EP0946724A2 - Polypeptides de la famille "basic helix-loop-helix" bhlh, sequences d'acides nucleiques correspondantes - Google Patents

Polypeptides de la famille "basic helix-loop-helix" bhlh, sequences d'acides nucleiques correspondantes

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Publication number
EP0946724A2
EP0946724A2 EP97952961A EP97952961A EP0946724A2 EP 0946724 A2 EP0946724 A2 EP 0946724A2 EP 97952961 A EP97952961 A EP 97952961A EP 97952961 A EP97952961 A EP 97952961A EP 0946724 A2 EP0946724 A2 EP 0946724A2
Authority
EP
European Patent Office
Prior art keywords
sequence
bhlh
nucleic acid
seq
vector
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.)
Withdrawn
Application number
EP97952961A
Other languages
German (de)
English (en)
French (fr)
Inventor
Christine Icard-Liepkalns
Jacques Mallet
Philippe Ravassard
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.)
Aventis Pharma SA
Original Assignee
Rhone Poulenc Rorer SA
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 Rhone Poulenc Rorer SA filed Critical Rhone Poulenc Rorer SA
Publication of EP0946724A2 publication Critical patent/EP0946724A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/028—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a herpesvirus

Definitions

  • the present invention relates to a new protein of the bHLH type and to the corresponding coding nucleic sequence. It also relates to expression vectors integrating said sequence and the use of this sequence or protein for therapeutic purposes.
  • the embryonic neural tube is originally composed of an undifferentiated neuroepithelium over its entire length.
  • the cells then differentiate according to their positions on the anterior / posterior, dorsal / ventral and medium / lateral axes generating
  • the proteins of this family have very conserved amino acid motifs and more particularly a basic domain followed by Helix 1 and Helix 2. These last two motifs are separated by a loop of variable size.
  • certain products of these bHLH genes participate in the various stages of neurogenesis during neuronal determination and differentiation.
  • the present invention specifically relates to the isolation, sequencing and characterization of a cDNA coding for a polypeptide belonging to a new family of proteins of the bHLH type. It also describes recombinant DNAs incorporating this sequence, vectors containing it and their use to activate expression of recombinant proteins
  • the present invention has for first object a bHLH type polypeptide endowed with a transcriptional activity characterized in that it shares a sequence homology with the bHLH type proteins only at the level of its bHLH domain.
  • the present invention relates to a polypeptide having a transc ⁇ ptional activity characterized in that it is represented in whole or in part by SEQ ID N ° 1 or one of its derivatives
  • drift is meant in the sense of the present invention products comprising for example modifications in the primary structure, such as deletions of one or more residues, substitutions of one or more residues, and / or modifications at the level of one or more residues
  • the number of residues affected by the modifications can be for example from 1 2 or from 3 to 10 20 or 30 residues
  • Le term de ⁇ ve also includes molecules comprising additional internal or terminal parts, of peptide or non-peptide nature II can be in particular active parts, markers, amino acids, such as a methionine in position -1
  • derivative includes also molecules with modifications in the tertiary structure (N-terminal end, etc.)
  • the derivatives share at least one structural homology with the claimed protein and more preferably at least at the level of the regions bordering the bHLH domain, and retain its biological property of transc ⁇ ptional activator
  • the derivatives can also bring new properties to the claimed polypeptides (labeling ,) or improve their properties (stability, biological activity, etc.)
  • it is a bHLH type polypeptide having the sequence represented in SEQ ID No. 1. It is more preferably a protein comprising an open reading frame, of 214 amino acids whose expression of the corresponding mRNA is detected in the rat embryo in the form of longitudinal bands.
  • This protein is designated below under the name Relax for "Rat Embryonic Longitudinal Axis" and constitutes one of the objects claimed according to the present invention.
  • the present invention also relates to a nucleic acid sequence capable of expressing a bHLH type polypeptide as defined above.
  • the present invention relates to a nucleic acid sequence characterized in that it is represented in whole or in part by SEQ ID No. 1, its complementary strand or its derivative
  • the claimed nucleic acids may be fragments of complementary DNA (cDNA), genomic DNA (gDNA) or fragments of RNA. It is more preferably cDNA or gDNA.
  • cDNA complementary DNA
  • gDNA genomic DNA
  • RNA fragments of RNA. It is more preferably cDNA or gDNA.
  • the term de ⁇ ve designates any sequence different from the sequence considered due to a degeneration of the genetic code obtained by one or more modifications of a genetic and / or chemical nature as well as any sequence hybridizing with these sequences or fragments thereof and whose product has the activity indicated
  • modification of a genetic and / or chemical nature one can understand any mutation, substitution, deletion, addition and / or modification of one or more residues.
  • Such derivatives can be generated for different purposes, such as in particular that of improving its production levels which increase and / or modify its activity, or that of giving it new pharmacokinetic and / or biological properties.
  • chimeric nucleic sequences comprising an additional heterologous part. linked to one end, of the type for example hybrid constructions consisting of a cDNA with which one or more introns would be associated
