MXPA01000438A - Treatment of diabetes with synthetic beta cells - Google Patents

Abstract

Hepatocytes transfected with a replication deficient adenovirus vector containing a gene cassette expressing the proinsulin gene in response to physiological levels of glucose provide novel beta islet replacement cells. The cassette comprises the structural gene for human proinsulin genetically altered to make it cleavable to active insulin, a promoter operably linked to the proinsulin gene, and a glucose regulatory response module located 5'to the promoter. Synthesis of proinsulin mRNA is ablated at less than 5mM glucose, and peaks at about 15 mM.

Description

BETA SYNTHETIC CELLS FOR THE TREATMENT OF THE DLABETES Field of the Invention This invention relates to the field of gene therapy and to a method of using cells other than normal islets transfected with a proinsulin gene inducibly expressed in such cells in the presence of glucose. The proinsulin synthesized in the cells is further processed in mature insulin.

Background of the Invention Insulin dependent diabetes mellitus (IDDM or Type I Diabetes) occurs when an autoimmune response destroys the beta cells of the islets of Langerhans, leading to the cessation of insulin production. For many years, and indeed for many patients still in the present, the only recourse to treat this fatal condition is the periodic administration of injectable insulin of the animal, or more recently, of recombinant human origin. Although the administration of exogenous insulin is lifesaving for a prolonged period, severe side effects, such as circulatory disturbances, leading to blindness, gangrene, and heart attack are common. The insulin doses injected into the diabetic patient are only approximate, even when careful diet controls are implemented. These continuous imbalances in blood glucose that result from deviations from optimal insulin levels are thought to cause, or contribute to, the side effects observed. There have been many attempts to minimize the side effects of insulin therapy. Home urine and blood glucose tests or sets, help the diabetic to check blood sugar levels. Some researchers have proposed pumping devices which dose insulin continuously and in more precise doses. An interesting device of them is described in U.S. Pat. No. 5,364,838 which provides insulin in the form of aerosol. The hormone is absorbed through the lung, thus avoiding the route of administration of the most invasive injection. In the field of diabetes there is a general recognition that the replacement of beta cell function could be a superior therapy with respect to the administration of insulin, because .A'tfe »M natural cells could secrete insulin in response to glucose levels in the micro environment. This fine tuning control could therefore eliminate the deleterious side effects of insulin administration. Actually, it has been shown in the relatively small group of patients who have successfully received pancreatic transplants that results in remission of side effects or cessation of progressive tissue damage. Unfortunately, pancreatic transplantation is available only to a few diabetics, compared to the high number of people afflicted. In addition, transplantation is associated with significant toxicity due to immunosuppressive therapy. There have been many proposed alternatives to provide the benefits of beta cell replacement without involving organ transplantation. These alternatives involve the replacement of beta cell function with real beta cells or other insulin-secreting pancreatic cell lines, as described in Lacy, et al., Ann. Rev. Med., 37:33 (1986). Since the introduction of exogenous cells into the body is perceived by the immune system as any other allograft, it is necessary to isolate cells from contact with cells and substances immunoactive. In particular, the cells of the donor islets must be protected from T cells and macrophages that have a mediating effect on the cytolytic processes. One approach is physical immunoisolation, either by microencapsulation or by a microporous chamber. The challenge in these technologies is to overcome the natural process of rejection of foreign bodies that results in the separation of the implant by a dense layer of the fibroblasts. The key to a successful implant is to provide both a biocompatible surface and sufficient porosity to allow some degree of microvascularization. For a general review of these approaches, see Brau er, et al., J. Biomed. Mat. Res., 29: No. 12, 1995. To date, immunoisolation still has significant problems including loss of viability and function of captured cell populations, rejection of foreign bodies, fissures and inflammation, and reactions immune. Another approach is to design the function of the beta cell in cells derived from autologous tissue or artificially constructed cell lines. The U.S. Patent No. 5,427,940 describes an artificial beta cell produced by the design of endocrine cells in the secretion cells of At-T-20 ACTH. A cell Stably transfected At-T-20 ins is obtained by the introduction of the cDNA encoding human insulin and the GLUT-2 glucose transporter gene excited by the constitutive CMV promoter. The cell line already expresses the correct isoform of the glucokinase required for the expression of the glucose response of the insulin gene. This cell line functions in response to glucose, but is regulated at a secretagogo level below the physiological range or range. Therefore, although the system is of interest, it is not of clinical significance because an animal in which these cells are introduced could be chronically hypoglycemic. Another disadvantage is that the cells, which are derived from a heterologous source, carry their distinctive foreign antigens, and must be used in the immunoisolation. A further disadvantage is that At-T-20 is a transformed cell line with potential for unlimited growth. The U.S. patent No. 5,534,404 describes another approach to obtain a cell line regulated by the secretagogue correctly. Starting from the beta-TC-6 cells, the populations of the cells are selected at an initial stage by a cell sorter capable of recognizing the cells having an increased internal concentration of the calcium ion, associated with the expression of insulin (fluorescence). activated with Ca ++). After successive passes, the populations of the cells are further selected, which respond to glucose in the physiological range of 4 to 10 mM in a typical sigmoidal curve. For therapeutic use, the cells were encapsulated in alginate bound by a selective hollow fiber membrane by PAN / PVC permeation according to the Dionne method (U.S. Patent Application No. PCT / US92 / 03327). 10 Valera et al., FASEB Journal, 8: 440 (1994) describes transgenic mouse hepatocytes expressing insulin under the control of the PEPCK promoter excited by P-enolpyruvate. The PEPCK promoter is sensitive to the glucagon / insulin ratio and is activated in elevated glucose states. The chimeric insulin / PECK gene was introduced into the fertilized mouse eggs. Under the conditions of suppression of severe islets by streptozotocin, the production and secretion of intact insulin by the liver compensated for the loss of islet function. The strategy of genetic therapy for the treatment of diabetes is complicated by the complexity of the regulation of insulin and the structure of the protein itself. The release in response to the insulin glucose from islet beta cells is a ^ • * & AÍ? "Complex event that involves the migration of the preprocessed protein from the cytoplasm to the Golgi apparatus where the secretory granules fully sprout and travel to and fuse with the plasma membrane prior to release. The product of the initial protein is preproinsulin which has a sequence of the N-terminal signal, which is segmented during transport to the crude endoplasmic reticulum. After this the resulting proinsulin is further processed to insulin by the removal of the C peptide that binds the two polypeptides of the mature molecule, the A and B chains. In the design of insulin production in a host cell, it is impossible to obtain a functional insulin only providing polypeptides A and B. The synthesis of an intact molecule is necessary for proper folding, and only after the correct conformation is obtained, the C-peptide can be removed by trimming. Accordingly, any designed cell expressing mature functional insulin must have the machinery of the Kex2 enzyme, including the endopeptidases PC1 / PC3 and PC2, or a functional substitute thereof, as suggested by Newgard, Biotechnology, 10: 1112 ( 1992). The control of the production and release of insulin is further complicated by the regulation of the glycolytic flow. It is believed that two proteins are used by the beta cell to detect changes in glucose levels: Glut-2 is a glucose transporter of the specific facilitated diffusion type, and a particular glucose phosphorylation enzyme, glucokinase IV. 5 Both enzymes have a higher FCm and Vmax than the other enzymes in their related families. Both also have high affinities towards glucose that lead to large changes or shifts in activity over the physiological range of glucose concentration. Although the reduction in GLUT-2 leads to depression in insulin production, the loss of glucokinase abruptly stops the production of insulin, and identifies the phosphorylation of glucose as the limiting step of true velocity. The transformation of cells with the genes expressible for these enzymes seems to restore the regulatory characteristics in response to glucose with respect to the production of insulin, but not infrequently outside the physiological range of control. The experiences of many researchers have had to underline the problems inherent in the complexity of the control of insulin production through the manipulation of the metabolic utilization of glucose. There is a need in the field of diabetes for a new model of regulation of insulin and replacement of beta cells.

