WO1998031397A1 - Traitement du diabete a l'aide de cellules beta de synthese - Google Patents

Traitement du diabete a l'aide de cellules beta de synthese Download PDF

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Publication number
WO1998031397A1
WO1998031397A1 PCT/US1998/001155 US9801155W WO9831397A1 WO 1998031397 A1 WO1998031397 A1 WO 1998031397A1 US 9801155 W US9801155 W US 9801155W WO 9831397 A1 WO9831397 A1 WO 9831397A1
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glucose
insulin
promoter
gene
proinsulin
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PCT/US1998/001155
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English (en)
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Debra A. Hullett
Tausif Alam
Hans W. Sollinger
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Wisconsin Alumni Research Foundation
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Priority to AU59269/98A priority Critical patent/AU5926998A/en
Publication of WO1998031397A1 publication Critical patent/WO1998031397A1/fr

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    • 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
    • C07K14/62—Insulins
    • 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
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • This invention relates to the field of gene therapy and to a method of utilizing normal non-islet cells transfected with a proinsulin gene inducibly expressed in such cells in the presence of glucose.
  • the proinsulin synthesized in the cells is further processed into mature insulin.
  • Insulin-dependent diabetes melitis occurs when an autoimmune response destroys the beta cells of the islets of Langerhans, resulting in cessation of insulin production.
  • IDDM Insulin-dependent diabetes melitis
  • the only recourse for treating this fatal condition is the periodic administration of injectable insulin of animal, or more recently, of recombinant human origin. While the administration of exogenous insulin is life- saving over the long term, severe side effects, such as circulatory disturbances resulting in blindness, gangrene, and heart attack are common. The doses of insulin injected into the diabetic patient are only approximate, even when careful dietary controls are implemented. These continual imbalances in blood glucose resulting from deviations from optimal levels of insulin are thought to cause, or contribute to, the observed side effects.
  • U.S. Patent No. 5,427,940 discloses an artificial beta cell produced by engineering endocrine cells of the At-T-20 ACTH secreting cells.
  • a stably transfected cell At-T-20 ins is obtained by introducing cDNA encoding human insulin and the glucose transporter gene GLUT-2 driven by the constitutive CMV promoter.
  • the cell line already expresses the correct isoform of glucokinase required for glucose responsive expression of the insulin gene. This cell line is responsive to glucose, but is regulated at a level of secretagogue below physiological range.
  • U.S. Patent No. 5,534,404 discloses another approach to obtaining a correctly secretagogue regulated cell line.
  • a cell sorter capable of recognizing cells having an increased internal concentration of calcium ion, associated with insulin expression (Ca++ activated fluorescence) .
  • cell populations are further selected which respond to glucose in the physiological 4 to lOmM range in a typical sigmoidal curve.
  • the cells were encapsulated in alginate bounded by a PAN/PVC permselective hollow fiber membrane according to the method of Dionne (U.S. Patent application No. PCT/US92/03327) .
  • the strategy of gene therapy for treatment of diabetes is complicated by the complexity of insulin regulation and the structure of the protein itself.
  • the responsive release of insulin from the beta islet cells is a complex event involving migration of preprocessed protein from cytoplasm to the Golgi apparatus where secretory granules bud off and travel to and fuse with the plasma membrane prior to release.
  • the initial protein product is preinsulin having an N- terminal signal sequence, which is cleaved during transport to the rough endoplasmic reticulum. Thereafter the resulting proinsulin is further processed to insulin by removal of the C-peptide joining the two polypeptides of the mature molecule, the A and B chains.
  • any engineered cell expressing mature functional insulin must have the Kex2 enzyme machinery, including the PC1/PC3 and PC2 endopeptidases, or a functional substitute thereof, as suggested by Newgard, Biotechnology, 10: 1112 (1992) .
  • the control of insulin production and release is further complicated by the regulation of glycolytic flux.
