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WO2006108201A1 - Promoteur en vue de l'expression de genes etrangers dans des cellules neuronales - Google Patents

Promoteur en vue de l'expression de genes etrangers dans des cellules neuronales Download PDF

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Publication number
WO2006108201A1
WO2006108201A1 PCT/AT2006/000140 AT2006000140W WO2006108201A1 WO 2006108201 A1 WO2006108201 A1 WO 2006108201A1 AT 2006000140 W AT2006000140 W AT 2006000140W WO 2006108201 A1 WO2006108201 A1 WO 2006108201A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
sequence
promoter
gene
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AT2006/000140
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German (de)
English (en)
Inventor
Manfred Windisch
Ulrike Bauer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
JSW-RESEARCH FORSCHUNGSLABOR GmbH
JSW Research Forschungslabor GmbH
Original Assignee
JSW-RESEARCH FORSCHUNGSLABOR GmbH
JSW Research Forschungslabor GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by JSW-RESEARCH FORSCHUNGSLABOR GmbH, JSW Research Forschungslabor GmbH filed Critical JSW-RESEARCH FORSCHUNGSLABOR GmbH
Priority to EP06721198A priority Critical patent/EP1869178A1/fr
Priority to AU2006235186A priority patent/AU2006235186A1/en
Priority to CA002604241A priority patent/CA2604241A1/fr
Publication of WO2006108201A1 publication Critical patent/WO2006108201A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • 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
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • 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
    • 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
    • C12N2830/40—Vector systems having a special element relevant for transcription being an insulator