Brief Description of the Invention Control of insulin production in synthetic beta cells can be effected by alternative regulatory routes rather than through the attempted restoration of natural control over a system of expression of transformed beta cells. Although the actual release of insulin in normal beta cells is modulated through metabolic intermediates, as is still poorly understood, an alternative control is at the level of transcription of the mRNA encoding the proinsulin precursor. Accordingly, it is an object of the present invention to provide a control system for the expression of the proinsulin gene in a suitable host cell, which is independent of the metabolic effectors and intermediates involved in normal regulation. It is a further object to provide an autologous replacement beta cell with the patient's own cells to avoid a requirement of immunoisolation of the cells that produce the insulin. Ideally, a population of cells will be selected which can be designed to synthesize insulin depending on the transcription of the regulated gene, without excision and extracorporal manipulation outside the body. It is a further object to provide a gene therapy that utilizes a cell population that has a glucose transport and phosphorylation system that functions normally and intact, so that control of insulin production is a function of transcriptional control alone. Consistent with this object is the provision of an enzyme system capable of generating an active insulin or an insulin-like analogue from proinsulin, without subjecting it to the feedback intervention of the glycolytic pathway. According to the present invention, a ette of the gene for the expression of proinsulin in autologous host cells comprises a nucleotide sequence encoding the proinsulin DNA of substantially complete length, more preferably the cDNA of insulin, operably linked to a promoter recognized by an RNA polymerase contained in the host cells, together with a glucose response regulator module having at least two glucose-inducible regulatory elements located upstream at the 5 'end of the promoter. The ette is integrated into a vector comprising a viral genome defective in replication, capable, when an appropriate target cell or target is infected in vitro, of packaging the vector in a t-afa ¿tafejfi ^ fea - f infectious viral particle for autologous host cells. The target host cell or preferred target is the hepatocyte because the liver cells already express GLUT-2 and glucokinase IV sufficiently to generate the appropriate intermediate compounds for the glucose-regulated transcriptional control of the proinsulin gene in the physiological interval. Hepatocytes also express the furine endopeptidase. A mutation can be introduced into the reading frame of the proinsulin gene that allows segmentation of the furin at the appropriate site to obtain substantially complete cleavage of the C-peptide with the appearance of essentially natural insulin activity. It is therefore an aspect of the present invention that in the transfection method, a vector is provided in which the transcriptionally controlled production of proinsulin is substantially completely converted to the active hormone, which is constitutively secreted in the parenchyma of the liver in response to the elevation in glucose concentration. * - & ~ Á (&9¡¡kt $ sM + * ¿s &amp - ^ Brief Description of the Drawings Figure 1 is a schematic view illustrating the nucleotide sequence of the glucose regulator modules C and F, respectively. Figure 2 is a genetic map of the 8.8 kb plasmid pACCMV.plpA containing the cloning sites for the expression cassette for proinsulin, and also several adenoviral genes. Figure 3a is a genetic map showing the insertion diagram of the expression cassette with respect to several markers in the plasmid pACCMV.p.A. Figure 3b shows the order of the genetic elements 5 'to 3'. Figure 4 is a genetic map of the large 40.3 kb plasmid pJM17, used to create the final recombinant vector for transfection. Figure 5 is a genetic map showing the recombination of the pACCMV.plpA and pJMl7 vectors to give the construction of AdC / FAM. Figure 6 is a gel reproduction of a Northern blot of RNA isolated from hepatocytes transfected with the recombinant plasmid vector containing the expression cassette, and cultured in the presence of various glucose levels.

^^. ^^ Ma > , ... Figure 7 is a gel reproduction of a Northern blotch identical to Figure 6, showing only the result of the longest exposure of the CMV control. Figure 8 is a duplicate experiment of that shown in Figure 6 showing only the migration position of a mRNA control band under the control of the constitutive CMV promoter. Figure 9 is a gel reproduction comparing the binding of rRNA with hepatocyte mRNA in the presence of various glucose levels. Figure 10 is a schematic diagram of the steps used to clone M2A and M2B. Figure 11 is a graph comparing the amount of insulin released from the Cos7 cells transfected with the recombinant plasmids. Figure 12 is a graph comparing the secretion and intracellular retention of insulin in Cos7 cells transfected with the recombinant plasmids. Figure 13 is an autoradiograph demonstrating the effect of time on the glucose-inducible up-regulation of hlns mRNA in transfected hepatocytes. (Strips 1, 2, and 3 - expression of hINs mRNA at 27.5 mM glucose for 0.5 h, 1 h, and 16 h, respectively, Strips 4, 5, and 6 - expression of hlns mRNA at 5.6 mM glucose for 0.5h, lh and 16h, respectively). Figure 14 is an autoradiograph demonstrating that human insulin production of glucose response is observed in cell lysates of transduced rat hepatocytes. (Cells were transfected with the glucose-inducible construct (Strips 1 and 2) and cultured in 3.3 mM glucose (Strip 1) or 27.5 mM glucose (Strip 2). Strip 3 is a control strip of cells transfected with Proinsulin under the control of a CMV promoter, Strip 4 represents the same material as in Strip 3, except that an excess of unlabeled insulin was added prior to the immunoprecipitation step with the insulin-specific antibody). Figure 15 is a bar graph comparing the insulin produced by the transfected hepatocytes with a construct containing 1 GIRE at different concentrations of glucose. Figure 16 is a bar graph comparing the insulin produced by the transfected hepatocytes with a construct containing 2 GIREs at different concentrations of glucose. Figure 17 is a bar graph comparing the insulin produced by the transfected hepatocytes with a construct containing 3 GIREs at different glucose concentrations.

