WO2000004171A1 - Traitement du diabete avec des cellules beta synthetiques - Google Patents

Traitement du diabete avec des cellules beta synthetiques Download PDF

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WO2000004171A1
WO2000004171A1 PCT/US1998/015189 US9815189W WO0004171A1 WO 2000004171 A1 WO2000004171 A1 WO 2000004171A1 US 9815189 W US9815189 W US 9815189W WO 0004171 A1 WO0004171 A1 WO 0004171A1
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insulin
glucose
promoter
proinsulin
gene
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PCT/US1998/015189
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English (en)
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Debra A. Hullett
Tausif Alam
Hans W. Sollinger
Amy L. Theron
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Wisconsin Alumni Research Foundation
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Priority to JP2000560268A priority Critical patent/JP2003526321A/ja
Priority to AU85083/98A priority patent/AU8508398A/en
Publication of WO2000004171A1 publication Critical patent/WO2000004171A1/fr

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    • 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
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • 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
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    • 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
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • 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
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • 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
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    • 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 mellitus occurs when an autoimmune response destroys the beta cells of the islets of Langerhans , resulting in cessation of insulin production.
  • IDDM Insulin-dependent diabetes mellitus
  • 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.
  • beta cell replacement without involving organ transplantation.
  • These alternatives involve replacement of beta cell function with actual beta cells or other insulin secreting pancreas-derived cell lines, as discussed in Lacy, et al . , Ann . Rev . Med. , 37:33 (1986). Since introduction of exogenous cells into the body is perceived by the immune system as any other allograft, it is necessary to isolate the cells from contact with immunoactive cells and substances. In particular, donor islet cells must be protected from T-cells and macrophages mediating cytolytic processes.
  • One approach is physical immunoisolation, either by microencapsulation or by a microporous chamber.
  • 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.
  • the system is of interest, it is not of clinical significance because an animal into which these cells are introduced would be chronically hypoglycemic .
  • U.S. Patent No. 5,534,404 discloses another approach to obtaining a correctly secretagogue regulated cell line. Starting with beta-TC-6 cells, subpopulations of ceils are selected in an initial stage by a cell sorter capable of recognizing cells having an increased internal concentration of calcium ion, associated with insulin expression (Ca++ activated fluorescence) . After successive passages, cell populations are further selected which respond to glucose in the physiological 4 to lOmM range in a typical sigmoidal curve.
  • the cells v/ere 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) .
  • Valera et al . , FASEB Journal, 8: 440 ;i994) describes transgenic mouse hepatocytes expressing insulin under control of the PEPCK promoter driven by P-enolpyruvate .
  • the PEPCK promoter is sensitive to the glucagon/insulin ratio and is activated in elevated glucose states.
  • the PECK/insulin chimeric gene was introduced into fertilized mouse eggs. Under conditions of severe islet suppression by streptozotocin, the production and secretion of intact insulin by the liver compensated for loss of islet function.
  • the glucose 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 preproinsuiin 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 ⁇ 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. It is believed that two proteins are used by the beta cell to sense changes in glucose levels: 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.
  • 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 DNA, most preferably insulin cDNA, 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 dia ram 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 a schematic diagram of the steps used to clone M2A and M2B.
  • Figure 11 is a graph comparing the amount of insulin released from Cos7 cells transfected with recombinant plasmids .
  • Figure 12 is a graph comparing the secretion and intracellular retention of insulin in Cos7 cells transfected with recombinant plasmids.
  • Figure 13 is a autoradiograph demonstrating the effect of time on glucose inducible upregulation of hlns mRNA in transfected hepatocytes.
  • Figure 14 is an autoradiograph demonstrating that glucose responsive human insulin production is observed in cell lysates from transduced rat hepatocytes.
  • Lane 3 is a control lane of cells transfected with proinsulin under the control of a CMV promoter.
  • Lane 4 represents the same material as in lane 3 , except that an excess of unlabelled insulin was added prior to the immunoprecipitation step with insulin specific antibody.
