EP0769062A2 - Neue interne ribosom eintrittstelle, vektor der diesen enthalter und dessen therapeutische verwendung - Google Patents

Neue interne ribosom eintrittstelle, vektor der diesen enthalter und dessen therapeutische verwendung

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Publication number
EP0769062A2
EP0769062A2 EP95925007A EP95925007A EP0769062A2 EP 0769062 A2 EP0769062 A2 EP 0769062A2 EP 95925007 A EP95925007 A EP 95925007A EP 95925007 A EP95925007 A EP 95925007A EP 0769062 A2 EP0769062 A2 EP 0769062A2
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EP
European Patent Office
Prior art keywords
vector
sequence
gene
dna fragment
interest
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EP95925007A
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English (en)
French (fr)
Inventor
Clarisse Berlioz
Sandrine Jacquemoud
Christophe Torrent
Jean-Luc Darlix
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Institut National de la Sante et de la Recherche Medicale INSERM
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Institut National de la Sante et de la Recherche Medicale INSERM
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Publication of EP0769062A2 publication Critical patent/EP0769062A2/de
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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
    • C12N15/63—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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13051—Methods of production or purification of viral material
    • C12N2740/13052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/70—Nanostructure
    • Y10S977/788—Of specified organic or carbon-based composition
    • Y10S977/802—Virus-based particle
    • Y10S977/803—Containing biological material in its interior
    • Y10S977/804—Containing nucleic acid
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/902—Specified use of nanostructure
    • Y10S977/904—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
    • Y10S977/915—Therapeutic or pharmaceutical composition
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/902—Specified use of nanostructure
    • Y10S977/904—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
    • Y10S977/92—Detection of biochemical

Definitions

  • New internal ribosome entry site New internal ribosome entry site. carrier vector and therapeutic use
  • the present invention relates to a DNA fragment isolated from a retrotransposon and comprising an internal ribosome entry site (IRES). More particularly, it relates to expression vectors comprising this DNA fragment and in particular polycistronic vectors allowing the efficient and stable expression of several genes of interest under the dependence of the same promoter.
  • the present invention finds an interesting application in the field of gene therapy vectors.
  • the vectors are obtained by deletion of at least part of the viral genes which are replaced by the genes of therapeutic interest.
  • Such vectors can be propagated in a complementation line which provides in trans the deleted viral functions to generate a viral vector particle defective for replication but capable of infecting a host cell.
  • retroviral vectors are among the most used, but mention may also be made of vectors derived from adenoviruses, viruses associated with adenoviruses, poxviruses and herpes viruses. This type of vector, their organization and their mode of infection are widely described in the literature accessible to those skilled in the art.
  • ribosomes enter the messenger RNA through the cap located at the 5 'end of all eukaryotic mRNAs.
  • IRES a certain number of these structures, called IRES because of their function, have been identified in the 5 ′ non-coding region of non-capped viral mRNAs such as that in particular of picornaviruses such as the poliomyelitis virus (Pelletier et al. ., 1988, Mol. Cell. Biol., ⁇ 9, 1103-1112) and EMCV (Encephalomyocarditis virus (Jang et al., J. Virol., 1988, 62, 2636-2643).
  • the genome of eukaryotic cells includes a number of mobile cellular genetic elements, called transposons, which have the ability to move from one site in the genome to another chosen at random. At present, their biological function and significance is unknown. Some of them, the retrotransposons, appear to be related to retroviral proviruses by their organization and their mode of transposition (by an RNA intermediary, reverse transcription and. integration into the cell genome). Among the various murine retrotransposons identified to date, there are the elements VL30. The murine genome contains 150 to 200 copies. They are approximately 6 kb in length and have direct repeats at their ends reminiscent of retroviral LTRs. They are defective for replication and do not contain coding sequences (codons for stopping translation in the different reading phases).
  • the IRES site of a murine VL30 is particularly advantageous. Firstly, it allows an effective and stable translation reinitiation rate in the long term and, secondly and unexpectedly, it can also, within the framework of a retroviral vector, fulfill the functions of dimerization and d packaging, regardless of its position in the vector. And finally, because of its weak homology with retroviral sequences, its use considerably reduces the risk of production of viruses competent for replication, an advantageous property in the context of gene therapy vectors intended for human use.
  • the present invention relates to an isolated DNA fragment comprising an internal ribosome entry site (IRES) and / or an encapsidation sequence, characterized in that it is derived from a retrotransposon.
  • IRS internal ribosome entry site
  • encapsidation sequence characterized in that it is derived from a retrotransposon.
  • isolated DNA fragment is meant a DNA fragment isolated from its context, that is to say not associated with another retrotransposon sequence other than that defined below.
  • retrotransposon refers to a genetic element mobile cell which exhibits characteristics of the retroviral type, in particular by the existence of direct repetitions at its two ends.
  • IRES is meant a sequence capable of promoting the entry of ribosomes into an RNA molecule in a manner independent of the cap, at an internal site of this RNA.