Detailed Description of the Preferred Modality In the present invention, replacement beta cells for the treatment of Type I diabetes are constructed by transfecting autologous cells, preferably hepatocytes, with a vector that expresses proinsulin genetically modified to be insulin-cleavable by an enzyme or endogenous enzymes for the transfected cells. Initially, a cassette of the gene is constructed, which contains the proinsulin gene and the appropriate control elements for its expression regulated by a secretagogue, preferably glucose. In the isolation of the natural proinsulin cDNA, the total RNA of normal human islet cells was extracted, and the mRNA fraction was isolated and used as a template in a reverse transcription reaction primed with oligo (dT) α 5. The insulin cDNA (-28 pb-443 bp) was amplified using the sense and antisense oligonucleotides which included the restriction sites for Kpnl and Salí, respectively. The sequences are shown in Table 1 designated TA423 and TA413, and are listed here as SEQ ID NOS: 1 and 2. Alternatively, the cDNA can be isolated according to the methods described in Bell, et al., Nature 282: 525 (1979) using the primers described there, but incorporating the restriction sites compatible with the selected cloning vehicle. In general, it is desirable to include in the amplified region a portion of the intron flanking the open reading frame of the proinsulin gene. The amplified DNA fragment (-28-443 bp) containing the complete coding sequence of human insulin and a portion of the untranslated region from the 5 'and 3' ends was subcloned into pBlueScript SK +. pBlueScript SK + is a phagemid producer of the 2.96 kb colony derived by the replacement of the PUC19 polylinker of pBS (+/-) with a synthetic polylinker. This cloning vehicle is described in Shon, et al., Nuc. Acids Res., 16: 7583 (1988). In the preferred embodiment of the invention, the hepatocytes are transfected with a vector having a gene of proinsulin regulated with glucose. The hepatocytes express an endogenous endopeptidase furine. Although furine is known to segment proinsulin at its B-C junction, it is very inefficient at cleavage at the C-A junction. Segmentation in both sites is required for the Cleavage of the C-peptide required for the conversion of the proinsulin to active insulin. A single point mutation (t267 to G) converts the amino acid sequence KQKR to RQKR producing a modified C-A binding compatible with the specificity of furin. Accordingly, the proinsulin protein can be processed to insulin using a unique endogenous enzyme. The mutation that creates the new C-A binding can be effected by standard methods known in the art. For example, the conversion of Lys to Arg can be to do in two steps. The sense oligonucleotide (TA403 designates SEQ ID NO: 3) which includes a point mutation corresponding to the desired change in target or target region, was used with the original insulin antisense oligonucleotide (TA413) to amplify a segment of insulin. Similarly, an antisense oligonucleotide (TA404) containing the Lys to Arg mutation was used with the original insulin sense oligonucleotide (TA423) to amplify the second modified insulin (MI) fragment. The two fragments, so produced, can be purified, and a mixture of them used as a template DNA in the amplification of insulin Ml modified in C-A with the oligonucleotides TA423 and TA413. Ml insulin modified with C-A can be subcloned into pBlueScript SK + at restriction sites Kpn I and Sal I. In practice, the InsMl DNA was sequenced and it was found to be free of errors. The TA413, TA404, and TA423 are shown in Table 1, and are listed here as SEQ ID NOS: 2, 4 and 1. The key aspect of the invention is the control elements which make the transcription of the proinsulin gene in response to extracellular glucose levels. Since enzymes of GLUT-2 and glucokinase are thought essential for the "detection" of glucose, the hepatocytes, which produce the enzymes, make a good candidate for a replacement beta cell. Although, the mechanism of "detection" is not known, the Applicants postulated that in addition to GLUT-2 and glucokinase, the rest of the "detection" machinery could be intact in the hepatocytes, including the formation of any substances that have a Mediating role in the expression of genes at the transcriptional level. To test this hypothesis, the Applicants used varying combinations of different regulatory elements inducible by glucose to determine the time course of up-regulation of insulin mRNA. When used herein, a glucose-inducible regulatory element (GIRE) contains two perfect CACGTG portions separated by five base pairs.

In the first set of experiments, the first module used, designated as sequence C, contained four perfect portions of CACGTG. The second module, designated sequence F (SEQ ID NO: 5), was based on the module found in the fatty acid synthetase. The first 21 bp are a perfect match of the inducible glucose module containing two perfect portions of CACGTG, but then a length segment of 11 bp (10-20 bp of the oligonucleotide), CACGTGGGCGC, is repeated a plurality of times (at least twice, and preferably three to six times), creating a series of glucose-inducible regulatory elements attached head to tail. These constructs were inserted upstream of the 5 'untranslated region of the human preproinsulin gene and then cloned into an adenovirus vector which was used to transfect the hepatocytes. Two sets of experiments were carried out. The first, the sensitivity of regulation in response to physiological levels of glucose was analyzed by Northern blotting. A strong induction of insulin mRNA synthesis observed at glucose concentrations greater than 5.5 mM. The gels showed that both glucose regulator modules, as described, are functional at approximately the same degree, although the construction of AdFAM using the F module seems to be somewhat more responsible. Next, the temporal response of the construction containing module C with respect to glucose was tested. This experiment showed that up-regulation of insulin mRNA occurs within 30 minutes of exposure to supraphysiological levels of glucose. Together, these experiments demonstrate that GIREs provide a transcriptional regulation of the synthesis of insulin mRNA in hepatocytes. In addition, the constructs respond within the period of time necessary for their use in the treatment of diabetes, in contrast to Shih et al., J. Biol. Chem., 269: 9380 (1994), which measures the total accumulation of CAT at 48 hours. Further analyzes of the construction described above indicated that the rat albumin promoter and the 5 'untranslated region contained an inverted repeat of approximately six pairs base. This inverted repeat has the potential to form a loop or circuit in the insulin mRNA, which inhibits processing and post-transcriptional translation. To solve this problem, two gene cassettes chimeric including the 5 'untranslated region of the Rat albumin (bases 153-188 of the sequence published as Accession No. M16825 of GenBank, incorporated herein for reference) fused to the cDNA for human preproinsulin, were amplified by PCR as described in Example 3. The cDNA The insulin was previously modified to be segmentable by furin as described above. The constructions were designated M2A and M2B. These constructs differ only in that M2A ended at the end of the human preproinsulin cDNA at base 382 while M2B includes 18 bases from the 3 'untranslated region of human preproinsulin. These promoter-free constructs were then cloned into pcDNA3 plasmid, which contains a CMV promoter. The expression of these 15 plasmids was evaluated in Cos7 cells. The data (presented in Figures 11 and 12) demonstrate that these constructions are capable of causing the production and secretion of insulin. The Applicants also compared the insulin production of the constructs containing either 1 (SEQ ID NO: 8), 2 (SEQ ID NO: 9) or three (SEQ ID NO: 10) GIREs in combination with the promoter of the rat albumin, the 5 'untranslated region of the rat albumin and M2B of the preproinsulin sequence. The complete sequences of these constructions, which were 'Í8SL ?. The vectors inserted into the adenovirus vectors are listed as SEQ ID NO: 11 (containing 1 GIRE), SEQ ID NO: 12 (containing 2 GIREs), and SEQ ID NO: 13 (which contains 3 GIREs). The production of insulin in the hepatocytes transfected with these vectors was analyzed. The hepatocytes transfected with the vector containing 1 GIRE showed only low levels of insulin production. Hepatocytes transfected with vectors containing either 2 or 3 GIREs demonstrated elevated levels of insulin production in response to physiologically relevant levels of hyperglycemia. It is expected that the production of the insulin baseline will be leveled with the increasing numbers of the GIREs. In addition, the construction containing 3 GIREs was more responsible at 10 mM glucose than the construction containing 2 GIREs. Shih, et al., Supra, describe different combinations of perfect and imperfect portions in combination with a L-PK TATA box, exciting a CAT gene. The first construct consists of the wild-type L-PK promoter that excites the CAT. The GIRE in this promoter consists of two imperfect portions, which have the sequences CACGGG and CCCGTG. Each of these portions contains an inequality of a base pair. The second construction consists of a GIRE of the wild type S14 in combination with the L-PK TATA box, -. •, - - ^^^^^^^^^^^^^. *** && . «FaSl, which excites a CAT gene. The GIRE in this construction consists of a perfect portion with the sequence CACGTG and an imperfect portion, which has an inequality of two base pairs, with the sequence CACGCG. The third construction consists of two GIREs of wild type S14, combined with the L-PK TATA box, which excites a CAT gene. This construction has two GIREs, each having a perfect CACGTG portion and an imperfect portion, which has an inequality of two base pairs, with the sequence of CACGCG. The CAT expression of these constructs was measured after the cells were cultured in 5.5 or 27.5 mM glucose for 48 hours. The activity of CAT in cell extracts was expressed as a conversion of the percentage of chloramphenicol to its acetylated form. The construction of L-PK demonstrated a high CAT expression after 498 hours in 27.5 mM glucose. Note that the portions in this construction contained an inequality of a base pair. The construction containing a GIRE of wild type of S14 consisting of a perfect CACGTG portion and an imperfect portion with an inequality of two base pairs exhibited only low levels of CAT expression. The vector consisting of two GIREs of the wild type S14, the GIREs consisting of a portion perfect with the CACGTG sequence and a portion with a ^^^^^ r ^^^^^^^^^^^ ñ inequality of two base pairs, also exhibited high levels of CAT expression. The reporter system used to evaluate the glucose-inducible regulation of transcription by the GIREs in Shih et al., Measured CAT activity after cells transfected with the vectors were cultured with glucose at concentrations of 5.5 or 27.5. mM for 48 hours. Therefore, this assay of gene expression measured the accumulation of the protein CAT in the cell extracts, and did not measure the synthesis of the mRNA directly. Therefore, the accumulation of CAT activity in cell extracts is consistent with the up-regulation of the transcription of the CAT gene. The applicant's data shows that the constructs containing two GIREs expressed high levels of insulin mRNA after only 30 minutes of glucose exposure. The regulatory module for the transcriptional responsiveness to glucose in this The invention is a synthetic oligonucleotide having at least two GIREs, each GIRE containing the two CACGTG segments of the operative regulatory portion flanking a linker segment of the nucleotide, conveniently of the sequence GGCGC. Preferably, the regulatory module contains from 2 to 8 GIREs, and more preferably the module contains from 3 to 5 GIREs. The additional GIREs are not believed to have an effect on the additional up-regulation because a leveling of the union efficiency is to be expected. However, additional GIREs above 5 are not expected to be detrimental to the regulation function. The ends of the double-stranded oligonucleotide module are synthesized to include the intermediate site restriction sequences to facilitate cloning. For example, for cloning into the preferred defective viral vector described hereinafter, each sense oligonucleotide starts with a mean Not I site on the 5 'end, and each antisense oligonucleotide includes a middle Eco IR site on the 5' end. . A functional cassette includes the proinsulin gene, the glucose regulator module, and a promoter. The promoter is preferably a relatively strong constitutive promoter normally operating only in the host cell of choice, and functions in response to the regulatory module located at its 5 'end. In the cassette contemplated herein, the rat albumin promoter turned out to be selected, although many other candidates are known in the art. Using the published sequence, (Heard, et al., "Determinants of Rat Albumin Promoter Tissue Specificity Analyzed by an Improved Transient Expression System", Mol.Cell.Biol., 7: 2425-2434 (1987), incorporated herein for reference). The PCR primers were synthesized containing Eco IR and Kpn I restriction sites, as indicated in Table 1 and designated SEQ ID NOS: TA420 (6) and TA421 (7). Nucleotides 1-184 were amplified after this. The fragment of the albumin promoter of the amplified rat was purified, and cut with the restriction enzymes Kpn I and Eco Rl. After cloning into pBlueScript, the sequence was verified by conventional sequencing techniques. Preferably the PCR amplification was carried out using the pfu polymerase that can be obtained from Stratagene, which has a significantly lower error rate than other polymerases. The 5 'untranslated region of the insulin is replaced with the 5' untranslated region of the rat albumin fused in the correct reading frame sequence (in the natural orientation) with the human total preproinsulin cDNA of substantially total length . Those skilled in the art will understand that other 5 'untranslated regions can be substituted for the 5' untranslated region of rat albumin. It is important that the appropriate spacing (approximately 25 base pairs) be provided between the GIRE module and the site ^^ íryí & ae ^^^^^ ^ & í ^ - ^^^ ris ^^^ .-. ? ^ - ^ - ^ f * ^^ * - ^^ Kt, starting from the transcription and that the 5 'untranslated region contains a secondary structure as small as possible so that there is the appropriate union to and processing by the ribosomes. The use of the 5 'untranslated region of the rat albumin can therefore be observed as a guide for the construction of other cassettes. The human complete preproinsulin cDNA of substantially complete length means the full-length human preproinsulin cDNA as well as other substituted or truncated preproinsulin cDNAs and genomic DNAs, the transcription and translation of which produces an insulin having biological activity. The cassette comprises, 5 'to 3', a glucose regulatory response module, a transcriptional promoter whose level of transcription can be further increased by the glucose response module, and the structural gene for the genetically modified proinsulin, which it will be segmentable by an endogenous endopeptidase of the host cell, they are spliced together and ligated by conventional techniques. The molecular ends of the polynucleotide cassette preferably have single-stranded sequences that define the average restriction site corresponding to the complementary middle sites on the vector in which it is to be inserted.

