EP0918875A1 - Novel internal ribosome entry site and vector containing same - Google Patents

Abstract

The invention concerns the use of a nucleotide sequence derived from all or part of the genomic RNA 5' end of a type C retrovirus except for Friend murine leukaemia virus (FMLV) and Moloney murine leukaemia virus (MoMLV) as internal ribosome entry site or as element enabling or improving retrovirus vector packaging. The invention also concerns a vector comprising said nucleotide sequence, a viral particle generated from this vector, a cell comprising this vector or infected by the viral particle, their therapeutic use and a pharmaceutical composition containing them. The invention further concerns the use of a vector, a viral particle or a pharmaceutical composition for transfecting or infecting pluripotent stem cells.

Description

 New internal ribosome entry site and vector containing it

The present invention relates to the use of a nucleotide sequence derived from the 5 ′ end of genomic RNA or the proviral DNA of a reticuloendotheliosis virus as internal ribosome entry site (IRES) and / or to improve retroviral packaging. More particularly, it relates to expression vectors comprising this sequence 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 feasibility of gene therapy applied to humans is no longer to be demonstrated and this concerns many therapeutic applications such as genetic diseases, infectious diseases and cancers. Many documents of the prior art describe the means of implementing gene therapy, in particular by means of viral vectors. In general, 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 particle defective for replication but capable of infecting a host cell. To date, 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 vectors, their organization and their mode of infection are widely described in the literature accessible to those skilled in the art.

As an indication, the retroviral genome consists of a linear, single-stranded RNA with positive polarity. In addition to the regulation sequences R and TJ5 and U3 and R present at the 5 'and 3' ends respectively, it carries three genes: gag coding for the proteins of the capsid, pol coding for the reverse transcriptase and the integrase and env coding for the proteins of the envelope. The packaging signals, located downstream of the U5 sequences up to the start of the coding region of the gag gene, participate in the dimerization and packaging of the viral RNA in the viral particles. The 5 'end of the genome includes a cap (cap) and the 3' end is polyadenylated. During the infectious cycle, the viral RNA is converted into a linear, double-stranded pro viral DNA provided at each end with reverse repeated sequences LTRs (for Long Terminal Repeat in English) necessary for the initiation of transcription. This, carried out by cellular machinery, allows the production of genomic and subgenomic RNAs from which viral proteins are synthesized. Retroviruses can be classified into 4 subfamilies A to D, based on their morphology. Type C includes the majority of retroviruses including the MLV (Murine Leukemia Virus) and MSV (Murine Sarcoma Virus) viruses used in most gene therapy vectors and the REV (Reticuloendotheliosis Virus) viruses from which the nucleotide sequence of the present invention.

It may be advantageous to have more efficient gene therapy vectors capable in particular of efficiently producing several proteins of interest. However, the presence of several promoters within the same vector very often results in a reduction or even a loss of expression over time. This is due to a well-known phenomenon of interference between the promoter sequences. In this context, the publication of international application WO93 / 03143 proposes a solution to this problem which consists in implementing an internal ribosome entry site (IRES). It describes a dicistonic retroviral vector for the expression of two genes of interest placed under the control of the same promoter. The presence of an IRES site of picornavirus between them allows the production of the expression product derived from the second gene of interest by internal initiation of the translation of the mRNA dicistronic.

Normally, ribosomes enter the messenger RNA (mRNA) through the cap located at the 5 'end of all eukaryotic mRNAs. The 40S ribosomal subunits move along the RNA until they meet an appropriate AUG codon to start protein synthesis. Generally, initiation takes place at the first AUG codon. But, if this is in an unfavorable context, the 40S subunits continue until a later AUG codon situated in a better translational context (Kozak, 1984, Nucleic Acid Res. 12, 3873-3893; Kozak, 1991, J. Biol. Chem. 266, 19867-19870; Pain, 1996, Eur. J. Biochem. 236, 747-771).