Definitions

  • the invention relates to a promoter for the expression of foreign genes in neuronal cells.
  • a promoter represents a regulatory start-up sequence important to gene expression within the genome of each organism; it determines if, how and to what extent the transcription of a gene into messenger RNA (mRNA) takes place.
  • mRNA messenger RNA
  • strong and weak promoters ie, those that cause the formation of numerous or less numerous mRNA transcripts of the gene
  • constitutive constantly active
  • inducible ie, those that control transcription depending on particular conditions
  • tissue-specific promoters tissue-specific promoters.
  • the neuron-specific Thyl promoter of the mouse has already been used several times to generate transgenic mice that express foreign genes in their central nervous system. Such mouse strains represent important instruments, in particular for the investigation of the molecular mechanisms of neurodegenerative diseases and for the development of related potential therapies.
  • the mThyl promoter is an atypical promoter without TATA box. It consists of two identical promoter nonamers, each followed by a short untranslated exon (exons Ia and Ib). Exon Ib is followed by intron A followed by the first translated exon (exon 2). This is followed by exons 3 and 4, each separated by another intron (introns B and C).
  • This sequence (first promoter-nonamer up to and including exon 4) is flanked by untranslated regions (5'- and 3'-UTR), with the 3'UTR containing the poly A signal. It is believed that the 5 'UTR, exons Ia and Ib, and intron A have regulatory properties that may also be responsible for neuron-specific expression of the promoter (E. Spanopoulou et al., Mol. Cell. Biol 8: 3847-3856 and 1991; 11 (4): 2216-2228). The original mThyl promoter also displayed expression in the thymus in addition to neuronal expression. By deleting the region from exon 2 to exon 4, thymus expression could be eliminated (HA Ingraham and GA Evans, Mol.
  • the mThyl deletion deletion promoter of exon 2-4 was and is used to direct transgene expression in the generation of transgenic animals to specifically express the corresponding transgene in the neurons.
  • transgenic mice that express a mutated alpha-synuclein protein found in a rare hereditary form of Parkinson's disease under mThyl promoter control exhibit similar symptoms as the corresponding patients (H. van der Putten et al. J Neurosci 2000; 20 (16): 6021-9; B. Sommer et al., Exp Gerontol 2000; 35 (9-10): 1389-403).
  • the thyl promoter has significant disadvantages.
  • the considerable size of the thyl promoter makes it difficult for genetic engineering work, especially for the installation of larger gene constructs, difficult to handle.
  • the promoter size considerably restricts the size of the gene to be transferred.
  • the thyl promoter is often not strong enough to effect expression of the foreign gene in the transgenic animals to the desirable extent. It was therefore the object of the invention to modify the known mThyl promoter structure so that
  • Promotors can be solved, the only certain (original or slightly different) partial sequences of the mThyl promoter in
  • the invention relates to a promoter comprising a nucleic acid sequence derived from Thy-1 mammals which effects the neuron-specific transcription of a heterologous, 3 'downstream promoter nucleic acid sequence in both mammalian and non-mammalian cells.
  • the invention further relates to a nucleic acid sequence consisting of the sections
  • the invention further relates to a nucleic acid construct comprising an expression cassette comprising the portions (a), (b), (c), (d), (e), (f) and (g) characterized according to claim 2 and a heterologous Nucleic acid sequence positioned between and operatively linked to sections (e) and (f) is associated with.
  • the invention further relates to advantageous embodiments of this nucleic acid construct as disclosed according to claims 4 to 8.
  • the invention relates to an isolated cell or cell line comprising the promoter according to claim 1.
  • the invention also relates to an isolated cell or
  • a cell line comprising the nucleic acid sequence of claim 2.
  • This isolated cell or cell line advantageously comprises
  • the invention relates to a transgenic non-human animal comprising the promoter according to claim 1.
  • the invention also relates to a transgenic non-human animal comprising the nucleic acid sequence according to claim 2.
  • the transgenic non-human animal advantageously comprises a nucleic acid construct according to any one of claims 3 to claim 8.
  • the invention relates to a method for gene expression in neuronal and non-neuronal cells, which comprises the transformation / transfection of the cells with a vector comprising the nucleic acid sequence according to claim 2 or a nucleic acid construct according to any one of claims 3 to 8.
  • the invention relates to advantageous embodiments of this method with the features according to claims 16 to 18.
  • the invention relates to the use of a vector comprising the nucleic acid sequence of claim 2 or a nucleic acid construct according to any one of claims 3 to 8 for the preparation of injection solutions, which are for injecting in the central nervous system or in the cerebrospinal fluid are suitable.
  • the invention relates to the use of a vector comprising the nucleic acid sequence according to claim 2 or a nucleic acid construct according to one of claims 3 to 8 for the preparation of injection solutions for the treatment of neuronal functional disorders, such as stroke, ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's Disease, Brain and Spinal Cord Trauma, Amyotrophic Lateral Sclerosis.