  • An packaging sequence is a sequence involved in the packaging process of retroviruses or retroviral vectors by promoting the dimerization of two copies of the retroviral genome and by allowing the dimer to be packaged in viral particles.
  • derivative refers to a structure having a retrotransposon origin but which may have undergone some modifications, have been obtained by chemical synthesis or else various elements comprising parts of retrotransposon, such as the HaMSV virus.
  • a DNA fragment according to the invention is capable of exerting an IRES function and an packaging function when it is introduced into an appropriate retroviral vector.
  • a DNA fragment according to the invention is isolated from the 5 'end of a retrotransposon and in particular from the region which directly follows the direct repeat located at its 5' end (LTR-like 5 ') and, in particular, the RNA transfer binding site (PBS).
  • LTR-like 5 ' the region which directly follows the direct repeat located at its 5' end
  • PBS RNA transfer binding site
  • it comprises at least 100 nucleotides of said region, advantageously at least 200 nucleotides, preferably at least 300 nucleotides, preferably at least 400 nucleotides and, most preferably, at least 550 nucleotides , this by counting the nucleotides apart from the 5 'direct repetition. But, of course, it can extend beyond in the 3 'direction up to at most 0.88 to 1.5 kb.
  • a DNA fragment according to the invention is derived from a VL30 element of rodent, preferably, of murine origin and, very particularly, of rat or mouse.
  • a DNA fragment having a sequence substantially homologous to the sequence presented in the identifier of sequence SEQ ID NO: 1, starting at nucleotide 1 and ending at nucleotide 590 or, optionally, starting at nucleotide 176 and ending at nucleotide 590.
  • the second variant (DNA fragment isolated from a mouse VL30) use will preferably be made of a DNA fragment having a sequence substantially homologous to the sequence presented in the sequence identifier SEQ ID NO: 2, starting at nucleotide 1 and ending at nucleotide 788.
  • substantially homologous refers to a degree of homology greater than 70%, advantageously greater than 80%, preferably greater than 90% and, most preferably, greater than 95%.
  • a DNA fragment according to the invention may have a sequence slightly different from one of the sequences described in the sequence identifiers 1 and 2, provided however that the substitution, the deletion or the addition of one or more several nucleotides does not affect its IRES function and / or the packaging function.
  • a DNA fragment according to the invention is intended to be integrated (in any orientation) into a transfer and expression vector for one or more gene (s) of interest.
  • the choice of such a vector is wide and the techniques of cloning into the selected vector are within the reach of those skilled in the art.
  • poxvirus canary pox or vaccinia virus
  • adenovirus baculovirus
  • l virus herpes virus associated with an adenovirus or retrovirus.
  • adenoviral vector when it is an adenoviral vector, it can be derived from a human adenovirus, preferably of type 2 or 5, animal, preferably canine or avian, or alternatively a hybrid between various species.
  • adenoviruses The general technology for adenoviruses is disclosed in Graham and Prevec (1991, Methods in Molecular Biology, Vol 7, Gene tran fer and Expression Protocols; Ed E.J. Murray, the human Press Inc, 109-118).
  • a DNA fragment according to the invention is preferably positioned upstream of a gene of interest to improve the translation of the expression product for which it codes. It can be included in an expression cassette of the monocistronic type (for the expression of a gene of interest placed under the control of a promoter) or polycistronic (for the expression of at least two genes of interest placed under the control of the same promoter).
  • the latter can contain several elements in tandem "IRES site-gene of interest" of which at least one of the IRES sites consists of a DNA fragment according to the invention. It is particularly preferred to use it in a dicistronic cassette into which it can be inserted either upstream of the first gene of interest or upstream of the second, the latter variant being the preferred.
  • a vector according to the invention comprises several expression cassettes, these can be inserted in any orientation relative to each other; in the same orientation (promoter acting in the same direction) or in reverse orientation (promoter acting in the opposite direction).
  • a vector according to the invention comprises several DNA fragments according to the invention, it is preferable that they come from retrotransposons of different origins. According to this particular embodiment, it is preferred that one of the fragments is derived from a rat VL30 and in particular has a sequence substantially homologous to SEQ ID NO: 1 and that the other is derived from a mouse VL30 and has in particular a sequence substantially homologous to SEQ ID NO: 2.
  • a vector according to the invention derives from a retrovirus.
  • retroviruses such as avian erythroblastosis virus (AEV), avian leukemia virus (AVL), avian sarcoma virus (ASV), necrosis virus of spleen (SNV) and Rous sarcoma virus (RSV), bovine retroviruses, feline retroviruses, murine retroviruses such as murine leukemia virus (MuLV), Friend virus (F-MLV) and murine sarcoma virus (MSV) and primate retroviruses.
  • retroviruses such as murine leukemia virus (MuLV), Friend virus (F-MLV) and murine sarcoma virus (MSV) and primate retroviruses.
  • other retroviruses can be used.
  • MoMuLV Moloney murine leukemia virus
  • the retroviral vectors which can be envisaged for the purposes of the present invention are shown diagrammatically in FIG. 1 (a, b and c). Of course, these examples are not limiting.