  • Glut-2 a specific facilitated diffusion type glucose transporter, and a particular glucose phosphorylating enzyme, glucokinase IV. Both enzymes have a higher Km and Vmax than the other enzymes in their related families. Both also have high affinities for glucose that result in large shifts in activity over the physiological range of glucose concentration. While reduction in GLUT-2 results in depression in insulin production, loss of glucokinase abruptly halts insulin production, and identifies glucose phosphorylation as the true rate limiting step. Transformation of cells with expressible genes for these enzymes appears to restore glucose responsive regulatory characteristics to insulin production, but not infrequently outside the physiological range of control. The experiences many researchers have had underscores the problems inherent in the complexity of the control of insulin production through manipulation of the metabolic utilization of glucose. There is thus a need in the field of diabetes for a new model of insulin regulation and beta cell replacement .
  • Control of insulin production in synthetic beta cells may be accomplished by alternative regulatory pathways than through attempted restoration of natural control over a transformed beta cell expression system. While the actual release of insulin in normal beta cells is modulated through metabolic intermediates, as yet poorly understood, an alternative control is at the level of transcription of the mRNA encoding the proinsulin precursor. It is thus an object of the present invention to provide a control system for expression of the proinsulin gene in a suitable host cell, which is independent of the metabolic effectors and intermediates involved in normal regulation.
  • a cell population is to be selected which can be engineered to synthesize insulin dependent on regulated gene transcription, without excision and extracorporeal manipulation outside the body.
  • a gene therapy utilizing a cell population having intact and normally functioning glucose transporter and phosphorylating system, so that control of insulin production is a function only of transcriptional control. Consistent with this object is provision of an enzyme system capable of generating active insulin or insulin-like analog from proinsulin not subject to feedback intervention of the glycolytic pathway.
  • a gene cassette for expression of proinsulin in autologous host cells comprises a nucleotide sequence coding substantially full length proinsulin operably linked to a promoter recognized by an RNA polymerase contained in the host cells, together with a glucose responsive regulatory module having at least two glucose inducible regulatory elements located upstream at the 5' end of the promoter.
  • the cassette is integrated into a vector comprising a replication defective viral genome capable, when infecting a suitable target cell in vitro, of packaging the vector in a viral particle infective for the autologous host cells .
  • the preferred target host cell is the hepatocyte because liver cells already express GLUT-2 and glucokinase IV sufficiently to generate the appropriate intermediates for glucose regulated transcriptional control of the proinsulin gene in the physiological range.
  • Hepatocytes also express the endopeptidase furin.
  • a mutation can be introduced into the reading frame of the proinsulin gene that permits furin cleaving at the appropriate site to obtain substantially complete excision of the C-peptide with appearance of essentially native insulin activity. It is therefore an aspect of the present invention that in the transfection method, a vector is provided in which transcriptionally controlled production of proinsulin is substantially completely converted to the active hormone, which is constitutively secreted into the liver parenchyma in response to elevation in glucose concentration.
  • Figure 1 is a schematic depicting the nucleotide sequence of the glucose regulatory modules C and F respectively.
  • Figure 2 is a genetic map of the pACCMV.plpA 8.8 kb plasmid 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 in relation to various markers on the pACCMV.p.A plasmid.
  • Figure 3b shows the order of genetic elements 5' to 3 ' .
  • Figure 4 is a genetic map of the large 40.3 kb pJM17 plasmid used to create the final recombinant vector for transfection.
  • Figure 5 is a genetic map showing the recombination of vectors pACCMV.plpA and pJM17 to yield the AdC/FAM construct .
  • 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 levels of glucose.
  • Figure 7 is a gel reproduction of a Northern blot identical to figure 6, only showing the result of a longer exposure of the CMV control .
  • Figure 8 is a duplicate experiment of that depicted in Figure 6 only showing the migration position of a control band of mRNA under control of the constitutive CMV promoter.
  • Figure 9 is a gel reproduction comparing rRNA bonding with mRNA from hepatocytes in the presence of various levels of glucose.
  • Figure 10 is an autoradiograph resulting from a Northern blot demonstrating the time frame of glucose induced synthesis of insulin mRNA.
  • Figure 11 is an autoradiograph resulting from an SDS-PAGE gel demonstrating the glucose induced synthesis of insulin in rat hepatocytes from an adenovirus vector under the control a promoter with glucose-inducible regulatory elements.