  • neuronal functional disorders such as stroke, ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, Huntington's Disease, Brain and Spinal Cord Trauma, Amyotrophic Lateral Sclerosis.
  • the invention relates to the use of a vector comprising the nucleic acid sequence of claim 2 or a nucleic acid construct according to any one of claims 3 to 8 for the preparation of Injection solutions for the treatment of neurogenic disorders.
  • the invention relates to a kit for the expression of recombinant gene products comprising isolated cells or cell lines according to one of claims 9 to 11.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a promoter according to claim 1.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a protein which is encoded by the nucleic acid sequence according to claim 2.
  • the invention further relates to a medicament comprising a nucleic acid construct according to one of claims 3 to 8.
  • Possible ways to carry out the invention can be represented as follows: starting from the mThyl expression cassette (Moechars et al., 1996, EMBO J. 15 (6), 126574), which has an original size of about 8.2 kb and which instead of the When mThyl Exons 2 to 4 contains an Xho I linker, larger deletions were made with the help of restriction endonucleases, followed by smaller insertions, resulting in a 1.6 kb promoter cassette.
  • the mThyl expression cassette was cleaved with Sma I, thereby removing the region adjacent to the 5 'end of the MThyl 5'-UTR.
  • the resulting construct into which 3 'terminal of mThyl intron A the eGFP reporter gene (coded for a green fluorescent protein, which is harmless for the mouse and serves as a fluorescence microscopic marker), was integrated by transfection into two neuronal cell lines (SH- SY5Y and Neuro-2A) for promoter activity (eGFP expression).
  • mTUB promoter mouse T_hylsable for brain expression; see Figure 3 for the sequence.
  • mTUB promoter mouse T_hylsable for brain expression; see Figure 3 for the sequence.
  • the functionality was transfected into SH-SY5Y and neuro-2A cells and control of eGFP expression compared to eGFP construction under the control of the unchanged mThyl promoter (s) for human platelet-derived growth factor (hPDGF ) and cytomegalovirus (CMV), as shown below.
  • s mThyl promoter
  • hPDGF human platelet-derived growth factor
  • CMV cytomegalovirus
  • Promoter properties may be different in vitro and in vivo.
  • the intrinsic neuron-specific mThyl promoter after transfection of the hamster ovarian cell line CHO-Kl with a mThyl / eGFP construct causes strong expression of the eGFP reporter gene.
  • mTUB-eGFP-tg transgenic mouse strain
  • the mTUB-eGFP transgenic mouse strain was generated as follows, whereby for microinjection the promoter-gene construct was incorporated into an insulator cassette (chicken- ⁇ -globin insulator sequences) which should facilitate expression and protect against foreign regulation (plasmid pC2xINS, JSW Research). 1. Preparation and Preparation of the Vector for Microinjection:
  • mTUB-eGFP construct has Not I or Nde I ends; Nde I end is filled in with polymerase I Klenow fragment
  • the linear insulator mTUB-eGFP insulator construct was isolated from a 0.8% TAE agarose gel without ethidium bromide, purified and diluted with microinjection buffer such that there were 1000 DNA molecules per injection volume. The injection was carried out according to the standard protocol (Brem et al., 1985). 3 weeks old hormone-treated female CB6F1 were used for this purpose
  • mice mated with C57BL / 6 males.
  • the resulting fertilized CB6F1 (B6) oocytes were removed and cultured until two clear pronuclei were visible.
  • the purified DNA construct was then injected into the pronucleus followed by overnight culture through to
  • the control gene eGFP was used to characterize promoter properties and transgene expression as quickly and easily as possible.
  • the eGFP gene can be replaced by any other neuron-specific gene to be expressed (see Fig. 3, sequence of the mTUB promoter cassette).
  • the functional regions are assigned to the following nucleotide regions in the sequence:
  • control gene eGFP (not part of the disclosure)
  • the coding sequence which extends from nucleotide 1393 to 2112, represents the reporter gene eGFP, which is not part of the disclosure. Following the reporter gene sequence is the late SV40 poly A
  • the following table shows a comparison of the mTUB promoter according to the invention with the unchanged murine thyl promoter, as well as the promoters of the human platelet-derived growth factor (hPDGF) and the cytomegalovirus (CMV).
  • hPDGF human platelet-derived growth factor
  • CMV cytomegalovirus
  • the mTUB promoter in both neuronal cell lines is the mThyl promoter both in terms of transfection efficiency as well as the expression intensity far superior.
  • mTUB achieved a 25-fold higher transfection efficiency than the hPDGF promoter with approximately the same average signal intensity
  • Neuro-2A cells a 1.5-fold higher transfection efficiency than the hPDGF promoter at approximately 6-fold average signal intensity.
  • the transfection efficiency is comparable to that of the CMV promoter, with significantly higher average signal intensity (in Neuro-2A).
  • FIG. 4 shows fluorescence microscopic images of brain sections of the transgenic mouse strain mTUB-eGFP-tg in comparison to the corresponding nontransgenic strain C57BI6 ntg.
  • the cerebral sections clearly show the eGFP-expressing neurons of the transgenic mouse compared to the eGFP-negative brain section of the non-transgenic mouse.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
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Abstract