The best available vector is a defective plasmid of free replication, helper, derived from the adenovirus genome, and described in Newgard, et al., "Glucose-Regulated Insulin Secretion", in Molecular Bioloqy of Diabetes, eds. Draznin, and collaborators, Humana Press. 1992. Figures 2-5 are a diagram of the genetic components and the construction of the vector that contains the cassette of the gene. The advantages of this vector include the absence of the helper virus, thus preventing the spread of the virus and a high efficiency of ineffectiveness of the host cells. It also has the disadvantage of being diluted out of the replication cells, since the adenovirus integrates the genome of the host cell with very low efficiency. Other transduction systems useful in the present invention include certain retroviral integration systems and another recombinant adenoviral system free of the helper, described in U.S. Pat. No. 5,436,146. Another advantage of virally derived vectors is that delivery to the target or target cells in the intact animal does not require excision of the tissue, infection in vitro, and re-establishment. However, nothing could prevent the use of an allogeneic source of cells under immunosuppressive conditions. A material in purified viral storage (2-40 infectious units "it ^ H ^ g" by target cell) can be injected into the hepatic portal vein, with efficient infection rates that can be obtained when the viral particles penetrate the liver capillary beds and come into contact with the hepatocytes Thus, the replacement beta sites are generated in situ without altering the normal cellular architecture The additional advantages of the present invention will become apparent from the following Examples.Alternatively, the hepatocytes can be transfected ex vivo and then transplanted to the human being It will be evident that any structural gene for which modulated glucose control is desired can be inserted into the cassette of the gene by conventional recombinant techniques and expressed in an appropriate host cell. do not require additional processing, the hepatocyte's fucine enzyme is, of course, superfluous. Metabolic diseases for which the present invention has therapeutic value in the restoration of a protein function mediated by the glucose response, can be identified. u &rx3 rk £ 5 > r £, A, * EXAMPLE 1 Generation and Cloning of the Insulin Gene Cassettes in the plasmid pACdeltaCMV.

The plasmid pACCMV.pLpA (Fig. 2), used as a vector for the generation of recombinant replication-defective adenovirus containing the genes of interest, was cut for the complement with the restriction enzyme Sal I and partially with the enzyme Not I. The 8.3 kb piece of KNA, which lacks the CMV promoter, was purified with the gel and used as the vector for the insertion of the insulin gene cassettes. The pair of oligonucleotides corresponding to one of the GIREs was mixed with the albumin promoter of Eco IR-Kpn I and the DNA fragments of Kpn I-Sal I InsMl, the mixture was ligated with the plasmid vector pAC? CMV. A combination of Modules C or F of Glucose Regulatory Response (Figure 1), the albumin promoter and the mutant insulin cDNA, produced a total of two constructs. The integrity of both constructions was confirmed by the analysis of the sequence. Each of the two constructs was cotransfected with the plasmid pJM17 in the host cell line 293, as described, to generate recombinant replication-defective adenovirus constructs, especially Ad.CAMI and Ad.FAMI (see Figure 5) .

EXAMPLE 2 Insulin expression in Hepatocytes at various concentrations of glucose.