  • Figure 15 is a bar graph comparing insulin produced by hepatocytes transfected with a construct containing 1 GIRE at different glucose concentrations.
  • Figure 16 is a bar graph comparing insulin produced by hepatocytes transfected with a construct containing 2 GIREs at different glucose concentrations.
  • Figure 17 is a bar graph comparing insulin produced by hepatocytes transfected with a construct containing 3 GIREs at different glucose concentrations.
  • 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 ID 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 .
  • conversion of Lys to Arg can be made in two steps.
  • the sense oligonucleotide (TA403 designates SEQ ID 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 SK+ at the restriction sites Kpn I and Sal I .
  • the InsMl DNA was sequenced and found to be error free.
  • TA413, TA404, and TA423 are shown in Table 1, and are listed herein 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 responsive to the levels of extracellular glucose.
  • hepatocytes which produce the enzymes, make a good candidate for a replacement beta cell.
  • the mechanism of " sensing” is not known, Applicants postulated that in addition to GLUT-2 and glucokinase, the rest of the "sensing" machinery would be intact in hepatocytes, including formation of any substances mediating gene expression at the transcriptional level.
  • a glucose inducible regulatory element contains two perfect CACGTG motifs separated by five base pairs.
  • the first module utilized contained four perfect CACGTG motifs.
  • the second module designated sequence F (SEQ ID NO: 5) , was based on the module found in fatty acid synthetase.
  • the first 21 bp constitute a perfect match of glucose inducible module containing two perfect CACGTG motifs 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 regulatory elements joined head to tail.
  • chimeric gene cassettes including the 5 ' -untranslated' region of rat albumin (bases 153-188 of the sequence published as GenBank Accession No. M16825, incorporated herein by reference) fused to the cDNA for human preproinsulin were amplified by PCR as described in Example 3.
  • the insulin cDNA was previously modified to be cleavable by furin as described above.
  • the constructs were designated M2A and M2B. These constructs differed only in that M2A terminated at the end of the human preproinsulin cDNA at base 382 while M2B includes 18 bases of the 3 ' -untranslated region of human preproinsulin.
  • SEQ ID NO: 11 containing 1 GIRE
  • SEQ ID NO: 12 containing 2 GIREs
  • SEQ ID NO: 13 containing 3 GIREs
  • Hepatocytes transfected with these vectors Insulin production in hepatocytes transfected with these vectors was analyzed. Hepatocytes transfected with the vector containing 1 GIRE demonstrated only low levels of insulin production. Hepatocytes transfected with the vectors containing either 2 or 3 GIREs demonstrated high levels of insulin production in response to physiologically relevant levels of hyperglycemia . It is expected that the baseline production of insulin will plateau with increasing numbers of GIREs. Furthermore, the construct containing 3 GIREs was more responsive to lOmM glucose than the construct containing 2 GIREs .
  • the first construct consists of the wild type L-PK promoter driving CAT.
  • the GIRE in this promoter consists of two imperfect motifs, having the sequences CACGGG and CCCGTG. Each of these motifs contains a one base pair mismatch.
  • the second construct consists of a S14 wild type GIRE in combination with the L-PK TATA box, driving a CAT gene.
  • the GIRE in this construct consists of one perfect motif with the sequence CACGTG and one imperfect motif, having a two base pair mismatch, with the sequence CACGCG.
  • the third construct consists of two S14 wild type GIREs, combined with the L-PK TATA box, driving a CAT gene.
  • This construct has two GIREs, each having a perfect CACGTG motif and an imperfect motif, having a two base pair mismatch, with the sequence CACGCG.
  • CAT expression from these constructs was measured after the cells were cultured in 5.5 or 27.5 mM glucose for 48 hours.
  • CAT activity in cell extracts was expressed as a percentage conversion of chloramphenicol to its acetylated form.
  • the L-PK construct demonstrated high expression of CAT after 48 hours in 27.5 mM glucose.
  • the motifs in this construct contained a one base pair mismatch.