  • replacement beta cells for treatment of diabetes Type I are constructed by transfection of autologous cells, preferably hepatocytes, with a vector expressing proinsulin genetically modified to be cleavable to insulin by an enzyme or enzymes endogenous to the transfected cells.
  • a gene cassette is constructed containing the proinsulin gene and control elements suitable for its expression regulated by a secretagogue, preferably glucose .
  • RNA from normal human islet cells was extracted, and the mRNA fraction was isolated and used as a template in an oligo (dT) 15 primed reverse transcription reaction.
  • Insulin cDNA ( -28bp-443bp) was amplified using sense and antisense oligonucleotides which included restriction sites for Kpnl and Sail, respectively. The sequences are shown in Table 1 designated TA423 and TA413, and are listed herein as Seq. I.D. Nos . 1 and 2.
  • cDNA can be isolated according to the methods described in Bell, et al .
  • pBlueScript SK+ is a 2.96 kb colony-producing phagemid derived by replacing pUC19 polylinker of pBS(+/-) with a synthetic polylinker.
  • hepatocytes are transfected with a vector having a glucose regulated proinsulin gene.
  • Hepatocytes express an endogenous endopeptidase furin.
  • furin is known to cleave proinsulin at its B-C junction, it is very inefficient at cleaving the C-A junction. Cleavage at both sites is required for excision of the C-peptide required for conversion of proinsulin to active insulin.
  • a single point mutation ( ⁇ 267 to G converts the amino acid sequence KQKR to RQKR producing a modified C-A junction compatible with the specificity of furin.
  • the proinsulin protein can be processed to insulin utilizing a single endogenous enzyme .
  • the mutation creating the new C-A junction may be effected by standard methods known in the art . For example, conversion of Lys to Arg can be made in two steps.
  • the sense oligonucleotide (TA403 designates Seq. I.D. No. 3) including a point mutation corresponding to the desired change in the target region was used with the original insulin antisense oligonucleotide (TA413) to amplify one segment of insulin.
  • an antisense oligonucleotide (TA404) containing the Lys to Arg mutation was used with the original insulin sense oligonucleotide (TA423) to amplify the second fragment of modified insulin (Ml) .
  • the two fragments, thus produced, can be purified, and a mixture of them used as template DNA in amplification of C-A modified insulin Ml with oligonucleotide TA423 and TA413.
  • the C-A modified insulin Ml may be subcloned in pBlueScript
  • the key aspect of the invention is the control elements which make the transcription of the proinsulin gene responsive to the levels of extracellular glucose. Since the enzymes GLUT-2 and glucokinase are believed essential for glucose "sensing", hepatocytes, which produce the enzymes, make a good candidate for a replacement beta cell.
  • GIRE consensus glucose-inducible regulatory element
  • the first 21 bp constitute a perfect match of glucose inducible module but then an 11 bp long segment (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 elements joined head to tail.
  • a preferred module is shown in figure 1 as TA418, and listed as Seq. I.D. No. 5, in which CACGTGGGCGC is repeated four additional times, creating five glucose inducible elements (CACGTG separated by five nucleotides) joined head to tail.
  • the preferred regulatory module for transcriptional responsiveness to glucose is a synthetic oligonucleotide having at least two glucose inducible regulatory elements containing the operative regulatory motif segments CACGTG flanking a nucleotide linker segment, conveniently of the sequence GGCGC.
  • the ends of the double stranded oligonucleotide module are synthesized to include half site restriction sequences to facilitate cloning.
  • each sense oligonucleotide starts with a Not I half site on the 5' end, and each antisense oligonucleotide includes an Eco RI half site on the 5' end.
  • a functional cassette includes the structural proinsulin gene, the glucose regulatory module, and a promoter.
  • the promoter is preferably a relatively strong constitutive promoter normally operative only in the host cell of choice, and responsive to the regulatory module located on its 5' end.
  • the rat albumin promoter happened to be selected, although many other candidates are known in the art.
  • PCR primers were synthesized containing Eco RI and Kpn I restriction sites, as indicated in Table 1 and designated Seq.
