WO2001029210A1 - Procede d'etablissement de connexions entre genes - Google Patents

Procede d'etablissement de connexions entre genes Download PDF

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Publication number
WO2001029210A1
WO2001029210A1 PCT/IL2000/000642 IL0000642W WO0129210A1 WO 2001029210 A1 WO2001029210 A1 WO 2001029210A1 IL 0000642 W IL0000642 W IL 0000642W WO 0129210 A1 WO0129210 A1 WO 0129210A1
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Prior art keywords
gene
regulatory
cell cycle
genes
regulatory region
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PCT/IL2000/000642
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English (en)
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WO2001029210B1 (fr
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Ben-Zion Vider
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Genena Ltd.
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Priority to AU78155/00A priority Critical patent/AU7815500A/en
Publication of WO2001029210A1 publication Critical patent/WO2001029210A1/fr
Publication of WO2001029210B1 publication Critical patent/WO2001029210B1/fr

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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • 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
    • 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
    • C12N15/67—General methods for enhancing the expression

Definitions

  • Cyclins are regulators of the cell cycle. Eukaryotic cell cycle progression is regulated by an orderly and sequential activation of cyclins and cyclin dependent kinases (Cdks) which phosphorylate key substrates to initiate DNA synthesis (1-4).
  • oncogenes including ras, myc, EGF-R, and C-met, are basically in their native form essential genes that play key roles in cell cycle events.
  • tumor suppressor genes like p53 and the retinoblastoma gene, while inactivated contribute to cancer development, and in their normal structure and in normal cells they are basically genes involved in central cell cycle checkpoints (5-6). When they are overexpressed or become over activated they result in cell cycle a ⁇ est and in the entry of the cells to an appoptotic phase. Transcriptional regulation of the p53 gene plays an important role in the expression of wild type p53. and alterations in p53 transcription levels occur in response to changes in the cell cycle. P53 itself activate the expression of a number of target genes including p21 WAFL GADD45. bax Fas/APOl and many others. These genes in addition to p53 itself might mediate p53 apoptotic effect.
  • Cyclin dependent kinase inhibitors play important roles in cell cycle control by coordinating internal and external signals and impending proliferation at several key checkpoints (7-11).
  • regulatory sequence regulatory element, and regulatory regions are synonyms describing all the known classes of cis -acting elements, promoters, enhancers, silencers, regulatory sequences within introns. and 3' regulatory sequences. In most instances it refers to the upstream regulatory element and unless stated otherwise in the following recombinant vectors it includes both enhancers and promoters.
  • genes that are cell cycle activators is a broad and unique term used in this patent application to describe any gene that is either a cell cycle gene like cyclin or Cdk. or an oncogene which is responsible in general terms to increased proliferation rate.
  • genes that are cell cycle suppressors is a broad and unique term comprising in this patent application two groups of genes one is that of inhibitory cell cycle genes that interact with cyclins or Cdks like p21 WAFl or INK4 family (pl5 INK4b, pl6 INK4a, pl8 INK4c, and pi 9 INK4d) and the other is that of tumor suppressor genes that have more general effect on cells terminal differentiation, or apoptosis like Rb. p53 and Bax genes.
  • recombinant construct or recombinant vector are synonyms describing any plasmid or any other vector used in biotechnology that contains either an inserted foreign regulatory element, or an inserted foreign gene, or an insertion of both foreign regulatory element and of an inserted foreign gene that can be both part of the same gene or can be originated out of two different genes.
  • heterologous gene or heterologous regulatory region describes in this patent application a situation in which the recombinant construct contains an insertion of a regulatory region positioned upstream to a gene and the two inserted elements are originated out of two different genes or genes' surrounding.
  • the vectors of the present invention contain all the genes necessary for selection by antibiotic resistance or by any other method known to those versed in the art those cells that incorporate the vectors, and only those aspects of the vectors that are unique to the method of invention are described in details.