  • the construct containing one S14 wild type GIRE consisting of a perfect CACGTG motif and an imperfect motif with a two base pair mismatch displayed only low levels of CAT expression.
  • the reporter system used for assaying glucose inducible regulation of transcription by GIREs in Shih et al measured CAT activity after cells transfected with the vectors had been cultured with glucose at concentrations of 5.5 or 27.5 mM for 48 hours. Therefore, this gene expression assay measured the accumulation of the CAT protein in cell extracts, and did not measure mRNA synthesis directly. Therefore, the accumulation of CAT activity in the cell extracts is consistent with the slow up-regulation of transcription from the CAT gene.
  • the Applicants' data demonstrates that constructs containing two GIREs expressed high levels of insulin mRNA after only 30 minutes of exposure to glucose.
  • the regulatory module for transcriptional responsiveness to glucose in the present invention is a synthetic oligonucleotide having at least two GIREs, each GIRE containing the two operative regulatory motif segments CACGTG flanking a nucleotide linker segment, conveniently of the sequence GGCGC.
  • the regulatory module contains from 2 to 8 GIREs, and most preferably the module contains from 3 to 5 GIREs. Additional GIREs are not believed to effect futher upregulation as a plateau of biding efficiency is to be expected. However, additional GIREs above 5 are not expected to be detrimental to regulating function.
  • 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 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. 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
  • PCR primers were synthesized containing Eco RI and Kpn I restriction sites, as indicated in Table 1 and designated SEQ ID NOS: TA420 (6) and TA421 (7) . Nucleotides 1-184 were thereupon amplified. The amplified rat albumin promoter fragment was purified, and cut with restriction enzymes Kpn I and Eco RI . After cloning into pBlueScript, the sequence was verified by conventional sequencing techniques. Preferably the PCR amplification is carried out utilizing the pfu polymerase obtainable from Stratagene, which has a significantly lower error rate than other polymerases .
  • the 5' untranslated region of insulin is replaced with the 5 ' -untranslated region of rat albumin fused in correct reading frame sequence (in native orientation) with substantially full-length human preproinsulin cDNA.
  • rat albumin fused in correct reading frame sequence (in native orientation) with substantially full-length human preproinsulin cDNA.
  • Those skilled in the art will understand that other 5 ' -untranslated regions may be substituted for the rat albumin 5 ' -untranslated region. It is important that adequate spacing (about 25 base pairs) be provided between the GIRE module and the transcription start site and that the 5' untranslated region contains as little secondary structure as possible so that there is proper binding to and processing by ribosomes. The use of the 5'- untranslated region of rat albumin may therefore be seen as a guide to the construction of other cassettes.
  • Substantially full-length human preproinsulin cDNA means the full-length human preproinsulin cDNA as well as other truncated or substituted preproinsulin cDNAs and genomic DNAs, the transcription and translation of which produce insulin having biological activity.
  • 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.
  • the best available vector is a helper- free replication defective plasmid derived from the adenovirus genome, and described in Newgard, et al . , "Glucose-Regulated Insulin Secretion, " in Molecular Biology of Diabetes, eds . Draznin, et al . , Humana Press: 1992.
  • Figures 2-5 diagram the genetic components and construction of the vector containing the gene cassette.
  • the advantages of this vector include the absence of helper virus, thus preventing propagation of virus and high efficiency of infectivity of host ceils. It has the disadvantage of being diluted out of replicating cells, since adenovirus integrates the host cell genome with very low efficiency.
  • Other transducing systems useful in the present invention include certain integrating retroviral systems and another helper- free recombinant adenoviral system disclosed in U.S. Patent No. 5,436,146.
  • 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. Further advantages of the present invention will become apparent from the Examples which follow.
  • hepatocytes may be transfected ex vivo and then transplanted into the human.
  • 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.
  • KNA lacking CMV promoter
  • 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.
  • 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.
  • 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 multiple 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 mM. A strong response is apparent at lOmM.
  • the normalized amount of insulin mRNA expressed at euglycemic level (5.6 mM glucose) is arbitrarily assumed to be one.