  • the 5 'and 3' retroviral LTRs are represented by a hatched box, the murine VL30 sequences (indifferently mouse and / or rat) by a bold line, the internal promoter by a dotted box, the genes of interest by a white box and finally the retroviral packaging region (Psi) by a thin line.
  • the retroviral LTR 5 ′ can be used as a promoter for the expression of one or more gene (s) of interest, but an internal promoter can also be used.
  • a retoviral vector according to the invention can, optionally, comprise a retroviral packaging region such as the Psi sequence of MoMuLV.
  • a DNA fragment according to the invention can also fulfill this function and this regardless of its position in the retroviral vector of the invention (upstream of a gene of interest and / or downstream of the 5 'LTR).
  • a gene of interest for use in the invention can be obtained from a eukaryotic or prokaryotic organism or from a virus by any conventional technique. It is preferably capable of producing an expression product having a therapeutic effect and it may be a product homologous to the host cell or, alternatively, heterologous.
  • the term expression product denotes a protein or a fragment thereof.
  • a gene of interest can encode a product (i) intracellular (ii) membrane present on the surface of the host cell or (iii) secreted outside the host cell. It can therefore include appropriate additional elements such as, for example, a sequence coding for a secretion signal. These signals are known to those skilled in the art.
  • a gene of interest can code for a protein corresponding to all or part of a native protein as found in nature. It can also be a chimeric protein, for example from the fusion of polypeptides of various origins or a mutant having improved and / or modified biological properties. Such a mutant can be obtained by conventional biological techniques by substitution, deletion and / or addition of one or more amino acid residues.
  • a vector according to the invention is particularly intended for the prevention or treatment of cystic fibrosis, hemophilia A or B, Duchenne or Becker's myopathy, cancer, AIDS and other bacteria or infectious diseases due to a pathogenic organism: virus, bacteria, parasite or prion.
  • the genes of interest which can be used in the present invention are those which code for the following proteins:
  • cytokine and in particular an interleukin, an interferon, a tissue necrosis factor and a growth factor and in particular hematopoietic
  • factor VIII a factor or cofactor involved in coagulation and in particular factor VIII, factor IX, von Willebrand factor, antithrombin III, protein C, thrombin and hirudin,
  • an enzyme inhibitor such as ⁇ l-antitrypsin and viral protease inhibitors
  • a suicide gene expression product such as HSV (herpes virus) type 1 thymidine kinase
  • a protein whose absence, modification or deregulation of expression is responsible for a genetic disease, such as the protein CFTR, dystrophin or minidystrophin, insulin, ADA (adenosine diaminose), glucocerebrosida e and phenylhydroxylase,
  • a protein capable of inhibiting the initiation or progression of cancers such as a product for expression of tumor suppressor genes (p53 genes,
  • Rb a toxin, an antibody, an immunotoxin,
  • a protein capable of inhibiting a viral infection or its development for example an antigenic epitope of the virus in question, an antibody or an altered variant of a viral protein capable of entering into competition with the native viral protein.
  • a gene of interest in use in the present invention can also code for a selection marker making it possible to select or identify the host cells transfected with a vector according to the invention.
  • Mention may be made of the neo gene (neomycin) conferring resistance to the antibiotic G418, the dhfr gene (dihydrofolate reductase), the CAT gene (Chloramphenicol Acethyl Transferase) or also the gpt gene (xanthine phosphoribosyl).
  • a functional promoter in the host cell considered and, preferably, a human cell.
  • the choice of promoter is very wide and within the reach of those skilled in the art. It can be a promoter naturally governing the expression of a gene of interest in use in the present invention or any other promoter (of eukaryotic or viral origin). Furthermore, it can be ubiquitous or regulable in nature, in particular in response to certain tissue-specific or event-specific cellular signals.
  • tissue-specific promoter when it is desired to target the expression of the gene (s) of interest in a particular cell or cell type, for example lymphocytes in for AIDS, lung cells for cystic fibrosis or muscle cells for myopathy.
  • lymphocytes in for AIDS, lung cells for cystic fibrosis or muscle cells for myopathy.
  • SV40 promoters By way of nonlimiting examples, mention may in particular be made of the SV40 promoters.
  • the late promoter MPL Major Late Promoter
  • HMG Hydroxy ethyl-Glutaryl Coenzyme A
  • TK Thymidine kinase
  • retroviral LTRs and, in particular, that of MoMuLV or MSV when using a retoviral vector
  • the late promoter MPL Major Late Promoter
  • the 7.5K and H5R promoters in particular intended for vectors derived from poxvirus and especially from the vaccinia virus
  • the PGK promoter Phosphoglycero kinase
  • the liver-specific promoters of the genes coding for ⁇ l-antitrypsin, factor IX, albumin and transferrin the promoters of the immunoglobulin genes which allow expression in lymphocytes
  • the promoters of the genes coding for the surfactant or the CFTR protein which have a certain specificity for lung tissue.
  • an expression cassette present in a vector according to the invention may comprise other sequences necessary for the expression of the gene (s) of interest, both at the level of transcription and of translation; for example enhancer-type transcription activator sequences, introns, transcription termination signals and, as indicated above, a secretion signal.
