Then, the cells were passaged in the new flasks.

MTT Assay

For assessment of antibiotic cytotoxicity, a common methodology is the MTT assay which has been widely used as a colorimetric approach based on the activity of living cells. MTT assay is a standard assay (an assay which measures changes in color) for measuring cellular proliferation. Yellow MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in the mitochondria of living cells. The absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually between 500 and 600 nm) by a spectrophotometer.

The pBudCE4.1 and pBluescript plasmids were employed for expression of insulin gene and neomycin gene, include zeocin and geneticin (neomycin) resistant gene respectively.

Therefore, the MTT assay was done for both antibiotics to determine the appropriate concentration of the antibiotic that kills the entire STC-1 cells lacking the antibiotic resistant gene. In this case, STC-1 cells (without any antibiotic resistant gene) were treated with different concentration of zeocin and geneticin antibiotic. The concentration of antibiotics in the culture media was in the range of 0 to 1 mg/ml in 12 wells (Table 7).

TABLE 7Concentration of zeocin and ampicilin antibiotic in the culture mediaCon. Antug/ml0501002003004005006007008009001000DMEM180179.5179178177176175174173172171170Serum202020202020202020202020Anti00.512345678910Total200200200200200200200200200200200200

Optical density of solutions was read at 560 nm on an ELISA plate reader. The absorbance of colored solution is directly proportional to the number of cells. Based on MTT assay result, the concentration of geneticin and zeocin antibiotic that are able to kill all the STC-1 cells (without antibiotic resistant gene) were 400 ug/ml and 500 ug/ml.

Transfection of pGLP-1/Neo/pBlu Plasmid

The L cell line was isolated from heterogeneous population of STC-1 cell line by pGLP-1/Neo/pBlu plasmids. This plasmid is able to express neomycin resistant gene under control of GLP-1 promoter. So, recombinant constructed plasmid (pGLP-1/Neo/pBlu plasmids) was transfected to the STC-1 cell line by transfection reagent (Lipofectamine), according to manufacturer's protocol. Selection of stable clones was performed by replacing medium the day after transfection with complete medium, supplemented with proper amount of G418 (Geneticin antibiotic) that measured in MTT assay (400 ug/ml). Medium was changed every 2-3 days, until individual clones of transfected cell appeared. Stable transfected cell clones were isolated for next step analysis.

RT-PCR for Mouse GLP-1 Gene

Expression of mouse GLP-1 mRNA was detected by reverse transcription reaction by PCR to confirm the success of transformation work that has been carried out on the L cell line. GLP-1 protein is expressed cell specifically, so just L cells are able to produce GLP-1 mRNA. In this case, the result of RT-PCR approved the present of GLP-1 mRNA in the mouse L cell line that was extracted from STC-1 cell line.

Total RNA was extracted by using RNA Extraction Kit, according to manufacturer's protocol. Then, extracted RNA was digested with DNase I (free RNase). RT-PCR was carried out with total RNA according to proposed step in RT-PCR kit. The PCR reaction was carried out in a 30 ul final volume containing primers for control mRNA (mouse β-actin) and mouse GLP-1 mRNA. Primers were designed to amplify nucleotides 204-762 of coding sequence for mouse β-actin and 265-515 of the coding sequence for mouse glucagon (GLP-1) mRNA. Theses primers bind within two different exons, therefore, products generated from mRNA and genomic DNA can be easily distinguished. The upstream and downstream primers are used to amplify β-actin and GLP-1 mRNA are listed in table 8 and 9 respectively.

The result of RT-PCR was analyzed on the electrophoresis gel in comparison to DNA ladder to check the correctness of products sizes. The products of β-actin and GLP-1 RT-PCR were 558 bp and 250 bp respectively (FIG. 12).

Transfection of pGLP-1/Ins/pBud Plasmid

To study the insulin expression in the L cell line, the GLP-1/Ins/pBud plasmid was transfected to the extracted L cell line according, to manufacturer's protocol. Selection of stable clones was performed by replacing medium the day after transfection with complete medium, supplemented with proper amount of zeocine antibiotic that has been measured and identified in the MTT assay (500 ug/ml). Medium was changed every 2-3 days, until individual clones of the transfected cells appeared. Stable transfected cell clones were isolated for the next step analysis.

The expression of the insulin protein into the L cell line was evaluated by immunocytochemistry test. In this method, mouse monoclonal antibody against human insulin as primary antibody and goat polyclonal antibody against mouse IgG conjugated with fluorescein isothiocyanate (FITC) as secondary antibody were used. The L cells were grown on 6 well tissue culture plates, containing sterilize glass coverslip before the day of transfection. After 48 h, transfected cells were fixed with 4% Paraformaldehayde. Then, the cells were incubated in 0.1% triton X-100 for permeabilization and then in 3% BSA (bovine serum albumin) for blocking. The slide was then overlaid with primary monoclonal antibody diluted at ratio 1:100 for overnight. Next, the slide was incubated with secondary antibody conjugated with FITC, diluted at 1:100 in TTBS, at RT for 2 h. Following that, the slide was incubated with DAPI nucleic acid stain to dye the nucleus of cells. Finally the slides were analyzed by an inverted phase contrast microscope with fluorescence light.