  • the chimeric insulin genes of Example 1 were further modified by eliminating the 5' untranslated region of human insulin mRNA and replacing that region with the 5' untranslated region of rat albumin (bases 153-188 as published in GenBank Accession No. M16825, herein incorporated by reference) . This modification was accomplished by PCR amplification of hInsM2 using three oligonucleotide primers (sequences listed in Table 3).
  • the oligonucleotide TA455 (5' -3') comprises a sequence corresponding to the restriction enzyme Kpn I recognition site, bases 153-188 of the albumin promoter corresponding to the 5 ' -untranslated region of albumin and a sequence corresponding to human preproinsulin mRNA (starting with the first amino acid) .
  • the reverse (antisense) oligonucleotide TA452 terminates at the end (base 392) of the translated sequence of the human insulin cDNA sequence.
  • the reverse (antisense) oligonucleotide TA454 corresponds to the 3' region of human preproinsulin CDNA plus 18 bases of the 3' untranslated region of human insulin (bases 393-410) .
  • Both of these reverse oligonucleotides also included a Sal I restriction site to facilitate cloning of the generated products into the small pACCMV based plasma described in Example 1.
  • the amplification product of TA455 and TA452 has been designated as mutant M2A.
  • the amplification product of TA455 and TA454 has been designated as the mutant M2B .
  • the modified chimeric insulin genes were then cloned without their promoter sequences into the commercial plasmids vector pcDNA3 containing the CMV promoter.
  • the steps involved in cloning and expression are schematically depicted in Figure 10.
  • pcDNA3 does not have a unique Sal I site, the orientation of insertion was controlled by using Kpn I and EcoRV.
  • the insulin gene mutants M2A and M2B were digested with Kpn I and separately ligated into the pcDNA3 vector. After transformation of E. coli using the ligated DNA, plasmids containing the two insulin mutants were prepared and used to transfect Cos7 cells.
  • Cos7 cells were used to test the ability of the constructs described in Example 3 to synthesize and secrete insulin.
  • the TRANS ITTM transfection reagent Pan Vera Corp., Madison, Wisconsin
  • the TRANS ITTM transfection reagent was used to transfect the Cos7 cells with plasmids containing the M2A and M2B mutants for transient expression.
  • a four hour period of incubation for transfection was followed by an overnight incubation of the cells and fresh DMEM supplemented with 10% fetal calf serum. The medium was then changed and the plates incubated for two days in medium containing either 10% or 5% fetal calf serum.
  • the medium and the cells were harvested separately, and the cells lysed in Tris-buffered saline (pH 7.6) containing 1% NP-40 and protease inhibitors (trypsin inhibitor and PMSF) . Both the medium and cell lysate were analyzed for the presence of insulin by antigen capture ELISA.
  • Example 5 Ouantification of glucose-dependent insulin release from hepatocytes transfected with adenovirus constructs containing insulin and 1, 2 or 3 GIREs.
  • GIREs glucose-inducible regulatory elements
  • each construct contains albumin promoter and human insulin cDNA (including the two sets of mutations to aid furin mediated processing of proinsulin to insulin) .
  • the general strategy of assembling the hlns constructs was essentially the same as described above.
  • the sense and antisense oligonucleotides corresponding to 1 or 2 GIREs were chemically synthesized.
  • Each set of oligonucleotide pairs was designed such that when annealed together, the double stranded DNA contained the sticky ends for the restriction enzymes Not I and EcoR I on 5' and 3' -ends, respectively.
  • An additional pair of oligonucleotides corresponding to one GIRE was made to contain EcoR I and Xbal sites on the 5' and 3'- ends, respectively.
  • the original sense oligonucleotide used for amplification of rat albumin promoter contains EcoR I site followed by Xba I site.
  • the last pair of oligonucleotide (IGIRE) was inserted into the construct containing 2 GIREs using EcoR I -Xba I, thus giving rise to the construct with 3 GIREs.
  • the rat albumin promoter sequence described earlier was extended by PCR to include the entire 5'- untranslated region of rat albumin.