  • the invention also covers the viruses and viral particles obtained by transfection of the viral vector according to the invention in an adequate complementation line.
  • the complementation lines that can be used to generate infectious viral particles as well as the method to be used.
  • Being an adenoviral vector use may be made of line 293 (Graham et al., 1977, J. Gen. Viral., 36, 59-72).
  • ecotropic cell lines such as the CRE line (Danos and Mulligan, 1988, Proc. Natl. Acad. Sci. USA, 85, 6460-6464 ) or GP + E-86 (Markowitz et al., 1988, J. Virol., 62, 1120-1124).
  • an amphotropic complementation line such as the PG13 line (Miller et al., 1991, J. Virol., 65, 2220-2224) or Psi Env-am (Markowitz et al., 1988, TAAP Vol. CI, 212-218).
  • PG13 line Miller et al., 1991, J. Virol., 65, 2220-2224
  • Psi Env-am Markowitz et al., 1988, TAAP Vol. CI, 212-218.
  • the invention also extends to eukaryotic cells comprising a DNA fragment according to the invention. They can be obtained by infection with infectious viral particles according to the invention or by introduction of a plasmid or viral vector either in vitro (in a cell taken from a patient or an animal) or directly in vivo.
  • the methods for introducing a vector into a cell are conventional. It is possible to use the precipitation technique with calcium phosphate, that with DEAE dextran, direct injection of the vector or of a portion thereof into a cell or even encapsulation in molecules of the liposome type.
  • the vectors according to the invention can be present in the host cell either in the form integrated into the cell genome or in episomal form both in the nucleus and in the cytoplasm of the cell.
  • the cell according to the invention is advantageously a mammalian cell and, preferably, a human cell.
  • the present invention also relates to the therapeutic use of a vector or a cell according to the invention, for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of a genetic disease or of a disease. acquired like cancer or an infectious disease.
  • a vector according to the invention can be used for other purposes such as the recombinant production in eukaryotic cells of expression products intended to be included after purification in said pharmaceutical composition.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising, as therapeutic or prophylactic agent, a vector or a cell according to the invention, in combination with a vehicle which is acceptable from a pharmaceutical point of view.
  • a pharmaceutical composition according to the invention can be manufactured in a conventional manner.
  • a therapeutically effective amount of such an agent is combined with an acceptable carrier, diluent or adjuvant.
  • She may be administered according to any route of administration and this in a single or repeated dose after a certain interval of interval.
  • the quantity to be administered will be chosen according to different criteria, in particular the use as a treatment or vaccine, the route of administration, the patient, the type of disease to be treated and its state of progress, the duration of treatment .... etc.
  • a pharmaceutical composition according to the invention comprises between 10 4 and 10 w pfu (unit forming plaques), advantageously between ÎO "1 and 10 ⁇ pfu and, preferably, between 10 6 and 10 n pfu of viral particles .
  • the invention relates to a method of treatment of genetic diseases, cancers and infectious diseases according to which a therapeutically effective amount of a vector or a cell according to the invention is administered to a patient in need of such treatment .
  • a therapeutically effective amount of a vector or a cell according to the invention is administered to a patient in need of such treatment .
  • they can be administered directly in vivo, for example by intravenous, intramuscular injection or by aerosolization in the lungs.
  • an ex vivo gene therapy protocol which consists in taking the cells from a patient, bone marrow stem cells or peripheral blood lymphocytes, transfecting them with a vector according to the invention and cultivate in vitro before re-implanting them in the patient.
  • the invention also relates to an isolated DNA fragment comprising an encapsidation sequence (Psi) derived from a Moloney murine leukemia virus (MoMuLV) as an internal ribosome entry site (IRES) and its implementation.
  • Psi encapsidation sequence
  • MoMuLV Moloney murine leukemia virus
  • IVS internal ribosome entry site
  • It will be a polycistronic vector preferably comprising two genes of interest under the control of the same promoter and having a Psi sequence of MoMuLV between the two genes.
  • the vector may include additional genes of interest, either in the form of a polycistronic cassette (several elements in tandem "IRES-gene of interest" of which at least one IRES consists of a Psi sequence derived from MoMuLV), or in the form of an independent cassette provided with its own promoter.
  • IRES-gene of interest consists of a Psi sequence derived from MoMuLV
  • the invention also covers viral particles and eukaryotic cells comprising such a vector, their therapeutic use as well as a pharmaceutical composition. It is indicated that an IRES site according to the invention is preferably carried by the sequences extending from nucleotides 210 to 1035 of the MoMuLV genome (+1, representing the site of initiation of transcription).
  • FIG. 1 represents some retroviral vectors usable within the framework of the invention a: of monocistronic type, b: of dicistronic type and c: of mixed type comprising cassettes of mono and dicistronic expression.
  • the 5 'and 3' LTRs are represented by a hatched box, the murine VL30 sequences (rat or mouse) by a bold line, the retroviral Psi sequence by a thin line, the internal promoter by a dotted box and finally the genes d interest by a white box.