  • the insert includes the regulatory sequence of an investigated gene, and the other insert that is joint to it includes the cDNA of a cell cycle activator gene, or a cell cycle a ⁇ est gene, and vice versa: one insert includes the regulatory sequence of a cell cycle activator gene, or a cell cycle a ⁇ est gene and the other insert that is joint to it includes the cDNA of the investigated gene. Each one of these recombinants is then transfe ⁇ ed into the cells and the activity of the regulatory element is measured. This activity reflects the functional connections that exist between the product of one gene and the regulatory region of a second gene while at least one of the two investigated genes or their regulatory regions are those of a cell cycle gene.
  • Figure 1 shows the four basic types of recombinant constructs: 1A. Type I IB. Type II 1C. Type III ID. Type IV
  • Figure 2 shows a recombinant construct that contains two identical regulatory elements and as it model for the explanation it uses Type I construct of figure 1.
  • Type I contains the regulatory sequence of a first gene categorized as a cell cycle activator gene indicated by 105. and linked downstream to it is the cDNA of a second gene which is the investigated gene A indicated by 110.
  • Type II contains the regulatory sequence of the investigated second gene which is gene A indicated by 115, and linked downstream to it is the cDNA of the first gene which is the cell cycle activator gene indicated by 120.
  • Type III contains the regulatory sequence of a third gene categorized as a cell cycle suppressor gene indicated by 125, and linked downstream to it is the cDNA of the second gene which is the investigated gene A indicated by 110.
  • Type IV contains the regulatory sequence of the investigated gene A indicated by 115, and linked downstream to it is the cDNA of the third gene which is the cell cycle suppressor gene indicated by 130.
  • the purpose of the cross reacted fragments of DNA inserted together into the same vector is to identify these recombinant vectors in which a positive regulatory loop is formed between the protein encoded by the cDNA and its heterologous regulatory sequence.
  • This loop might be a very direct and short loop that can occurs whenever the transcribed gene is a transcription factor, that binds and activates the recombinant heterologous regulatory sequence that is located upstream to it, thus causing the augmentation of its own transcription.
  • Positive loops might be the result of an indirect and more complicated loop.
  • a proposed mechanism for the recombinant vector mode of action might include the following steps and alternatives:
  • the cloned recombinant vector is transfe ⁇ ed into the cells.
  • the recombinant vector is composed for example from the regulatory sequence of a cell cycle activator gene, and linked downstream to it is the cDNA of a second gene, for example a cDNA encoding a transcription factor known to bind and activate the cell cycle activator gene regulatory sequence.
  • Basal transcription of the gene encoding the transcription factor exist as a result of activation of the native gene within the cell.
  • a positive auto-regulation loop is then established enhancing the recombinant cDNA transcription.
  • a cascade of genes is activated sequentially initiated by the heterologous protein encoded cDNA.
  • the last gene in the cascade is a transcription factor that binds and activates the regulatory sequence.
  • An indirect circle of positive feedback is thus established, that result in enhancement of the heterologous cDNA expression.
  • the regulatory regions of the new recombinant construct are those of the cell cycle activator gene of figure 1 and they are indicated by 105 and the heterologous gene which is linked downstream to one of them is indicated by 110.
  • Linked downstream to the second regulatory region is a reporter gene like the chloramphenicol acetyl transferase indicated by 140, that measures the extent to which the regulatory region is activated and the time course of the activation.
  • This system enables continuous measurements of the regulatory region activity not dependent on intermittent measurements of the specific gene " s RNA transcripts.
  • this vector that is designated the '"double regulatory region vector" contains in two different parts of the costruct the same regulatory region.
  • Table 1 demonstrates an illustration of results, obtained with the four basic recombinant vectors transfe ⁇ ed into a specific cell type.
  • the four rows in table 1 are equivalent to and are present in the same order as the four types (types I -IV) of recombinant vectors presented in page 4 and described in figure 1 .
  • the product of the cell cycle activator gene directly or indirectly activates the regulatory sequence derived from the investigated gene, gene A (raw 1), and thus results in a positive feedback and in its up regulation.