  • the human insulin cDNA containing two sets of mutations, corresponding to the B/C and C/A junction of proinsulin, from constructs previously described was modified to eliminate the 5'- untranslated region arising from hlns cDNA by PCR.
  • the two fragments of DNA were joined together by overlap extension in a PCR reaction.
  • the product of this reaction contained (5'->3') albumin promoter, 5 ' -UTR of albumin and translated sequence of hlns modified to be compatible with furin.
  • the culture medium was changed 16h post- transfection, with medium containing 2.5, 5.6, 10 or 27.5 mM glucose, as indicated.
  • the insulin present in medium was assayed by antigen capture ELISA 32h later (the numbers shown in Table 4 represent ng Insulin/ml + SD) .
  • the results are presented in Figs. 15, 16 and 17 and in Table 4.
  • 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 13 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. Quantitation of the bands revealed that the upregulation of insulin message observed at 27.5 mM is roughly 10 fold as compared to the 5.6 mM treatment.
  • Freshly prepared rat hepatocytes were transfected with two different adenovirus constructs containing the Ml mutated insulin gene: AdSAMl (containing the rat Albumin promoter modified to contain 2 GIREs) and AdCMVInsMl (containing the constitutive and highly active CMV promoter) .
  • AdSAMl containing the rat Albumin promoter modified to contain 2 GIREs
  • AdCMVInsMl containing the constitutive and highly active CMV promoter
  • 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 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, -galactosidase, and untransfected cells. To further confirm the identity of the immunoprecipitated material, in a separate set of tubes 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).
  • a 15 ⁇ l aliquot of SDS- 3ME-treated immunoprecipitated material from each sample was resolved on the gel along with peptide size markers from BioRad.
  • the gels were fixed, stained, destained, soaked in "Amplify” solution (Amersham) , dried under vacuum, and exposed to X-ray film at -80OC.
  • Figure 14 show the presence of an anti-insulin antibody-binding band in cell extracts of hepatocytes transduced with both of the insulin cDNA- containing constructs, AdSAMl (glucose-inducible) and AdCMV.InsMl (constitutive).
  • both constructs contain the Ml 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 cytomegalovirus immediate/early promoter.
  • AdSAMl was used, the insulin band appeared only in the hepatocytes exposed to high glucose and not the low glucose.
  • the insulin- positive band was present in approximately equal amounts regardless of the glucose concentration. 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 versus 27.5 mM glucose (10-15% lower viability at low glucose) , by co-precipitating rat serum albumin and using it as an internal standard.
  • the size of the insulin positive band was determined to be 7,700 Daltons . This differs from the sizes of mature insulin B and A chains and most likely contains C+B peptides of insulin as a result of incomplete processing proinsulin.
  • 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.

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Abstract

Cette invention se rapporte à des hépatocytes transfectés avec un vecteur d'adénovirus difficient en réplication contenant une cassette génique exprimant le gène de pro-insuline en réponse à des niveaux physiologiques de glucose, ces hépatocytes fournissant de nouvelles cellules de remplacement sécrétrices d'insuline bêta. Cette cassette comprend le gène structurel pour la pro-insuline humaine génétiquement modifiée pour pourvoir être clivée en insuline active, un promoteur lié opérationnellement au gène de pro-insuline, et un module de réponse régulatoire de glucose situé en position 5' par rapport au promoteur. La synthèse de l'ARNm de pro-insuline est arrêtée à moins de 5mM glucose et sa crête se situe à environ 15mM.