  • Figure 2 shows schematically the vectors pVL-CT2, pCBTl, pCBT2, pMLV-LacZ +, pSJEl and pSJE2.
  • Figure 3 shows diagrammatically the vectors pMLV-CBT, pVL-CBT2-E + and pVL-CBT5 (plap represents the alkaline phosphatase gene hereinafter designated PA).
  • the constructions described below are produced according to the general techniques of genetic engineering and molecular cloning detailed in Maniatis et al. (1989, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or as recommended by the manufacturer when using a commercial kit.
  • the filling of the protruding 5 ′ ends can be carried out using the Klenow fragment of the DNA polymerase of E. coli and destruction of the protruding 3 'ends in the presence of phage T4 DNA polymerase.
  • PCR techniques are known to those skilled in the art and extensively described in PCR Protocols, a guide to methods and applications (Ed: Innis, Gelfand, Sninsky and White, Académie Press, Inc.).
  • the position of the VL30 sequences of rats and mice is indicated by reference to the RNA molecule, the position + 1 corresponding to the first nucleotide of the RNA molecule, that is to say at the site of initiation of the transcription in the DNA molecule (first nucleotide of the R sequence).
  • the RNA positions 362-575 and 362-1149 correspond respectively to the DNA positions 631-844 and 631-1418 of the sequence described in Adams et al. (1988, Mol. Cell. Biol., (9, 2989-2998)
  • the vector pCB28 is obtained in the following way
  • the EcoRI-Nhel fragment from pLNPOZ (Adam et al., 1991, J. Virol. 65, 4985- 4990) is cloned into Bluescript II KS + digested with EcoRI and Spel to give pCB25.
  • the HindIII-Xbal fragment of pCB25 containing the sequences coding for neomycin (neo), the IRES site of the poliovirus and the ⁇ -galactosidase gene (LacZ) is cloned in the vector pRc / CMV (Invitrogen) deleted from positions 1284 to 3253 and digested with HindIII and Xbal.
  • a fragment carrying the viral sequences of the F-MLV virus from positions 1 to 651 is generated by PCR using primers 6 and 7 (SEQ ID NO: 3 and 4).
  • a DNA preparation obtained from this virus by conventional techniques can be used as a template.
  • the PCR fragment is digested with Xhol and BamHI and then inserted between the neo and LacZ genes of pCB27 partially digested with Xhol and BamHI.
  • VL30 sequences were generated by PCR from the pVL-CG20 matrix
  • PVL-D 1-794, pVL-D205-794 and pVL-D380-794 are obtained according to the inserted VL30 sequence.
  • the neo-VL30-LacZ dicistronic cassette is under the control of the T7 RNA polymerase promoter for expression in vitro and of the early cytomegalovirus (CMV) promoter for expression in eukaryotic cells. It is indicated that the AUC initiator of the lacZ gene is placed in a context favorable to the initiation of translation according to the Kozak rule (A / G CCAUGG; Kozak, 1986, Cell - / - /, 283-292).
  • Dicistronic RNAs are generated by in vitro synthesis from 5 ⁇ g of recombinant plasmids pVL linearized by Xbal. The transcription reaction takes place for 3 h at 37 ° C. in 0.1 ml of 40 mM Tris-HCl (pH 7.5), 6 mM MgCl 2 , 2 mM spermidine, 10 mM dithiothreitol, 10 mM NaCl, 0.5 mM of each of the ribonucleoside triphosphate in the presence of 40 U of T7 RNA polymerase and 80 U of RNAsin (RNase inhibitor).
  • RNAs After treatment with DNase (RQ1), the RNAs are extracted with phenol-chloroform and precipitated with ethanol. They are then redissolved in sterile double-distilled water and subjected to an agarose gel electrophoresis to verify their integrity (single band migrating to the expected size of 4.6 to 5 kb depending on the plasmids).
  • RNAs (10 ⁇ g of RNA / ml) are then translated in vitro into a system of rabbit reticulocyte lysates (RRL system, Proméga) at 50% of its original concentration in the presence of 1 mCi [ 33 S] methionine / ml (Amersham) (reaction time lh at 31 ° C).
  • the reaction mixture is supplemented with potassium acetate and potassium chloride to a final concentration of 60 mM and 40 mM respectively.
  • the tubes are placed at 100 ° C. in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% / 3-mercaptoethanol and 0.02% bromophenol blue and the labeled proteins. at 3 'S are analyzed by gel electrophoresis polyacrylamide 10% - 0.2% SDS. The quantification of the translation products of the neo and LacZ genes is carried out by scanning. The neo protein serves as standardization of the level of translation and the quantity of ⁇ galactosidase synthesized (PM 110 kDa) is evaluated.
  • RNAs produced from each of the vectors allow the synthesis of the expression products of the two genes (neo and ⁇ galactosidase) which indicates that they are dicistronic and suggests the existence of an IRES in the VL30 sequences used. , ensuring the initiation of translation of the second cistron, hence the production of / 3-galactosidase.
  • Retroviral vectors comprising a sequence of a rat VL30.