  • the claimed nucleic acids may comprise promoter, activation, regulation sequences, etc.
  • the term derivative also includes sequences homologous to the sequence considered, derived from other cellular sources and in particular from cells of human origin, or from other organisms, and having an activity of the same type or substantially similar type. Such homologous sequences can be obtained by hybridization experiments Hybridizations can be carried out from nucleic acid libraries, using as probe the native sequence or a fragment thereof, under conventional conditions of st ⁇ ngence (Maniatis et al, Cf general molecular biology techniques), or, preferably, under high conditions
  • the present invention also intends to cover the corresponding antisense sequences, the expression of which makes it possible to control the transcription of cellular mRNAs.
  • sequences may consist of all or part of the nucleic sequence considered transcribed in the reverse orientation. They are Complementary or significantly complementary to the nucleic acid sequence in whole or in part
  • Another subject of the present invention is the use of the claimed polypeptides to control and / or participate in the expression of genes.
  • These transcriptional activators can be very particularly advantageous for targeting the expression of a protein at the level of the central nervous system.
  • polypeptides of the invention can be obtained by expression in a cellular host of a nucleotide sequence as described above, incorporated or not in a recombinant DNA, using techniques known to those skilled in the art, or by a combination of these techniques
  • the nucleic acid sequences according to the invention form part of a vector useful for inducing in vivo, ex vivo and / or in vitro the expression of the claimed polypeptides.
  • the vector used can be of various origins, since it is capable of transforming animal cells, preferably human nerve cells
  • a viral vector is used, which can be derived from adenovirus, retrovirus, adeno-associated virus (AAV), virus herpes, cytomegalovirus (CMV), vaccinia virus, etc.
  • the recombinant virus according to the invention is a defective virus
  • the term "defective virus designates a virus incapable of replicating in the target cell.
  • the genome of the defective viruses used in the context of the present invention is therefore devoid of at least sequences necessary for the rep cation of said virus in the infected cell These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the nucleic acid of the invention
  • the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.
  • nucleic acid sequences of the invention in the form incorporated into an adenovirus, an AAV or a defective recombinant retrovirus. According to a preferred embodiment, it is an adenovirus
  • adenovirus serotypes whose structure and properties vary somewhat.
  • human adenoviruses of type 2 or 5 Ad 2 or Ad 5
  • adenoviruses of animal origin see application WO94 / 26914.
  • adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of murine bovine canine origin (example Mavl Beard et al, Virology 75 (1990) 81) avian or even simian porcine ovine (SAV example)
  • the adenovirus of animal origin is a canine adenovirus, more preferentially an adenovirus CAV2 [Manhattan strain or A26 / 61 (ATCC VR- 800) for example]
  • adenovirus of human or canine or mixed origin Preferably used in part of the invention of adenovirus of human or canine or mixed origin
  • the viral gene considered can be made non-functional by any technique known to those skilled in the art, and in particular by total deletion, substitution, partial deletion, or addition of one or more bases in the gene or genes considered Other regions can also be modified, and in particular the region E3 (WO95 / 02697), E2
  • the adenovirus comprises a deletion in the El and E4 regions According to another preferred embodiment, it comprises a deletion in the El region at which the region is inserted
  • the deletion in the region E 1 preferably extends from nucleotides 455 to 3329 on the sequence of the adenovirus
  • the exogenous nucleic acid sequence is inserted at the level of the deletion in the El region
  • the recombinant defective viruses of the invention can be prepared by homologous recombination between a defective virus and a plasmid carrying, inter alia, the nucleotide sequence as defined above (Levrero et al, Gene 101 (1991) 195, Graham, EMBO J 3 (12) (1984) 2917) Homologous recombination occurs after co-transfection of said viruses and plasmid into an appropriate cell line.
  • the cell line used should preferably (î) be transformable by said elements, and (u), include the sequences capable of complementing the part of the defective virus genome, preferably in integrated form to avoid the risks of recombination
  • a line usable for the preparation of defective recombinant adenoviruses mention may be made of the human embryonic kidney line 293 (Graham and al J Gen Virol 36 (1977) 59) which contains in particular integrated in its genome, the left part of the genome of an Ad 5 adenovirus (12%)
  • the CRIP line Danos and Mulligan, PNAS 85 (1988) 6460 )
  • the viruses that have multiplied are recovered and purified according to conventional molecular biology techniques
  • the vector, the nucleic acid or the polypeptide of the invention can be in an isolated or purified form
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one nucleotide sequence, a vector or a polypeptide according to the invention
  • the invention also extends to any use of the claimed polypeptides to control and / or participate in the expression of genes
  • FIG. 1 CIG235 cDNA nucleotide sequence This sequence shows the different oligonucleotides used to isolate the complete cDNA The arrows indicate the 5 'to 3' orientation of these oligonucleotides