PCT/US1998/015189 1998-07-15 1998-07-22 Traitement du diabete avec des cellules beta synthetiques WO2000004171A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070940A1 (fr) * 2000-03-24 2001-09-27 National Cancer Centre Of Singapore Pte Ltd Constructions genetiques pour l'expression regulee de l'insuline
WO2002072875A1 (fr) * 2001-03-14 2002-09-19 The Chinese University Of Hong Kong Methodes et compositions d'evaluation du risque de developper un diabete de type 2 chez des individus de descendance chinoise
WO2004031372A1 (fr) * 2002-10-02 2004-04-15 Naoya Kobayashi Lignee cellulaire hepatique immortalisee secretant de l'insuline, modifiee par une sensibilite au glucose
AU2007253212B2 (en) * 2006-05-22 2012-09-27 Consejo Superior De Investigaciones Cientificas Use of proinsulin for the preparation of a neuroprotective pharmaceutical composition, therapeutic composition containing it and applications thereof
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
EP3272877A1 (fr) * 2016-07-18 2018-01-24 ETH Zurich Cellules mimétiques de lymphocyte b
WO2018015330A1 (fr) * 2016-07-18 2018-01-25 Eth Zurich Cellules mimétiques de cellules bêta

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998031397A1 (fr) * 1997-01-21 1998-07-23 Wisconsin Alumni Research Foundation Traitement du diabete a l'aide de cellules beta de synthese

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998031397A1 (fr) * 1997-01-21 1998-07-23 Wisconsin Alumni Research Foundation Traitement du diabete a l'aide de cellules beta de synthese

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALAM ET AL: "Glucose inducible expression of human insulin in hepatocytes", ACTA DIABETOLOGICA, vol. 34, no. 2, September 1997 (1997-09-01), pages 115, XP002098527 *
KOLODKA ET AL: "Gene therapy for diabetes mellitus in rats by hepatic expression of insulin", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 92, April 1995 (1995-04-01), pages 3293 - 3297, XP002098531 *
MUZZIN ET AL: "Hepatic Insulin Gene Expression as Treatment for Type 1 Diabetes Mellitus in Rats", MOLECULAR ENDOCRINOLOGY, vol. 11, no. 6, 1997, pages 833 - 837, XP002098528 *
SHIH ET AL: "Two CACGTG Motifs with Proper Spacing Dictate the Carbohydrate Regulation of Hepatic Gene Transcription", JOURNAL OF BIOLOGICAL CHEMSITRY, vol. 270, no. 37, 15 September 1995 (1995-09-15), pages 21991 - 21997, XP002098529 *
VALERA ET AL: "Regulated expression of human insulin in the liver of transgenic mice corrects diabetic alterations", FASEB JOURNAL, vol. 8, 1994, pages 440 - 447, XP002098530 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001070940A1 (fr) * 2000-03-24 2001-09-27 National Cancer Centre Of Singapore Pte Ltd Constructions genetiques pour l'expression regulee de l'insuline
WO2002072875A1 (fr) * 2001-03-14 2002-09-19 The Chinese University Of Hong Kong Methodes et compositions d'evaluation du risque de developper un diabete de type 2 chez des individus de descendance chinoise
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
US9221896B2 (en) 2001-12-21 2015-12-29 Human Genome Sciences, Inc. Albumin fusion proteins
US9296809B2 (en) 2001-12-21 2016-03-29 Human Genome Sciences, Inc. Albumin fusion proteins
WO2004031372A1 (fr) * 2002-10-02 2004-04-15 Naoya Kobayashi Lignee cellulaire hepatique immortalisee secretant de l'insuline, modifiee par une sensibilite au glucose
US7312077B2 (en) 2002-10-02 2007-12-25 Naoya Kobayashi Insulin-secreting immortalized liver cell line modified by glucose sensitivity
US7919080B2 (en) 2002-10-02 2011-04-05 Naoya Kobayashi Immortalized hepatocyte cell line secreting modified insulin with glucose sensitivity
AU2007253212B2 (en) * 2006-05-22 2012-09-27 Consejo Superior De Investigaciones Cientificas Use of proinsulin for the preparation of a neuroprotective pharmaceutical composition, therapeutic composition containing it and applications thereof
EP3272877A1 (fr) * 2016-07-18 2018-01-24 ETH Zurich Cellules mimétiques de lymphocyte b
WO2018015330A1 (fr) * 2016-07-18 2018-01-25 Eth Zurich Cellules mimétiques de cellules bêta

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