The hepatocytes of the rat were prepared by in situ perfusion of 0.5 mg / ml of collagenase in the balanced Hanks solution, supplemented, as described (Kreamer et al. (1986) In Vitro 22, 201-211). The viability of the isolated hepatocytes was 90% or better. Six 60 mm plates or boxes coated with collagen, each containing lxlO6 hepatocytes, were transfected with 5xl07 pfu / plate. Transfected hepatocytes were exposed to three concentrations of glucose, 3.3 mM, 5.6 mM and 27.5 mM, in RPMI supplemented with 10% fetal bovine serum, 30 μg / ml proline, 5 μg / ml insulin, 5 μg / ml of transferrin and 5 μg / ml of selenium. After 36 hours, one of the two plates in each of the glucose concentrations tested was used to prepare the RNA and the other plate was used to verify the viability of the hepatocytes. The viability , »« & •,, --b & toS &iíí d tiijB *. **. , -Jjjto, ... > ,. - "¿,. .-y, J of the hepatocytes at all tested concentrations of glucose, was no more than 10% different. Ten micrograms of the RNA of each sample were electrophoretically resolved on a 2% formaldehyde agarose gel, the RNA was transferred to a Nylon membrane, crosslinked with UV rays and hybridized with the cDNA of digoxin-labeled insulin. The detection of the probe bound to the membrane was carried out by chemiluminescence, the results were recorded as í? multiple exposures on the X-ray films for various time intervals and quantified by the analysis of the digital images. With reference to Figures 6 and 7, Northern analysis reveals that the RNA that migrates in the position of Polynucleotides of approximately 1.35 kb, corresponding to the predicted size of proinsulin transcription, is evident only when the transfected hepatocytes are cultured in the presence of 27.5 mM glucose. Importantly, no induction of the gene proinsulin on the bottom is indicated at 3.3 or 5.5 mM glucose. Unlike other attempts in the construction of a replacement cell for the production of inducible, artificial insulin, in which induction occurs at the subphysiological levels of glucose, those present transfectanate hepatocytes show a response only in the physiological or supraphysiological interval. Strong induction is observed at glucose concentrations greater than 5.5 mM. A strong response is evident at 10 mM. The gels show that both glucose regulator modules, as described, are functional to approximately the same degree, although the AdFAM construction using module F seems to be somewhat more responsible. As a control, the Ad.CMP-Ins in which the gene of interest (proinsulin or beta-galactosidase) is under the control of the constitutive CMV promoter, generates RNA of the distinctive size without taking into account the concentration of glucose and it is used to quantify the amount of insulin mRNA (Figures 6, 7 y). The quantification by phosphoformation of images is summarized in Table 2. The results show only a slight difference in the relative expression between 3.3 and 5.6 mM glucose, in contrast to a value of 3.06 to 27.5 M. Figure 9 shows the rRNA and the normalization mRNA in a gel to reach the value of 3.06. ! ¿& ^ st £ te2 ¿£ Table 1 «, < ^^ ^ - ^. ^ s. ^. S £ & £ ^% agsTs * L "'rf Table 2 Quantification of Insulin mRNA in Hepatocytes • For comparison purposes, the normalized amount of insulin mRNA expressed at the euglycemic level (5.6 mM glucose) was arbitrarily assumed to be one.

EXAMPLE 3: CLONING OF THE CONSTRUCTION OF INSULIN WITH THE UNTRANSLATED REGION 5 ^ OF THE ALBUMIN OF THE RAT The chimeric insulin genes of Example 1 were further modified by the elimination of the 'untranslated region of human insulin mRNA and replacing this region with the 5' untranslated region of rat albumin (bases 153-188 as published in Access No. M16825 of GenBank, incorporated herein by reference). This modification was effected by the PCR amplification of hInsM2 using primers of three oligonucleotides (sequences listed in Table 3). Oligonucleotide TA455 (5 '-3') comprises a sequence corresponding to the Kpn I recognition site of the restriction enzyme, bases 153-188 of the albumin promoter corresponding to the 5 'untranslated region of the albumin and a sequence which corresponds to human preproinsulin mRNA (starting from the first amino acid). The reverse oligonucleotide (antisense) TA452 ends at the end (base 392) of the translated sequence of the human insulin cDNA sequence. The reverse oligonucleotide (antisense) TA454 corresponds to the 3 'region of the human preproinsulin cDNA plus 18 bases of the 3' untranslated region of human insulin (bases 393-410). Both of these inverse oligonucleotides also included a Sal I restriction site to facilitate cloning of the small pACCMV-based plasma-generated products described in Example 1. The amplification product of TA455 and TA452 has been designated as the mutant M2A. The product of the amplification of TA455 and TA454 has been designated as the M2B mutant. After amplification, the modified chimeric insulin genes were then cloned without their promoter sequences in the commercial plasmid vector poDNA3 containing the CMV promoter. The steps involved in cloning and expression are shown schematically in Figure 10. Since pcADNA3 does not have a unique Sal I site, the orientation of the insertion was controlled using Kpn I and EcoRV. The M2A and M2B mutants of the insulin gene were digested with Kpn I and ligated separately into the pcDNA3 vector. After the transformation of E. coli using the ligated DNA, the plasmids containing the two insulin mutants were prepared and used to transfect Cos7 cells.

EXAMPLE 4: INSULIN SECRETION FROM COS 7 CELLS TRANSFERRED WITH INSULIN CONSTRUCTIONS CONTAINING THE 5 'NON-TRANSLATED REGION OF RAT ALBUMIN Cos7 cells were used to test the capacity of the constructs described in Example 3 to synthesize and secrete insulin. The TRANS IT® transfection reagent (Pan Vera Corp., Madison, Wisconsin) was used to transfect Cos7 cells with the plasmids containing the M2A and M2B mutants for temporal expression. A four hour incubation period for transfection was followed by an overnight incubation of the cells and fresh DMEM supplemented with 10% fetal bovine serum. The medium was then changed and the plates were incubated for two days in medium containing either 10% or 5% fetal bovine serum. The medium and cells were collected separately, and the cells were used in a saline solution buffered with Tris (pH 7.6) containing 1% NP-40 and protease inhibitors (trypsin inhibitor and PMSF). Both the medium and the used cell were analyzed to verify the presence of insulin by ELISA with capture of the antigen. Preliminary results show that clone arrivals are capable of causing the synthesis and secretion of insulin. M2B demonstrates a trend towards higher insulin production (see Figures 11 and 12). It is also evident that most, if not all, of the synthesized insulin is secreted (Figure 12). The control cells transfected with the pcDNA 3 of the vector without the insert did not produce insulin. The Measured levels of insulin in these controls were not significantly different more than in the baseline of the standard insulin curve.

TABLE 3 Oligonucleotides Used for the Amplification of M2A and M2B Insulin Mutants Quantification of the insulin-dependent release of glucose from hepatocytes transfected with adenovirus constructions containing insulin and 1, 2 or 3 GIREs.