However, there are exceptions to this universal rule. The absence of a cap in certain viral mRNAs suggested the existence of alternative structures allowing entry of ribosomes to an internal site of these RNAs. To date, 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. 8, 1103-1112) and EMCV (Encephalomyocarditis virus (Jang et al., 1988, J. Virol. 62, 2636-2643). Cellular mRNAs having IRES elements have Also described have been those coding for the protein BIP (for Immunoglobulin heavy chain binding protein; Macejak and Sarnow, 1991, Nature 353, 90-94), certain growth factors (Teerink et al., 1995, Biochem. Biophy Acts 1264, 403-408; Vagner et al., 1995, Mol. Cell. Biol. 15, 35-44), the translation initiation factor eIF4G (Gan and Rhoads, 1996, J. Biol. Chem. 271, 623-626) and two yeast transcription factors TFIID and HAP4 (lizuka et al., 1994, Mol. Cell. Biol., 14, 7322-7330). IRES sites have also highlighted in murine retrotransposons of the VL30 type (Berlioz et al., 1995, J. Virol. 69, 6400-6407) and, more recently in the mRNAs coding for the gag precursor of the Friend (FMLV) and Moloney murine leukemia viruses (MoMLV) (Berlioz and Darlix, 1995, J. Virol. 69, 2214-2222; Vagner et al., 1995, J. Biol. Chem. 270, 20376-20383).

A new internal ribosome entry site has now been found in the 5 'non-coding region of avian reticuloendotheliosis virus (REV) RNA type A (REV-A) and shown to be effective in initiating the translation of coding sequences placed after it in a monocistronic or dicistonic manner.

The IRES site of the present invention is particularly advantageous compared to those already described in the literature. First, it allows a high level of expression of the cistron it controls. In addition and, unexpectedly, it can also, in the context of a retroviral vector, contribute or improve, in association with an appropriate packaging region, the dimerization or packaging functions, allowing an increase in the viral titer . And finally, because of its weak homology with the murine retroviral sequences used in most gene therapy vectors intended for human use, its use considerably reduces the risk of production of viruses competent for replication.

Most gene therapy protocols approved by the RAC (Recombinant DNA Advisory Committee) in the United States use vectors derived from the MoMLV virus. At the present time, the choice of a specific retroviral vector for a given therapeutic application remains empirical and the factors influencing the viral titer and the expression of the genes have not yet been clearly elucidated. Studying the cis-acting sequences that control packaging and establishing the relative forces of various IRES elements can help optimize gene therapy vectors in terms of titer and gene expression. One of the aims of the present invention is to propose new retroviral vectors capable of being propagated at high titer and of allowing optimal expression of one or more genes of interest.

This is why the present invention relates to the use of a nucleotide sequence derived from all or part of the 5 'end of the genomic RNA of a type C retrovirus with the exception of the murine leukemia viruses of Friend (FMLV) and Moloney (MoMLV), as internal ribosome entry site (IRES) in a vector and / or to allow or improve the packaging of a retroviral vector.

By nucleotide sequence is meant a sequence composed of ribo (RNA) or deoxyribonucleotides (DNA). In the context of the present invention, the 5 ′ end of the genomic RNA of a retrovirus corresponds to the 5 ′ quarter of said RNA which extends from the site of initiation of transcription (nucleotide +1) to approximately 2 kb in direction 3 '. The term retrovirus is widely defined in basic virology works accessible to those skilled in the art and the essential characteristics have been summarized by way of indication above. The term "derivative" refers to a sequence having a type C retroviral origin, but which may have undergone at least one modification with respect to the native sequence. The possible modification (s) include the deletion, addition, substitution and / or mutation of one or more nucleotides (nt). Such modifications may have the aim, for example, of increasing the IRES functions, of packaging, or of introducing adequate restriction sites to facilitate the subsequent cloning steps. The term "derivative" also includes the DNA equivalent of genomic RNA in a modified or unmodified form.