  • the plasmid pCB71 is obtained in the following way:
  • neomycin gene (neo; positions 4 to 844) is amplified by
  • PCR from the vector pLNPOZ (Adam et al., 1991, supra) and using primers 10 and 11 (SEQ ID NO: 5 and 6) provided at their ends with restriction Sali, Spel and BamHI.
  • the PCR fragment generated is digested with SalI and BamHI and introduced with the EcoRV-SalI fragment from pCB70 carrying the alkaline phosphatase gene into the vector pLNPOZ digested by Bail and BamHI.
  • a 0.59 kb DNA fragment comprising the sequences of the rat VL30 (positions 205 to 794) is isolated by PCR and provided at its 5 'and 3' ends with an Nhel site.
  • the plasmid pVL-CG20 (Torrent et al., 1994, supra) is used as a matrix and the oligonucleotides 12 and 13 reported in SEQ ID NO: 7 and 8.
  • the PCR fragment is digested with Nhel before be inserted into the vector pCB71 digested with Spel, to give pCBT2.
  • an oligonucleotide 13 has included a translation initiator ATG codon placed in a favorable Kozak context (A / GCCATGG) which will then make it possible to introduce a coding sequence devoid of initiator codon.
  • the final construction pCBT2 ( Figure 2) contains the 5 'LTR of MoMuLV, the gene for human alkaline phosphatase, a 0.59 kb fragment isolated from rat VL30 (positions 205 to 794) followed by the neomycin gene and LTR 3 'of MoMuLV.
  • the construction of the plasmid pCBTl is carried out as follows
  • a DNA fragment comprising the rat VL30 sequences (positions 205 to 379) is generated by PCR from the vector pVL-CG20 and oligonucleotides 8 and 9 (SEQ ID NO: 9 and 10). After digestion with Nhel, this is inserted into the vector pCB28 also cleaved by Nhel, to give pCB57. This Nhel fragment is isolated from pCB57 and introduced into the vector pCB71 digested with Spel. As before, this fragment includes a translation initiator codon placed in a Kozak context.
  • PCBT1 (FIG. 2) is generated which is identical to pCBT2 except for the length of the VL30 fragment of rat (0.175 kb instead of 0.59 kb).
  • a DNA fragment comprising the HaMSV sequences is isolated by PCR
  • the plasmid pVL-CG20 (Torrent et al, 1994, supra) is used as a matrix and the oligonucleotides 16 and 13 reported in SEQ ID NO: 11 and 8.
  • the PCR fragment is digested with Smal and Ncol before being inserted into the vector pLNPOZ (Adam et al., 1991, supra) digested with Ncol and partially with Smal, to give pVL-CT2 ( Figure 2).
  • the GP + E-86 ecotropic complementation line (Markowitz et al., 1988, J. Virol., 62, 1120-1124) and the target cells NIH3T3 (mouse fibroblastic cells) available at ATCC, are cultured at 37 ° C in the presence of 5% C0 2 in DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 5% newborn calf serum.
  • DMEM medium Dulbecco's Modified Eagle's Medium
  • the day before transfection and infection the GP + E-86 cells and the NIH3T3 target cells are cultured at the rate of 5 ⁇ 10 5 cells per 10 cm dish and 1.5 ⁇ 10 D cells per well, respectively.
  • Viral infections are carried out according to the conventional protocol described in the literature.
  • the titration method is that of the limit dilution point.
  • the vectors pCBTl, ⁇ CBT2 and pVL-CT2 as well as the reference vector pMLV-LacZ are transfected in parallel in the GP + E-86 cells according to the method of Chen and Okyama (1987, Mol (Cell. Biol., 7, 2745-2753).
  • D + 1 the cells are washed according to the methods of the art and the culture supernatant is harvested on D + 2.
  • Different dilutions are used to infect the target cells NIH3T3.
  • the cells are cultured in a selective medium (800 g / ml of G418) 24 hours after transfection or infection.
  • LacZ gene after X-Gal staining is measured on a culture aliquot of cells infected and transfected with pMLV-LacZ and pVL-CT2. This coloring technique is described in the basic works accessible to those skilled in the art. It is also possible to use a commercial kit (Promega).
  • alkaline phosphatase is regularly determined over time in the transfected GP + E-86 cells and in the NIH3T3 cells infected with pCBT1 and pCBT2.
  • the cells are rinsed in PBS 1 buffer and fixed 5 min at room temperature with a solution containing 2% formaldehyde and 0.2% glutaraldehyde in PBS x 1.
  • the cells were then rinsed twice in PBS x 1 buffer and then incubated for 30 min at 65 ° C in PBS x 1. They are washed in AP buffer (0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 50 mM MgCl,).
  • This buffer is then replaced by the staining solution (containing 0.1 g / ml of 5-bromo-4-chloro-3-indolyl phosphate 1, 1 mg / ml of Nitoblue terazolium salt 1, Levamisol 1 mM in of the AP buffer).
  • the cells are incubated for 6 hours at room temperature, protected from light.