The above examples included data showing that the glucose-inducible transcription of hlns in the hepatocytes of the rat is achieved by means of a genetic construct with hns cDNA under the control of a chimeric serum albumin promoter containing regulatory elements inducible by glucose (GIREs). Information regarding the ability of hlns mRNA to produce and secrete the insulin protein was also provided using COS7 cells. To study expression in hepatocytes, three constructs of chimeric human insulin in the adenovirus, containing 1, 2 or 3 GIRE units were constructed. Followed by the specific number of GIRE (s), each construct contains an albumin promoter and the human insulin cDNA (including both sets of mutations to aid the insulin-mediated processing of proinsulin to insulin). The general strategy of assembling the hlns constructs was essentially the same as described above. The sense oligonucleotides and "rJ.'qaaMLrJ-gT, ~. ^ l? h? á? 'JßUc & Mtm antisense that correspond to 1 or 2 GIREs were chemically synthesized.Each set of oligonucleotide pairs was designated in such a way that when annealed together, the double-stranded DNA contained the sticky ends for the restriction enzymes Not I and EcoR I on the 5 'and 3' ends, respectively.An additional pair of the oligonucleotides corresponding to a GIRE was made to contain the EcoR I and Xbal sites on the 5 'and 3' ends, respectively The original sense oligonucleotide used for the amplification of the rat albumin promoter contains the EcoR I site followed by the Xba I site. Oligonucleotides (1 GIRE) was inserted into the construct containing 2 GIREs using EcoR I-Xba I, consequently causing construction with 3 GIREs The sequence of the rat albumin promoter initially described was extended by PCR to which includes the complete 5 'untranslated region of the rat albumin. The cDNA of human insulin containing two sets of mutations, corresponding to the B / C binding and C / A of proinsulin, of the constructs previously described, was modified to eliminate the 5 'untranslated region arising from hlns cDNA by PCR. The two DNA fragments were joined together by the extension of the overlap in a PCR reaction. He - & & * £ & I? ^ & itÁ * M¿ r &, product of this reaction contained the albumin promoter (5 '-> 3'), 5'-UTR of the albumin and the translated sequence of the modified hlns so that be compatible with furina. This DNA fragment containing the restriction enzyme EcoR I and SalI sites on the 5 'and 3' ends, respectively, were digested with the two enzymes, mixed with the annealed pair of the oligonucleotides corresponding to 1 or 2. GIREs, described above, and ligated into the vector of the plasmid used to produce the adenovirus (pACCMV from which the CMV promoter has been deleted). The Applicants then determined whether the glucose-induced up-regulation of hlns mRNA is effected by a corresponding glucose-induced secretion of the hlns protein and whether the extent of insulin secretion induced by glucose varies depending on the number of GIRE units used by construction. The peptides corresponding to the 2 or 3 GIREs were individually mixed with the plasmid pJM17 and the mixture of the two plasmid DNAs were co-transfected into the 293 HEK cells to generate the recombinant replication defective adenovirus (as initially described ). Each recombinant hlns construct was sequenced to find out that no errors were introduced by PCR or during the td ^ ig ^ g ^^ - í. subsequent cloning procedures. The newly prepared hepatocytes were placed in plates on 30 mm plates or plates coated with collagen and transfected (MOI = 4) with the adenovirus containing the insulin construct with 1 (AdlSAM2B), 2 (Ad2SAM2B) or 3 (Ad3SAM2B) GIREs, as indicated. The culture medium was changed 16 h after transfection, with a medium containing 2.5, 5.6, 10 or 27.5 mM glucose, as indicated. The insulin present in the medium was evaluated by ELISA with aen capture 32 h later (the numbers shown in Table 4 represent ng Insulin / ml + DS). The results are presented in Figures 15, 16 and 17 and in Table 4. A glucose dependent increase in insulin secreted from the transduced hepatocytes is clearly observed when the gene construct contains either 2 (Figure 16) or 3 (Figure 17) GIREs. However, in the case of the construction that has 1 (Figure 15) GIRE, the total insulin secretion as well as the glucose induction is minimal. In addition, the construct contains 3 GIREs, in addition to a maximum maximum induction at 27.5 mM glucose (an increase of about 9 times over the level to 2.5 or 5.6 mM glucose as opposed to 6.5 times the increase in the case of the construction with 2 GIREs under conditions ideal), it also shows an increase > 2 times in the secretion of insulin at 10 mM glucose.

TABLE 4 Insulin secreted from Hepatocytes transfected with vectors containing 1, 2, or 3 GIREs Example 6, Temporary response of GIRE constructs to the physiological levels of glucose.

The 60 mm plates or discs coated with collagen, each containing 1 x 106 hepatocytes, were transfected with 3.5 x 10 6 pfu / plate of the test adenovirus containing the insulin gene and two GIREs. The control plates either did not receive a virus, or received a virus that codes for B-galactosidase ## & - ?? The bacterial cells were then exposed for 16 h at 5.6 mM glucose in RPMI supplemented with 10% fetal bovine serum, 30 μg / ml proline, 5 μg / ml insulin, 5 μg / ml transferrin and 5 μg / ml of selenium, at 37 degrees Celsius.The plates containing the transfected cells were then divided into two groups, one group receiving the fresh medium containing 5.6 mM glucose, the second group receiving the fresh medium with 27.5 mM glucose From each of these two groups, the individual plates were removed after 30 minutes, 1 h, 2 h, 4 h, 8 h, and 16 h, the medium was decanted, and the cells were frozen in liquid nitrogen. Total RNA was extracted and analyzed to verify the hlns mRNA by Northern blotting.The Northern spotting in Figure 13 demonstrates that after exposure to 27.5 mM glucose, the hlns mRNA was detectable at the first time point of 30 minutes and it increased after this d e a time-dependent manner. At the level of normal glucose (5.6 mM) the signal was much lower. The quaication of the bands revealed that the ascending regulation of the insulin message observed at 27.5 mM is approximately 10 times when compared with the treatment 5.6 mM. These data demonstrate that the construction of the vector containing two GIREs initiates transcription in response to elevated glucose levels in a time frame comparable to islet cells. This rapid temporary response to elevated glucose levels confers a level of control of insulin synthesis in addition to the control mechanisms that arise from the detection of glucose by the GLUT-2 and glucokinase pathways. These data also demonstrate an accurate and correct response to the physiological levels of glucose. The production of insulin mRNA is not up-regulated in the presence of 5.6 mM glucose, the concentration of the permanent state of glucose in the bloodstream. The synthesis of insulin mRNA is stimulated by high levels of glucose (above 5.6 mM).

Example 7 Insulin synthesis in physiological intervals by hepatocytes transfected with the construction containing 2 GIREs.