  • the product of the investigated gene (gene A) activates in a direct or indirect manner its own transcription as a result of an activated protein somewhere in the cascade that binds to the regulatory sequence derived from the cell cycle activator gene (raw 2).
  • the other two recombinants vectors presented in rows 3 and 4 do not result in positive auto regulation.
  • gene A and the chosen cell cycle activator gene activate each other and establish a connection that support cell cycle progression. It is reasonable to presume that these recombinants, types I and II, might also accelerate the proliferation rate of the transfected cells.
  • two recombinant constructs can be co-transfected into a cell revealing the final effect of the two genes and their crossed regulatory regions present together and augmenting the transcription activation effect they possess on one another as exemplified in table 1. This might be even more important when one of the genes or both of them are suspected of having a positive auto regulatory effects, meaning that in their native state the gene product activates its own transcription. In this situation combining the two recombinants together enable the genes to activate their own regulatory regions though these regulatory regions are not positioned in cis to their own genes, and in fact might contribute to the activation of the heterologous gene that is positioned downstream to the regulatory region.
  • this new method of invention might be directed toward transcription factors, for example those encoded by large gene families like the homeobox genes and like the MADS box genes, especially when a direct role of these genes in the cell cycle events is suspected. Indeed several reports point for example to the possible role of several homeobox genes in the process of cell cycle regulation (17-22). On the other hand it is also well known that these genes are expressed in a differential manner in different cells and tissues. Projecting these two pieces of information to these genes " sequences makes it temptating to speculate that these genes' conserved homeobox motives serve in this conserved function of participating in the cell cycle network. Thus the biological system that is based on this method of invention offers better understanding of these genes * function in the context of the cell cycle.
  • a niinimal promoter element should be inserted in such a recombinant vector.
  • the final recombinant vector will include the following inserts: 1) upstream is the gene family conserved region segment acting as the regulatory element. 2) down stream to it is the n ⁇ iimal promoter region 3) linked downstream to the ⁇ nal promoter region is the heterologous gene sequence which is of a cell cycle activator gene in one recombinant construct, and a cell cycle suppressor gene in a second recombinant construct.
  • upstream is the gene family conserved region segment acting as the regulatory element.
  • down stream to it is the n ⁇ iimal promoter region 3
  • linked downstream to the ⁇ nal promoter region is the heterologous gene sequence which is of a cell cycle activator gene in one recombinant construct, and a cell cycle suppressor gene in a second recombinant construct.
  • the method can be used to classify any gene from a cDNA gene library as either related to cell cycle activation or to cell cycle suppression.
  • the cDNA molecules of an expression Ubrary are inserted into a library in which the vectors are those of type I or III only in relation to the regulatory sequences they have.
  • One library contains the regulatory region of a selected cell cycle activator gene and another library the regulatory region of a cell cycle suppressor gene.
  • the construct can be one of the basic forms described in figure 1 or the double regulatory region vectors as exemplified in figure 2.
  • Selection for the cells that expresses the introduced gene is then performed by detecting and picking those cells which highly expresses the reporter gene.
  • the selection can also be performed as a result of the effect that occurs within the cells between the gene its regulatory region and the general circuit of the cell cycle. If the gene that was introduced causes significant activation of the heterologous regulatory region that is of a cell cycle activator gene, and also augments the cell cycle circuit in general then the final effect might be an accelerated proliferation of the cells and thus an easy selection of the rapidly dividing cells.
  • the gene that was introduced causes activation of the heterologous regulatory region that is of a cell cycle suppressor gene, and also causes cell cycle a ⁇ est then the final effect might be a decreased proliferation rate of the cells that expresses this recombinant construct relative to the non transfected cells.
  • selection for the cells with the inhibited proliferation rate can be performed after exposing the cells to an anti mitogenic agent and picking those cells that survived the treatment as they had a slower proliferation rate.