  • PCR amplification is carried out utilizing the pfu polymerase obtainable from Stratagene, which has a significantly lower error rate than other polymerases .
  • the cassette comprising, 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 proinsulin genetically modified to be cleavable by a host cell endogenous endopeptidase is spliced together and ligated by conventional techniques.
  • the molecular ends of the polynucleotide cassette preferably have single stranded sequences defining the half restriction site corresponding to complementary half sites on the vector into which it is to be inserted.
  • viral -derived vectors delivery to target cells in the intact animal does not require excision of tissue, invitro infection, and reimplantation. None however, would preclude the use of an allogenic source of cells under conditions of immunosuppression.
  • a purified viral stock (2-40 infective units per target cell) may be injected into the hepatic portal vein, with efficient infective rates obtainable as viral particles penetrate the hepatic capillary beds and come into contact with the hepatocytes. In this way, replacement beta sites are generated insitu without disturbing the normal cellular architecture.
  • hepatocytes transfected with an adenovirus vector containing insulin under the control of glucose-inducible response elements are subject to control at physiological levels of glucose.
  • Figures 6 and 7 demonstrate that the transfected hepatocytes only initiate insulin mRNA synthesis in response to physiological or supraphysiological levels of glucose.
  • Figure 10 demonstrates that a vector construct containing 2 GIREs initiates transcription in response to elevated glucose levels in a time-frame comparable to islet cells.
  • Figure 11 demonstrates the synthesis of insulin in response to physiological levels of glucose in hepatocytes transfected with the proinsulin gene under the control of GIREs.
  • any structural gene for which glucose modulated control is desired may be inserted into the gene cassette by conventional recombinant techniques and expressed in an appropriate host cell .
  • the fucin enzyme of hepatocytes is, of course, superfluous.
  • a number of metabolic diseases for which the present invention has therapeutic value in restoring a glucose response mediated protein function can be identified.
  • the plasmid pACCMV.pLpA (Fig. 2) , used as a vector for generation of replication defective recombinant adenovirus containing genes of interest, was cut to completion with the restriction enzyme Sal I and partially with the enzyme Not I.
  • the 8.3 kb piece of KNA lacking CMV promoter, was gel purified and used as vector for inserting insulin gene cassettes.
  • the oligonucleotide pair corresponding to one of the GIREs was mixed with gel purified Eco RI - Kpn I albumin promoter and Kpn I - Sal I InsMl DNA fragments, the mixture was ligated with the above described plasmid vector pAC ⁇ CMV.
  • a combination of a Glucose Regulatory Response Modules C or F ( Figure 1) , albumin promoter and the mutant insulin cDNA, produced a total of two constructs. Integrity of both the constructs was confirmed by sequence analysis . Each of the two constructs was cotransfected with the plasmid pJM17 in the host 293 cell line, as described, to generate recombinant replication-defective adenovirus constructs, namely Ad.CAMl and Ad.FAMl (see Figure 5) .
  • Rat hepatocytes were prepared by in si tu perfusion of 0.5 mg/ml collagenase in supplemented balanced Hank's solution as described (Kreamer et . al . (1986) In Vitro 22, 201-211) . The viability of isolated hepatocytes was 90% or better.
  • the 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 calf serum, 30 ⁇ g/ml proline, 5 ⁇ g/ml insulin, 5 ⁇ g/ml transferrin and 5 ⁇ g/ml selenium. After 36 h, one of the two plates at each of the tested glucose concentrations, was used to prepare RNA and the other plated was used to check the viability of the hepatocytes. Hepatocyte viability at all the tested concentrations of glucose were no more than 10% different.
  • RNA from each sample was electrophoretically resolved on a formaldehyde-2% agarose gel, the RNA transferred to a Nylon membrane, UV-crosslinked and hybridized with digoxygenin-labeled insulin cRNA. Detection of the membrane-bound probe was performed by chemiluminescence, results recorded as ultiple exposures on X-ray films for various lengths of time and quantitated by digital image analysis.