Promoteur comprenant une séquence d'acide nucléique dérivée de Thy-1 de mammifères, qui provoque la transcription spécifique des neurones d'une séquence d'acide nucléique hétérologue se trouvant en position 3' en aval par rapport au promoteur, aussi bien dans des cellules de mammifères que dans des cellules de non-mammifères.
PCT/AT2006/000140 2005-04-14 2006-04-06 Promoteur en vue de l'expression de genes etrangers dans des cellules neuronales Ceased WO2006108201A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP06721198A EP1869178A1 (fr) 2005-04-14 2006-04-06 Promoteur en vue de l'expression de genes etrangers dans des cellules neuronales
AU2006235186A AU2006235186A1 (en) 2005-04-14 2006-04-06 Promoter for the expression of foreign genes in neuronal cells
CA002604241A CA2604241A1 (fr) 2005-04-14 2006-04-06 Promoteur en vue de l'expression de genes etrangers dans des cellules neuronales

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ATA637/2005 2005-04-14
AT6372005A AT501628B1 (de) 2005-04-14 2005-04-14 Promotor zur expression von fremdgenen in neuronalen zellen

Publications (1)

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WO2006108201A1 true WO2006108201A1 (fr) 2006-10-19

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PCT/AT2006/000140 Ceased WO2006108201A1 (fr) 2005-04-14 2006-04-06 Promoteur en vue de l'expression de genes etrangers dans des cellules neuronales

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EP (1) EP1869178A1 (fr)
AT (1) AT501628B1 (fr)
AU (1) AU2006235186A1 (fr)
CA (1) CA2604241A1 (fr)
WO (1) WO2006108201A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9398761B2 (en) 2012-03-19 2016-07-26 Brainco Biopharma, S.L. Transgenic animal model of mood disorders
WO2020168111A1 (fr) 2019-02-15 2020-08-20 Exhaura, Ltd. Inhibiteurs de kinase à double glissière à leucine pour thérapie génique

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006927A1 (fr) * 1994-09-01 1996-03-07 Merck & Co., Inc. Animal transgenique exprimant une forme familiale d'une proteine precurseur de l'amyloide humaine
US6504080B1 (en) * 1999-10-15 2003-01-07 Novartis Ag Transgenic animal model for neurodegenerative disorders
WO2003016495A2 (fr) * 2001-08-20 2003-02-27 Merck & Co., Inc. Rongeurs transgeniques comme modeles animaux pour la modulation de la proteine du recepteur b1 de la bradykinine

Family Cites Families (4)

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US6610287B1 (en) * 1990-04-16 2003-08-26 The General Hospital Corporation Transfer and expression of gene sequences into nervous system cells using herpes simplex virus mutants with deletions in genes for viral replication
CA2290039A1 (fr) * 1997-05-14 1998-11-19 Merck & Co., Inc. Animal transgenique exprimant une proteine preseniline 1 mutante non native de type sauvage de la maladie d'alzheimer familiale dans un contexte depourvu de preseniline 1 native
AU2001255613B2 (en) * 2000-04-24 2005-08-04 Wyeth Transgenic animal
WO2004033661A2 (fr) * 2002-10-11 2004-04-22 Advanced Research And Technology Institute Acides nucleiques codant un variant de sous unite legere de ferritine, polypeptides, animaux transgeniques les contenant, anticorps associes, et procedes d'utilisation correspondant

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006927A1 (fr) * 1994-09-01 1996-03-07 Merck & Co., Inc. Animal transgenique exprimant une forme familiale d'une proteine precurseur de l'amyloide humaine
US6504080B1 (en) * 1999-10-15 2003-01-07 Novartis Ag Transgenic animal model for neurodegenerative disorders
WO2003016495A2 (fr) * 2001-08-20 2003-02-27 Merck & Co., Inc. Rongeurs transgeniques comme modeles animaux pour la modulation de la proteine du recepteur b1 de la bradykinine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHIN LIH-SHEN ET AL: "Neuron-specific expression of the synapsin II gene is directed by a specific core promoter and upstream regulatory elements", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 28, 1994, pages 18507 - 18513, XP002389205, ISSN: 0021-9258 *
INGRAHAM H A ET AL: "CHARACTERIZATION OF TWO ATYPICAL PROMOTERS AND ALTERNATE MESSENGER RNA PROCESSING IN THE MOUSE THY-1.2 GLYCOPROTEIN GENE", MOLECULAR AND CELLULAR BIOLOGY, vol. 6, no. 8, 1986, pages 2923 - 2931, XP002389203, ISSN: 0270-7306 *
SPANOPOULOU E ET AL: "THE FUNCTIONAL DOMAINS OF THE MURINE THY-1 GENE PROMOTER", MOLECULAR AND CELLULAR BIOLOGY, vol. 11, no. 4, 1991, pages 2216 - 2228, XP002389204, ISSN: 0270-7306 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9398761B2 (en) 2012-03-19 2016-07-26 Brainco Biopharma, S.L. Transgenic animal model of mood disorders
WO2020168111A1 (fr) 2019-02-15 2020-08-20 Exhaura, Ltd. Inhibiteurs de kinase à double glissière à leucine pour thérapie génique
US12270033B2 (en) 2019-02-15 2025-04-08 Exhaura, Ltd. Dual leucine zipper kinase inhibitors for gene therapy

Also Published As

Publication number Publication date
EP1869178A1 (fr) 2007-12-26
AU2006235186A1 (en) 2006-10-19
CA2604241A1 (fr) 2006-10-19
AT501628A1 (de) 2006-10-15
AT501628B1 (de) 2007-09-15

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