The newly prepared rat hepatocytes were transfected with two different adenovirus constructs containing the mutated insulin gene Ml: AdSAMl (containing the rat Albumin promoter). modified to contain 2 GIREs) and AdCMVInsMl (containing the constitutive and highly active CMV promoter). The hepatocytes transfected with AdCMV. β-Gal and untransfected hepatocytes were used as the 5 controls. Four plates of the hepatocytes were transfected with each adenovirus preparation; two plates were exposed to a low glucose level (3.3 mM) and the other plates were exposed to high glucose concentration (27.5 mM). After 36 h, the hepatocytes were exposed for 16 hours to glucose 5.6 mM or 27.5 mM in RPMI supplemented with 2 mg / ml of bovine serum albumin with omitted leucine, 30 μg / ml of proline, 5 μg / ml of insulin, 5 μg / ml of transferrin and 5 μg / ml selenium at 37 degrees Celsius. 15 Following a 6 h leucine depletion, a 2 ml aliquot of the low or high glucose containing the defined medium was added to the appropriate plates. For each adenovirus used, one plate for each glucose concentration received 0.2 mCi3H-leucine (500 Ci / mmoles). The remaining plate received the equivalent amount of unlabelled leucine and at the end of all incubations it was used for the determination of viability. The incorporation of leucine was carried out for 16 h, followed by a 4 h chase with leucine not labeled. The culture medium was aspirated, the cellular debris was removed, and the supernatant was used for the analysis of the secreted products. The cells on each plate were used with 0.8 ml of a solution containing 20 mM of the buffer solution of Tris-HCl at pH 7.6, 2 mM EDTA, 5 μg / ml of the trypsin inhibitor, 50 μm of sulfonyl phenylmethane fluoride ( PMSF), and 1% Triton-XlOO. The lysate was centrifuged at 16,000xg for 10 minutes in a microcentrifuge machine, the microspheres were discarded, and the supernatant solution was used for the analysis of the labeled intracellular products. For each insulin assay secreted by immunoprecipitation, 0.8 ml of the culture supernatant was used, and 0.4 ml of the cell lysate supernatant was used for each intracellular insulin assay. The samples were pre-cleared with Staph. Aureus in the absence of specific antibodies, to reduce non-specific precipitation of the proteins tagged during the procedure: 50 μl of the 10% suspension of Staphylococcus cells fixed in formalin (from Calbiochem) were added to each tube; the tubes were kept at room temperature for 30 minutes with continuous mixing, centrifuged (4 min, 16,000xg) and the supernatant was used for an analysis ? & feF - ~ --additional. For the immunoprecipitation of insulin and insulin-related products, 2.5 μl of the polyclonal guinea pig anti-human insulin (from Sigma Chemical Co.) were added to the supernatant solution from the previous step, mixed and maintained at room temperature for 45 minutes, followed by the addition of staphylococcus cells with 30 minutes of incubation plus centrifugation exactly as previously done. The supernatant was discarded, and the microspheres were washed 4-5 times with 1 ml of 20 mM Tris-HCl at pH 7.6, 0.15 M NaCl and 0.1% Triton X-100. The microspheres are suspended in 40 μl of the solution containing 60 mM Tris-HCl at pH 6.8, 1.2% SDS, 2% β-mercaptoethanol, heated in a boiling water bath for 4 minutes, centrifuged, and the supernatant was analyzed by SDS-polyacrylamine gel electrophoresis. As an internal control for the variation of sample to sample, in a second set of tubes, 2 μl of the rabbit anti-rat polyclonal albumin antiserum was included in the company of the anti-insulin antiserum, making possible the oo-preoxidation of the human insulin and the endogenous rat albumin in the single step subsequent. The specificity of the immunoprecipitated material was established by the use of control cells transduced with an unrelated gene, β-galactosidase, and untransfected cells. To further confirm the identity of the iniuunoprecipitated material, unlabeled insulin and rat serum were added to a separate set of tubes to provide competition with the labeled insulin and albumin respectively, and the tubes were processed simultaneously. The optimal gel system for the resolution of the A and B chains of insulin and albumin of the rat was found to be a linear polyacrylamine gradient of 10-20% SDS / Tris-Tricine based on the description from Schagger and Jagow (Analyt. Biochem. 166: 368-379, 1987). An aliquot of 15 μl of immunoprecipitated material treated with SDS-ßME from each sample is resolved on the gel in the company of BioRad peptide size markers. The gels were fixed, stained, destained, soaked in an "Amplify" solution (Amersham), dried under vacuum, and exposed to an X-ray film at -80 ° C. The results of the invention (Figure 14) show the presence of an ani-insulin antibody binding band in cell extracts of hepatocytes transduced with both of the constructs containing the insulin cDNA, AdSAMl (inducible by glucose) and AdCMV. InsMl (constitutive). As noted in the above Methods, both constructs contain the Mi-insulin cDNA, mutated in the coding region at the C / A junction. AdSAMl contains two glucose-inducible regulatory elements coupled to the rat albumin promoter, and AdCMV. InsMl contains the initial / immediate promoter of cytomegalovirus. When AdSAMl was used, the insulin band appeared only in hepatocytes exposed to a high glucose level and not at low glucose levels. When the AdCMV was used. InsMl, the positive insulin band was present in approximately equal amounts without taking into account the concentration of glucose. It should be noted that these results were controlled for possible differences in gel loading and other sources of sample to sample variation, including a difference in hepatocyte survival after incubation at 3.3 mM against 27.5 M glucose ( 10-15% of viability lower than a low glucose level), by the coprecipitation of rat serum albumin and using it as an internal standard. The size of the positive band of insulin was determined to be 7,700 Daltons. This differs from the sizes of the B and A chains of mature insulin and most likely contains the C + B peptides of insulin as a result of incompletely processed proinsulin. The size of the albumin of the rat was determined to be 67,000 Daltons, which compares favorably with the known size. The identities of the observed bands such as insulin and albumin were confirmed by the fact that the signals were almost completely removed when the unlabelled insulin in excess and the normal rat serum were included during the immunoprecipitation. A preliminary determination using digital densitometry revealed that the expression of the intracellular insulin protein at a high glucose level was only about 20 times lower when driven or excited by the chimeric albumin promoter used in AdSAMl than when it is driven or excited by the CMV promoter in AdCMV. InsMl. This is highly encouraging since the CMV promoter is the most active known promoter in most mammalian systems in vivo and ex vivo. It may not be desirable to express insulin at the levels achieved by the CMV promoter. These data demonstrate the synthesis of insulin in response to the physiological levels of glucose in hepatocytes transfected with a vector containing the proinsulin gene under the control of two GIREs. At 3.3 mM glucose, there is no detectable synthesis of insulin, at 27.5 mM glucose, insulin synthesis is easily . »?» Fc, »< .a t f .- »J.-ltÜ? detectable By adding two GIREs to the vector, which provides an additional control mechanism at the transcriptional level, the synthesis of insulin is regulated correctly in the appropriate physiological range. Insulin and insulin mRNA were synthesized at glucose concentrations exceeding 5.6 mM and are not synthesized at glucose concentrations of 5.6 mM. This characteristic makes the vectors containing the GIREs appropriate only for the treatment of Type 1 diabetes and superior to the vectors of the prior art.