  • RNA migrating at the position of polynucleotides of approximately 1.35 kb, corresponding to the predicted size of the proinsulin transcript, is evident only when transfected hepatocytes are cultured in the presence of 27.5 mM glucose. Importantly, no induction of the proinsulin gene over background is indicated at 3.3 or 5.5 mM glucose. Unlike other attempts at constructing an artificial inducible insulin-producing replacement cell, in which induction occurs at subphysiological levels of glucose, the present transfected hepatocytes show a response only in physiological or supraphysiological range. Strong induction is seen at glucose concentrations of greater than 5.5 M. A strong response is apparent at lOmM.
  • the gels show that both glucose regulatory modules, as described, are functional to about the same degree, although the AdFAM construct using the F module appears to be somewhat more responsive.
  • the normalized amount of insulin mRNA expressed at euglycemic level (5.6 mM glucose) is arbitrarily assumed to be one.
  • the plates containing transfected cells were then divided into two groups, one group receiving fresh medium containing 5.6 mM glucose, the second group receiving fresh medium with 27.5 mM glucose. From each of these two groups, individual plates were removed after 30 min, lh, 2h, 4h, 8h, and 16h, the medium decanted, and the cells frozen in liquid nitrogen.
  • Total RNA was extracted and analyzed for hlns mRNA by Northern blotting.
  • the Northern blot in Figure 10 demonstrates that after exposure to 27.5 mM glucose, hlns mRNA was detectable at the first time point of 30 min and increased thereafter in a time-dependent manner. At the normal glucose level (5.6 mM) the signal was much lower.
  • EXAMPLE 4 Glucose induced synthesis of human insulin in rat hepatocytes from an adenovirus vector with human insulin (hlns) cDNA under the control of a chimeric serum albumin promoter containing glucose-inducible regulatory elements (GIREs)
  • GIREs glucose-inducible regulatory elements
  • AdSAMl containing the rat Albumin promoter modified to contain 2 copies of the "S14" glucose-inducible regulatory element
  • AdCMVInsMl containing the constitutive and highly active CMV promoter
  • Hepatocytes transfected with AdCMV.3-Gal and untransfected hepatocytes were used as controls.
  • Four plates of hepatocytes were transfected with each adenovirus preparation; two plates were exposed to the low (3.3 mM) glucose and the other two plates to the high (27.5 mM) glucose concentration.
  • hepatocytes were exposed for 16h to 5.6 mM or 27.5 mM glucose in RMPI supplemented with 2 mg/ml bovine serum albumin with leucine omitted, 30 ⁇ g/ml proline, 5 ⁇ g/ml insulin, 5 ⁇ g/ml transferrin and 5 ⁇ g/ml selenium, at 37 degrees Centigrade.
  • the leucine incorporation was carried out for 16h, followed by a 4h chase with unlabeled leucine.
  • the culture medium was aspirated, cell debris removed, and the supernatant used for analysis of secreted products.
  • the cells on each plate were lysed with 0.8 ml solution containing 20 mM Tris-HCL buffer at pH 7.6 , 2 mM EDTA, 5 ⁇ g/ml trypsin inhibitor, 50 ⁇ M phenylmethane sulphonyl fluoride (PMSF) and 1% Triton-XlOO.
  • the lysate was centrifuged at 16,000xg for 10 min in a microcentrifuge, the pellet discarded, and the supernatant solution used for analysis of labeled intracellular products .
  • the pellet was suspended in 40 ⁇ l solution containing 60 mM Tris-HCl at pH 6.8 , 1.2% SDS , 2% ⁇ - mercaptoethanol, heated in a boiling water bath for 4 min, centrifuged, and the supernatant analyzed by polyacrylamide-SDS gel electrophoresis .
  • 2 ⁇ l of rabbit anti-rat albumin polyclonal antiserum was included along with anti-insulin antiserum, enabling co-precipitation of human insulin and the endogenous rat albumin in the subsequent single step.
  • immunoprecipitated material was established by the use of control cells transduced with an unrelated gene, 3-galactosidase, and untransfected cells.
  • unlabeled insulin and rat serum were added to provide competition with labeled insulin and albumin respectively, and tubes processed simultaneously.
  • the optimum gel system for resolution of insulin B and A chains and rat albumin was found to be an SDS/Tris-Tricine 10-20% linear polyacrylamide gradient based on the description of Schagger and Jagow (Analyt . Biochem. 166:368-379, 1987).