  • Figure 2 Comparison of amino acid sequences of the bHLH domain of Relax with other proteins in the family. In black are the homologous amino acids, in gray the amino acids of similar structure.
  • rat GCS Rat Superior Cervical Ganglia
  • the proteins of this family have very conserved acid motif motifs, in particular a basic domain followed by Helix 1 and Helix 2
  • degenerate oligonucleotides are chosen encoding these various conserved regions. They were therefore selected so as to conserve the basic regions and the Helix II domain of the Drosophilia achaete-scute complex and of the mammalian bHLH genes such as Mash-1, Mash-2 and M-Twist.
  • Such oligonucleotides have been used respectively as sense and antisense primer on the one hand, to carry out a direct screening of a GCS library constructed by PCR, and on the other hand to carry out PCR on cDNA-ss of GCS.
  • the sense oligonucleotide was chosen from the basic domain of these bHLH proteins, more precisely it is the oligonucleotide corresponding to the following polypeptide fragment: 5'-AATKHGMGIGAGCGCIDKCGCRYG-3 '(SEQ ID No. 2)
  • the antisense oligonucleotide was determined from the Helix II domain of the same bHLH proteins. It is the oligonucleotide corresponding to the following polypeptide fragment: 5'-GGCSRDTYTCAGGGTSYBGAYCTT-3 '(SEQ ID N ° 3)
  • the single-strand cDNA is prepared from 500 ng of denatured RNApolyA + from the GCS of rats, two days old
  • the PCR amplification is carried out on a mixture comprising, in addition to the PCR buffer (10 mM Tris, pH 8.3, 50 mM Kcl, 2.5 mM MgCl 2), 200 ⁇ M dNTP, 12.5 ⁇ M of each oligonucleotide and approximately 20 ng of ss-cDNA
  • La PCR is carried out by successively performing 3 minutes of denaturation at 94 ° C, 2 cycles (94 ° C for 30 sec, 50 ° C for 45 sec, 72 ° c for 1.5 min) followed by 38 cycles (94C for 30 sec , 62 ° C for 45 sec, 72) C for 1.5 min)
  • the PCR products are cloned at the SmaI site of pUC19 and the 1500 clones thus obtained, analyzed by sequencing We looked for the presence of a Helix I motif in the amino acid sequence deduced from the nucleotide sequence of these different cDNAs A large proportion of these cDNAs (670) contain such
  • the new isolated cDNA corresponds to a fragment of 76 nucleotides (excluding the sequences corresponding to the oligonucleotides introduced by the PCR).
  • the amino acid sequence alignments were carried out while not allowing either insertions or deletions. These alignments show that this fragment codes for a motif homologous to the Helix I motif of the bHLH proteins.
  • the isolation of the complete cDNA was then undertaken.
  • the isolation of the complete cDNA corresponding to CIG235 was extremely difficult, because the mRNA corresponding to CIG235 is very little expressed in the GCS II even seems that its level of expression is close to that of illegitimate transcription
  • the technique adopted consists on the one hand in isolating the 3 'end of the cDNA corresponding to CIG235 by anchored PCR and on the other hand in isolating the 5' end of this cDNA from spinal cord of rat embryos at the El stage 2, 5
  • GCS ss-cDNAs are prepared with the primer RA3 'NV (random reverse transcription primer carrying the anchor 3' NV) according to the Dumas method Milne Edwards et al (1995, A Pratical Approach Ed McPherson et al , pp89-1-18, IRL Piess Oxford UK)
  • Two specific nested primers are selected from the 76 nucleotide bHLH positive clone CIG5 5'-AACCTTAACTCCGCGCTGGATGCGC-3 '(SEQ ID NO: 4) and CIG5'-2 5'-CGCGGTGTCCTGCCCACC- 3 '(SEQ ID N ° 5)
  • CIG3 'and CIG5' cDNA overlap over a region of 120 nucleotides and form a total cDNA whose size is 1491 nucleotides
  • the proteins of the bHLH family form homo or heterodimeres and bind specifically to DNA (Murre et al, 1989, Cell, 558, 537-44)
  • C ANNTG consensus hexanucleotide sequence
  • box E box E
  • the purified recombinant Relax protein is capable of specifically binding to a sequence containing an E box
  • the DNA coding for the Relax protein is inserted between the NdeI and Kpnl sites in a pFLAG-CTC vector.
  • the Relax-Flag fusion protein is purified on a column. affinity then used in a binding reaction in the presence of an oligonucleotide containing an E box 2
  • the transcriptional activity of the Relax protein was tested by cotransfection experiments in the PC 12 cell line, derived from rat pheochromocytomas
  • luciferase reporter gene placed under the control, on the one hand, of a promoter containing an E box (5kb of the TH promoter) and, on the other hand, of the same promoter containing a mutated E box
  • the cDNA coding for the Relax protein (from position 389 to 1235) is inserted between the EcoRI and Xhol sites in the expression vector pcDNA3 downstream of the CMV promoter.
  • the TH promoter of the rat carrying an E box CAGGTG (SEQ ID N ° 6) at position 197 on a 5kbp fragment is inserted at the HindIII site made blunt-ended in the plasmid pKSLuc which becomes 5kbTH-Luc
  • the site is mutated into TCCGTG (SEQ ID No. 7) and the resulting plasmid designated 5kbTH (Delta E) -Luc
  • the plasmid pcDN A3 -Relax is cotransfected in different amounts varying from 0 to 5pmole, and brought back to 5pmole with empty pcDNA3 vector, in PC 12 cells (10 6 cells / plate) mixed with lpmole of plamide 5kbTH-Luc, 5kb -TH (deltaE) - Luc or pKSLuc
  • 0.2pmol of an expression vector of the gene coding for the CAT protein (chloramphenicol acetyltransferase) under the control of the RSV promoter are also cotransfected
  • This mRNA is only present in the embryo, between days 1 1 5 and 18 5 of development Its expression is restricted to the central nervous system In fact, no expression was detected, neither in the peripheral nervous system, nor outside the nervous system Inside the CNS, Relax mRNA is expressed only in the spinal cord, the posterior brain and the anterior brain
  • Relax mRNA is expressed in longitudinal compartments in the spinal cord and the posterior brain
  • NAME RHONE-POULENC RORER S.A.