  • the colored cells correspond to the positive phosphatase cells.
  • the rate of packaging of each of the vectors is estimated by calculating the ratio of number of infected colored cells (PA or LacZ positive) to the number of transfected colored cells (PA or LacZ positive) x 100. The results are shown in Table 1 next.
  • the data indicate that the rat VL30 DNA fragment (positions 205 to 794) includes an packaging signal at least as effective as the Psi sequence of MoMuLV. Su ⁇ renantly, the localization of the rat VL30 sequence between two genes has little effect on the packaging efficiency of the retroviral genome (vector pCBT2 compared to pVL-CT2).
  • the vast majority of cells expressing the neo gene (resistance to G418) simultaneously express the phosphatase gene.
  • the expression of the two genes is stable over time, since more than 90% of the cells resistant to G418 are also phosphatase positive after 40 days of culture in selective medium.
  • RNA precipitates are recovered using a glass rod while the RNAs are precipitated for 1 hour at -20 ° C and then centrifuged for 30 minutes at 10,000 ⁇ m at 4 ° C.
  • the purified cellular RNA is taken up in 150 ⁇ ⁇ of sterile water.
  • RNAs are incubated at 65 "C for 5 min in a MOPS buffer (20 mM mo ⁇ holinopropanesulfonic acid, 5 mM sodium acetate, ImM EDTA, pH7) containing 6% formaldehyde, 50% formamide, 30% blue glycerol.
  • MOPS buffer 20 mM mo ⁇ holinopropanesulfonic acid, 5 mM sodium acetate, ImM EDTA, pH7
  • the denatured RNAs are deposited on 0.7% agarose gel under denaturing condition (MOPS IX, 6% formaldehyde), the RNAs are then transferred to a nitrocellulose membrane in a 25mM sodium phosphate buffer for 1 hour 30 min at 800mA.
  • UV (252 nm; 0.32 J / cm 2 ) to fix the RNAs.
  • the membrane is incubated for 4 hours at 42 ° C in a prehybridization solution (50% formamide, 1M NaCl, 50mM NaP04 pH7, 10% Dextran sulfate, 1% SDS, 250 ⁇ g / ml denatured salmon sperm DNA 5 min at 100 ° C.
  • a prehybridization solution 50% formamide, 1M NaCl, 50mM NaP04 pH7, 10% Dextran sulfate, 1% SDS, 250 ⁇ g / ml denatured salmon sperm DNA 5 min at 100 ° C.
  • the membrane is then hybridized for 14 hours at 42 ° C. in 50% formamide, 0.8M NaCl, 50mM NaP04 pH7, 10 % Dextran sulfate and 1% SDS
  • the neomycin probe used at 0.5.10 ° cpm / ⁇ g is denatured for 5 min at 100 ° C and added to the hybridization solution.
  • the membrane is washed in successive baths: 2 x SSC, 1% SDS (2 times 10 min, room temperature), 2 x SSC, 0.1% SDS (30 min, 65 ° C) and 1 x SSC , 0.1% SDS (30 min, 65 ° C).
  • the membrane is then dried and exposed to -80 ° C for 72 hours.
  • the neomycin probe is complementary to the neomycin gene between positions 213 and 596 and corresponds to the Pstl-Ncol fragment from pLNPOZ (Adam et al., 1991).
  • the probe was labeled by primer extension with the Nonaprimer kit kit I (Appligene).
  • RNAs were extracted from GP + E-86 cells 72 hours after transfection of the plasmid pCBT2, or else from NIH3T3 cells infected with pCBT2 virions after 30 days of selection. Hybridization of these RNAs with a probe complementary to the neo gene reveals the presence of a single dicistronic RNA of identical size in the transfected and infected cells. Consequently, the simultaneous expression of the phosphatase and neomycin genes in 90-95% of the infected cells after 30 days of selection is due to the presence of a single dicistronic RNA.
  • This example describes the construction of dicistronic vectors containing an encapsidation signal in normal position (downstream of the 5 'LTR) and the second between two cistrons.
  • Two retroviral vectors (1) pVL-CBT2-E + are generated in which the MoMuLV signal is placed downstream of the 5 'LTR and the rat VL30 sequence (205-794) between the genes coding for alkaline and neo phosphatase and ( 2) pVL-CBT5 in which their respective positions are reversed.
  • pVL-CBT2-E + results from the cloning of the EcoRI fragment isolated from pLNPOZ (Adam et al., 1991, supra) carrying the 5 ′ LTR sequences and from packaging of MoMuLV in pCBT2 previously digested with this same enzyme (FIG. 3).
  • pVL-CBT5 is obtained in the following way: the sequences corresponding to the 5 'LTR of MoMuLV followed by the VL30 sequences (positions 205 to 794) are amplified by PCR from the pVL-CT2 matrix. It is within the capacity of those skilled in the art to determine the primers which are suitable and to provide them with EcoRI sites at their 5 ′ ends. The amplified fragment is digested with EcoRI and cloned into the vector pMLV-CTB (see below) also treated with EcoRI to give pVL-CBT5 ( Figure 3).