  • rat albumin was determined to be 67,000 Daltons, which compares favorably with the known size.
  • the identities of the observed bands as insulin and albumin were confirmed by the fact that the signals were almost completely ablated when excess unlabeled insulin and normal rat serum were included during the immunoprecipitation.
  • a preliminary determination using digital densitometry revealed that intra-cellular insulin protein expression at high glucose was only about 20 -fold lower when driven by the chimeric albumin promoter employed in AdSAMl then when driven by the CMV promoter in AdCMV.InsMl. This is highly encouraging since the CMV promoter is the most active known promoter in most in vivo and ex vivo mammalian systems. It would not be desirable to express insulin at the levels achieved by the CMV promoter.
  • the promoter and gene construct may further be characterized as a physiological acute synthesis promoter, meaning that acute synthesis of insulin and insulin mRNA is triggered by increases in glucose concentration of above the physiological setpoint of 5.6 mM, but not below the setpoint.
  • prior art references such as Stewart et al . (J. Mol .
  • Insulin mRNA and insulin are synthesized at glucose concentrations exceeding 5.6 mM and are not synthesized at glucose concentrations of 5.6 mM. This feature makes vectors containing GIREs uniquely appropriate for treatment of Type 1 diabetes and superior to the prior art vectors .

Abstract

L'invention concerne des hépatocytes transfectés à l'aide d'un vecteur d'adénovirus de réplication déficient renfermant une cassette de gènes exprimant le gène de proinsuline en réponse à des taux physiologiques de glucose, qui produisent de nouveaux îlots de cellules bêta de remplacement. La cassette renferme le gène de structure de proinsuline humaine modifié génétiquement de façon à le rendre clivable pour permettre d'activer l'insuline, un promoteur lié exploitable au gène de proinsuline, et un module de réponse régulatoire de glucose situé à 5' du promoteur. La synthèse d'ARNm de proinsuline est supprimée à moins de 5 mM de glucose, et présente un pic à environ 15 mM.
PCT/US1998/001155 1997-01-21 1998-01-21 Traitement du diabete a l'aide de cellules beta de synthese WO1998031397A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000004171A1 (fr) * 1998-07-15 2000-01-27 Wisconsin Alumni Research Foundation Traitement du diabete avec des cellules beta synthetiques
WO2000062862A1 (fr) * 1999-04-15 2000-10-26 South Eastern Sydney Area Health Service Technique de prophylaxie et de traitement des diabetes
WO2001070940A1 (fr) * 2000-03-24 2001-09-27 National Cancer Centre Of Singapore Pte Ltd Constructions genetiques pour l'expression regulee de l'insuline
EP2185696A1 (fr) * 2007-08-10 2010-05-19 University Of Technology, Sydney Cellules génétiquement modifiées pour comprendre la glucokinase d'un îlot pancréatique et leurs utilisations

Citations (2)

* Cited by examiner, † Cited by third party
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WO2000004171A1 (fr) * 1998-07-15 2000-01-27 Wisconsin Alumni Research Foundation Traitement du diabete avec des cellules beta synthetiques
WO2000062862A1 (fr) * 1999-04-15 2000-10-26 South Eastern Sydney Area Health Service Technique de prophylaxie et de traitement des diabetes
AU783594B2 (en) * 1999-04-15 2005-11-10 South Eastern Sydney Area Health Service Method of prophylaxis and treatment of diabetes
WO2001070940A1 (fr) * 2000-03-24 2001-09-27 National Cancer Centre Of Singapore Pte Ltd Constructions genetiques pour l'expression regulee de l'insuline
EP2185696A1 (fr) * 2007-08-10 2010-05-19 University Of Technology, Sydney Cellules génétiquement modifiées pour comprendre la glucokinase d'un îlot pancréatique et leurs utilisations
EP2185696A4 (fr) * 2007-08-10 2011-04-13 Univ Sydney Tech Cellules génétiquement modifiées pour comprendre la glucokinase d'un îlot pancréatique et leurs utilisations

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