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  • Health & Medical Sciences (AREA)
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Abstract

Cette invention a trait à une série de dosages et de produits de recombinaison établissant des connexions entre la région régulatrice d'un premier gène et le produit d'un autre gène. La région régulatrice du premier gène est introduite dans un produit de recombinaison et liée en aval à l'ADN complémentaire du second gène, la région régulatrice du second gène est ensuite introduite dans un produit de recombinaison et liée en aval à l'ADN complémentaire du premier gène. Dans chaque produit de recombinaison, l'ADN complémentaire du gène ou la région régulatrice est celui/celle d'un gène à cycle cellulaire. Les produits de recombinaison sont alors transférés dans les cellules et le degré d'activation des éléments régulateurs est régularisé.
PCT/IL2000/000642 1999-10-18 2000-10-12 Procede d'etablissement de connexions entre genes WO2001029210A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU78155/00A AU7815500A (en) 1999-10-18 2000-10-12 A method for establishing connections between genes

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IL132446 1999-10-18
IL13244699A IL132446A0 (en) 1999-10-18 1999-10-18 A method for establishing connections between genes

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997035992A1 (fr) * 1996-03-27 1997-10-02 Vical Incorporated Systemes de tetracycline inductibles/repressibles
WO1997040379A2 (fr) * 1996-04-23 1997-10-30 Cold Spring Harbor Laboratory EPREUVES ET REACTIFS PERMETTANT L'IDENTIFICATION DE MODULATEURS D'ACTIVATION DE LA MITOSE INDUITE PAR Cdc25
EP0848063A2 (fr) * 1996-09-24 1998-06-17 Hoechst Aktiengesellschaft Contruction d'acides nucléiques containant des promoteurs hybrides pour la thérapie génique
EP0848061A2 (fr) * 1996-12-11 1998-06-17 Hoechst Aktiengesellschaft Auto-augmentation de l'expression, par des systèmes controlâble pharmacologiquement
EP0864651A2 (fr) * 1997-03-14 1998-09-16 Hoechst Aktiengesellschaft Promoteur du gène cdc25B, sa préparation et son usage
WO1999011795A1 (fr) * 1997-09-05 1999-03-11 Icos Corporation Materiels de proteine-kinase point de controle du cycle cellulaire, effecteur de chk1 mammalien et methodes

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997035992A1 (fr) * 1996-03-27 1997-10-02 Vical Incorporated Systemes de tetracycline inductibles/repressibles
WO1997040379A2 (fr) * 1996-04-23 1997-10-30 Cold Spring Harbor Laboratory EPREUVES ET REACTIFS PERMETTANT L'IDENTIFICATION DE MODULATEURS D'ACTIVATION DE LA MITOSE INDUITE PAR Cdc25
EP0848063A2 (fr) * 1996-09-24 1998-06-17 Hoechst Aktiengesellschaft Contruction d'acides nucléiques containant des promoteurs hybrides pour la thérapie génique
EP0848061A2 (fr) * 1996-12-11 1998-06-17 Hoechst Aktiengesellschaft Auto-augmentation de l'expression, par des systèmes controlâble pharmacologiquement
EP0864651A2 (fr) * 1997-03-14 1998-09-16 Hoechst Aktiengesellschaft Promoteur du gène cdc25B, sa préparation et son usage
WO1999011795A1 (fr) * 1997-09-05 1999-03-11 Icos Corporation Materiels de proteine-kinase point de controle du cycle cellulaire, effecteur de chk1 mammalien et methodes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BLAYDES ET AL: "TOLERANCE OF HIGH LEVELS OF WILD-TYPE P53 IN TRANSFORMED EPITHELIAL CELLS DEPENDENT ON AUTO-REGULATION BY MDM-2", ONCOGENE,GB,BASINGSTOKE, HANTS, vol. 15, no. 14, 17 April 1997 (1997-04-17), pages 1859 - 1868, XP002075048, ISSN: 0950-9232 *
GALAKTIONOV K ET AL: "CDC25 CELL-CYCLE PHOSPHATASE AS A TARGET OF C-MYC", NATURE,GB,MACMILLAN JOURNALS LTD. LONDON, vol. 382, no. 6591, 8 August 1996 (1996-08-08), pages 511 - 517, XP002041131, ISSN: 0028-0836 *
ROTH F P ET AL: "FInding DNA regulatory motifs within unaligned noncoding sequences clustered by whole-genome mRNA quantitation", NATURE BIOTECHNOLOGY,NATURE PUBLISHING,US, vol. 16, October 1998 (1998-10-01), pages 939 - 945, XP002153325, ISSN: 1087-0156 *

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WO2001029210B1 (fr) 2001-10-18
IL132446A0 (en) 2001-03-19
AU7815500A (en) 2001-04-30

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