  • Dicistronic retroviral vector comprising the packaging signal of MoMuLV in the central position.
  • the equivalent of pCBT2 is constructed with the difference that the packaging signal of MoMuLV (positions 210 to 1035) replaces that of rat VL30. It is isolated from the vector pLNPOZ by PCR using suitable primers having at their ends an Nhel site. After Nhel digestion, the amplified fragment is introduced between the PA and neo genes of pMLV-CB71 (Berlioz and Darlix, 1995, J. Virol. 69, 2214-2222) digested with Spel. PMLV-CBT is obtained ( Figure 3).
  • the viral titers are determined in a transient or stable manner according to the technology previously applied by evaluation of the number of cells expressing alkaline phosphatase. The results are shown in Table 2 below and compared with the dicistronic vector pCBT2.
  • the GP + E-86 cells are transfected with each of the retroviral vectors and the expression levels of the PA gene are evaluated in the presence and in the absence of neomycin.
  • Neomycin resistant and PA positive cells are counted after 15 days of selection.
  • the values obtained are slightly greater for the dicistronic vectors carrying the two packaging signals than for their counterparts carrying only one, which suggests that the presence of two packaging sequences, one of retroviral origin. and the other of VL30 does not influence the gene expression of cistrons. Furthermore, in all cases, the expression of the neo gene is effective.
  • Retroviral vector comprising an encapsidation sequence isolated from a mouse VL30.
  • a DNA fragment containing the mouse VL30 sequence (positions 362 to 1149) is amplified by PCR from the vector pKT403 (Adams et al, 1988, supra) and using the oligonucleotide primers 3 and 4 (SEQ ID NO : 12 and 13).
  • the fragment generated is digested with Bail and Ncol before being inserted between the same sites of the vector pLNPOZ (Adam et al., 1991, supra).
  • the vector pSJE2 is obtained.
  • the vectors pSJEl and pSJE2 comprise the 5 'LTR of MoMuLV, the fragment
  • Mouse VL30 indicated, the LacZ gene and the 3 'LTR of MoMuLV.
  • the vector pSJE3 is constructed which is identical to the previous two but in which the VL30 sequence is replaced by a polylinker. It is obtained by introduction of oligonucleotide 5 (SEQ ID NO: 16) between the Bail and Ncol sites of pLNPOZ.
  • the technology used is comparable to that described in Example 1. Briefly, the vectors pSJEl, 2 and 3 are transfected into murine complementation lines, GP + E-86 or CRIP (Danos and Mulligan, 1988, Proc. Natl. Acad. Sci. USA, 85, 6460-6464). The culture supernatant is used to infect NIH3T3 murine target cells. The expression of the LacZ gene is measured in the infected and transfected cells after X-Gal staining. The viral titer corresponds to the ratio of the number of blue cells (LacZ +) infected to the transfection rate multiplied by the volume of viral supernatant used.
  • Table 3 gives an estimate of the viral titers obtained for each of the pSJE vectors as a function of the starting complementation line.
  • the vector pMLVLacZ + (Torrent et al., 1994, supra) comprising the conventional packaging region of MoMuLV is used for reference.
  • transient long term transient pSJEl 0.1 x 10 3 0.3 x 10 3 0.7 x 10 4
  • pSJE2 1 x 10 5 1, 3 x 10 5 6.5 x 10
  • pSJE3 ⁇ 0.001 x 10 3 ⁇ 0.001 x 10 3 ⁇ 0.01 x 10 4
  • pMLVlacZ 0.85 x 10 3 2 x 10 3 7 x 10 4
  • mice VL30 sequence between nucleotides 362 and 1149 comprises an packaging sequence at least as effective as that of MoMuLV.
  • the vector pSJE3 devoid of any packaging region, is incapable of generating viral particles.
  • the rat VL30 sequence is replaced in the vector pCBT2 by one of the mouse VL30 sequences (either the 0.78 kb fragment going from positions 362 to 1149 or the 0.21 kb fragment going from positions 362 to 575), an effective packaging rate and a correct expression of the alkaline phosphatase gene are measured.
  • TYPE OF MOLECULE DNA (genomics)
  • HYPOTHETIC NO
  • ANTI-SENSE NO
  • TTGTTTGTCT TCTTTGTGAC CTGACTGTGG TTTTCTGGAC GTGTTGTGTC TGTTAGTGTC 360
  • TYPE OF MOLECULE DNA (genomics)
  • HYPOTHETIC NO
  • ANTI-SENSE NO
  • ORGANISM Murine leukemia virus
  • ORGANISM Murine leukemia virus
  • TYPE OF MOLECULE DNA (genomics)
  • HYPOTHETIC NO
  • ANTI-SENSE NO
  • TYPE OF MOLECULE DNA (genomics)
  • HYPOTHETIC NO
  • ANTI-SENSE NO
  • ORGANISM element VL30 from rat
  • C INDIVIDUAL ISOLATED: oligo 8
  • TYPE OF MOLECULE DNA (genomics)
  • HYPOTHETIC NO
  • ANTI-SENSE NO

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