LIST OF THE SEQUENCES < 110 > Hullett, Debra A. Alam, Tausif 5 Sollinger, Hans W. Theron, Amy < 120 > Treatment of Diabetes with Synthetic Beta Cells 10 < 130 > CIP Application 96429/9003 < 140 > < 141 > 15 < 150 > 08/786625 < 151 > 1997-01-21 < 160 > 13 20 < 170 > Patentln View < 210 > 1 < 211 > 29 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: oligonucleotide that corresponds to human insulin < 400 > 1 ggggtaccat cagaagaggc catcaagca 29 < 210 > 2 < 2il > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: oligonucleotide that corresponds to human insulin < 400 > 2 cggagtcgac catctctctc ggtgcaggag gcgg 34 < 210 > 3 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: oligonucleotide that corresponds to the human insulin that contains the mutation of lysine and 2 arginine < 400 > 3 gaggggtccc ggcagaagcg tggca 25 < 210 > 4 < 2il > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: oligonucleotide that corresponds to the human insulin that contains the mutation of lysine and 2 arginine < 400 > 4 acgcttctgc cgggacccct ccagg 25 < 210 > 5 < 211 > 66 < 212 > DNA < 213 > Artificial Sequence »Kfe - ¿?«. »&»., »*? Aii ^^ í l ^? Í ^ ta» 4 íi *? & ^ íí¿¿ 'A? Í .... -. t .-, g '.:. .;. < 220 > < 223 > Description of the Artificial Sequence: glucose-inducible regulatory element < 400 > 5 ggccgctgtc acgtgggcgc cacgtgggcg ccacgtgggc gccacgtggg 50 cgccacgtgg gcgccg 66 < 210 > 6 < 2il > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: oligonucleotide corresponding to the rat albumin promoter < 400 > 6 ggaattctct agagggattt agttaaacaa ett 33 < 210 > 7 < 2il > 29 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: oligonucleotide corresponding to the rat albumin promoter < 400 > 7 ggggtaccag aggcagtggg ttgacaggt 29 < 210 > 8 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: 1 glucose-inducible regulatory element < 400 > 8 cacgtggtgg ccacgtg 17 < 211 > 52 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: 2 glucose-inducible regulatory elements < 400 > 9 cacgtggtgg ccacgtgctt gggcacgcca gttctcacgt ggtggccacg 50 tg 52 < 210 > 10 < 211 > 91 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: 3 regulatory elements inducible by glucose < 400 > 10 cacgtggtgg ccacgtgctt gggcacgcca gttctcacgt ggtggccacg 50 tgcttgggca cgaattccag ttctcacgtg gtggccacgt g 91 < 210 > 11 < 211 > 598 < 212 > DNA < 213 > Artificial Sequence \ * & * > < 220 > < 223 > Description of the Artificial Sequence: 1 glucose-inducible regulatory element fused to the promoter of rat albumin and the 5 'untranslated region and human insulin < 400 > 11 gcggccgcca gttctcacgt ggtggccacg tgcttgggca cgaattctct agagggattt 60 agttaaacaa cttttttttt tctttttggc aaggatggta tgattttgta atggggtagg 120 atgaaaggtt aaccaatgaa agtgtggtta atgatctaca gttattggtt agagaagtat 180 attagagcga gtttctctgc acacagacca cctttcctgt caacccactg cctctggcac 240 AATG? CCCTG tggatgcgcc tcctgcccct gctggcgctg ctggccctct ggggacctga 300 gcctttgtga cccagccgca accaacacct gtgcggctca cacctggtgg aagctctcta 360 cctagtgtgc ggggaacgag gcttcttcta cacacccagg accaagcggg aggcagagga 420 cctgcaggtg gggcaggtgg agctgggcgg gggccctggt gcaggcagcc tgcagccctt 480 ggccctggag gggtcccggc agaagcgtgg cattgtggaa caatgctgta ccagcatctg 540 ctccctctac cagctggaga actactgcaa ctagacgcag cctgcaggca gcgtcgac 598 < 210 > 12 < 211 > 633 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: 2 glucose-inducible regulatory elements fused to the promoter of rat albumin and the 5 'untranslated region and human insulin < 400 > 12 gcggccgcca gttctcacgt ggtggccacg tgcttgggca cgccagttct cacgtggtgg 60 ccacgtgctt gggcacgaat tctctagagg gatttagtta aacaactttt ttttttcttt 120 ctggcaagga tggtatgatt ttgtaatggg gtaggaacca atgaaatgaa aggttagtgt 180 ggttaatgat ctacagttat tggttagaga agtatattag agcgagtttc tctgcacaca 240 gaccaccttt cctgtcaacc cactgcctct ggcacaatgg ccctgtggat gcgcctcctg 300 cccctgctgg cgctgctggc cctctgggga cctgacccag ccgcagcctt tgtgaaccaa 360 cacctgtgcg gctcacacct ggtggaagct ctctacctag tgtgcgggga acgaggcttc 420 ttctacacac ccaggaccaa gcgggaggca gaggacctgc aggtggggca ggtggagctg 480 ggcgggggcc ctggtgcagg cagcctgcag cccttggccc tggaggggtc ccggcagaag 540 cgtggcattg tggaacaatg ctgtaccagc atctgctccc tctaccagct ggagaactac 600 tgcaactaga cgcagcctgc aggcagcgtc sac 633 < 210 > 13 < 211 > 666 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: 3 glucose-inducible regulatory elements fused to the promoter of rat albumin and the 5 'untranslated region and human insulin < 400 > 13 gcggccgcca gttctcacgt ggtggccacg tgcttgggca cgccagttct cacgtggtgg 60 ccacgtgctt gggcacgaat tccagttctc acgtggtggc cacgtgcttg ggcactctag 120 agggatttag ttaaacaact tttttttttc tttttggcaa ggatggtatg attttgtaat 180 ggggtaggaa ccaatgaaat gaaaggttag tgtggttaat gatctacagt tattggttag 240 & < & agaagtatat tagagcgagt ttctctgcac acagaccacc tttcctgtca acccactgcc 300 tctggcacaa tggccctgtg gatgcgcctc ctgcccctgc tggcgctgct ggccctctgg 360 ggacctgacc cagccgcagc ctttgtgaac caacacctgt gcggctcaca cctggt? gaa 420 gctctctacc tagtgtgcgg ggaacgaggc ttcttctaca cacccaggac caagcgggag 480 gcagaggacc tgcaggtggg gcaggtggag ctgggcgggg gccctggtgc aggcagcctg 540 cagcccttgg ccctggaggg gtcccggcag aagcgtggca ttgtggaaca atgctgtacc 600 agcatctgct ccctctacca gctggagaac tactgcaact agacgcagcc tgcaggcagc 660 gtcgac 666 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (9)

1. A cassette of the gene for the expression of a proinsulin gene in a host cell, characterized in that it comprises: a nucleotide sequence encoding a proinsulin that can be cleaved into active insulin and operably linked to a promoter sequence recognized by an RNA polymerase contained in the host cell; and a regulatory modulus that functions in response to glucose, located 5 'to the promoter, comprising four or more portions of CACGTG, the portions separated by a linker sequence.

2. The cassette of the gene according to claim 1, characterized in that it further comprises a nucleotide sequence that encodes a 5 'untranslated region of the rat albumin located in a natural orientation between the promoter sequence and the proinsulin sequence.

3. A vector for the transfection of a host cell capable of transcribing a proinsulin gene contained in the vector, the vector is characterized in that it comprises: a cassette of the gene consisting of a nucleotide sequence encoding a proinsulin that can be cleaved into active insulin and operably linked to a promoter that can be transcribed in the host cell and a regulatory module located 5 'of said promoter, and a viral genome defective in the replication, able to express the genes required to pack the vector in vivo in a viral particle infectious for said host cell.

4. The vector according to claim 3, characterized in that the host cell is a hepatocyte.

5. A regulatory module that functions in response to synthetic glucose, characterized in that it comprises: four or more CACGTG portions, the CACGTG portions separated by a linker segment.

6. The glucose response regulator module according to claim 5, ^ £ ^ r ^^ ÉÉ ^^^^^^ -j - »& * < * & , ^ m ^ f ^^ si & A characterized in that the linker segment joining the portions of CACGTG is 5'-GGCGC-3 '.

7. A cassette of the gene for the glucose-modulated expression of a structural gene in a host cell, characterized in that it comprises: a sequence of nucleotides encoding the structural gene operably linked to a promoter recognized by an RNA polymerase contained in said host cell; and a glucose response regulator module located 5 'to the promoter comprising four or more CACGTG portions, the portions separated by the linker sequence.

8. A method of treating diabetes, characterized in that it comprises: transfecting the cells expressing the glucose transporter 2, the glucokinase, and an enzyme capable of cleaving a proinsulin genetically modified to give a protein having insulin activity, with a vector comprising: a cassette of the gene consisting of a nucleotide sequence encoding a proinsulin gene of substantially complete length operably linked to a promoter that can be transcribed into said cells, and a regulatory module located 5 'to said promoter, a viral genome defective in replication capable of expressing the genes required to package said vector in vivo in a viral particle infectious for said cells.

9. The method according to claim 4, characterized in that the cells are hepatocytes.