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EP97952961A 1996-12-19 1997-12-19 Polypeptides de la famille "basic helix-loop-helix" bhlh, sequences d'acides nucleiques correspondantes Withdrawn EP0946724A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR9615651A FR2757524B1 (fr) 1996-12-19 1996-12-19 Polypeptides de la famille bhlh, sequences d'acides nucleiques correspondantes
FR9615651 1996-12-19
PCT/FR1997/002368 WO1998027206A2 (fr) 1996-12-19 1997-12-19 Polypeptides de la famille 'basic helix-loop-helix' bhlh, sequences d'acides nucleiques correspondantes

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US (1) US6998474B1 (cs)
EP (1) EP0946724A2 (cs)
JP (1) JP2001510464A (cs)
KR (1) KR20000057698A (cs)
AU (1) AU732438B2 (cs)
BR (1) BR9713968A (cs)
CA (1) CA2275454A1 (cs)
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HU (1) HU224305B1 (cs)
IL (1) IL130302A0 (cs)
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AUPP495598A0 (en) 1998-07-30 1998-08-20 Walter And Eliza Hall Institute Of Medical Research, The A novel regulatory molecule and genetic sequences encoding same
DE60034877T2 (de) * 1999-04-06 2008-01-17 The Regents Of The University Of California, Oakland MENSCHLICHE, FüR NEUROGENIN 3 KODIERENDE NUKLEOTIDSEQUENZEN
EP1354951B1 (en) * 2000-12-27 2008-06-04 Sumitomo Chemical Company, Limited Method of examining ability to control nerve cell plasticity
US20150315607A1 (en) * 2014-01-15 2015-11-05 Academia Sinica Mutated nucleotide molecule, and transformed plant cells and plants comprising the same

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US5695995A (en) 1994-05-06 1997-12-09 Fred Hutchinson Cancer Research Center Neurogenic differentiation (neurod) genes
EP0759938A4 (en) * 1994-05-06 1999-10-27 Hutchinson Fred Cancer Res PROTEINS AND NEUROGENIC DIFFERENTIATION GENES (NEURO D)
US6566496B1 (en) * 1996-09-27 2003-05-20 California Institute Of Technology Neurogenin

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IL130302A0 (en) 2000-06-01
BR9713968A (pt) 2000-04-11
WO1998027206A2 (fr) 1998-06-25
JP2001510464A (ja) 2001-07-31
AU732438B2 (en) 2001-04-26
HUP0001184A3 (en) 2002-01-28
WO1998027206A3 (fr) 1998-10-01
FR2757524A1 (fr) 1998-06-26
HU224305B1 (hu) 2005-07-28
FR2757524B1 (fr) 1999-01-29
NO993029L (no) 1999-08-16
ZA9711401B (en) 1998-06-25
CA2275454A1 (fr) 1998-06-25
HUP0001184A2 (hu) 2000-08-28
AU5667398A (en) 1998-07-15
US6998474B1 (en) 2006-02-14
SK81899A3 (en) 2000-05-16
CZ219499A3 (cs) 1999-10-13
KR20000057698A (ko) 2000-09-25
NO993029D0 (no) 1999-06-18

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