The term IRES denotes a site capable of promoting the entry of ribosomes into an RNA molecule in a manner independent of the cap. In accordance with the aims of the present invention, the IRES function can be exercised in any vector or expression cassette. A sequence used in the context of the present invention can also act as an activating element for the packaging of retroviruses or retroviral vectors by promoting the dimerization of two copies of the retroviral genome and / or the packaging of the dimer in the particles. viral. According to a preferred embodiment, said sequence is capable of exercising an IRES function and of improving the function encapsidation when introduced into an appropriate retroviral vector.

A nucleotide sequence as used in the context of the present invention can be isolated from the 5 ′ end of the genomic RNA or of the proviral DNA of a type C retrovirus or of any plasmid of the state of the technique carrying the retroviral fragment of interest. It goes without saying that it can be generated by any technique in use in the field of art, for example by cloning using appropriate probes, by PCR (Polymerase Chain reaction) or by chemical synthesis. Advantageously, said sequence comprises all or part of the region which follows the U3 domain of the 5 'LTR, up to the codon AUG initiator of the gag gene. For the purposes of the present invention, it comprises at least 50 nucleotides, advantageously at least 100 nucleotides, preferably at least 200 nucleotides and preferably at least 300 nucleotides included in said 5 ′ end. But, of course, it can extend beyond in the 5 ′ or 3 ′ direction or include additional sequences. Advantageously, said sequence comprises from 100 to 1500 nucleotides and, in particular, from 300 to 800 nucleotides.

It is preferred to use in the context of the present invention a type C retrovirus with the exception of the FMLV and MoMLV viruses. A type C retrovirus which is more particularly suitable is selected from the REV viruses (Reticuloendotheliosis virus), MSV (Murine sarcoma virus) and in particular that of Moloney (MMSV), MHV (Mus hortulanus virus), MEV (Mouse endogenous retrovirus) , FMOV (FBR murine osteosarcoma virus), AMLV (AKV murine leukemia virus), MEELV (Mouse endogenous ecotropic murine leukemia virus), SFFV (Friend spleen focus-forming virus), RAS V (rat sarcoma virus), FLV (Feline leukemia virus ), FSV (feline sarcoma virus), EFLV (Cat endogenous proviral feline leukemia virus), SSV (Simian sarcoma virus), GALV (Gibbon ape leukemia virus) and BAEV (Baboon endogenous virus).

According to an entirely preferred embodiment, a nucleotide sequence in use in the present invention derives from all or part of the 5 'end of the genomic RNA of a reticuloendotheliosis virus (REV). The REV viruses include in particular different subtypes A, B and T as well as the DIAV viruses (Duck infectious anemia virus), SNV (spleen necrosis virus) and CSV (Chick syncytial virus) (see for example Encyclopedia of Virology, 1994, Enrietto , Reticuloendotheliosis viruses, p 1227-1232 Ed. R. Webster and A. Granoff, Académie Press, Hartourt Brace § Company Publishers). A REV virus which is very particularly suitable is the avian reticuloendotheliosis virus, in particular that of type A (REV-A).

According to this last variant, use will preferably be made of a nucleotide sequence comprising at least 100 nucleotides and at most 800 nucleotides (nt) of the 5 ′ non-coding end of the REV-A virus and more particularly a substantially homologous or identical nucleotide sequence to all or part of the sequence presented in the sequence identifier SEQ ID NO: 1. Mention may be made, as preferred examples, of a nucleotide sequence substantially homologous or identical to the sequence presented in the sequence identifier SEQ ID NO : 2:

(i) starting at nucleotide 1 and ending at nucleotide 578, (ii) starting at nucleotide 265 and ending at nucleotide 578, or (iii) starting at nucleotide 452 and ending at nucleotide 578.

The term 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%. As already indicated, said nucleotide sequence may have a sequence slightly different from that described in SEQ ID NO: 1 or 2, provided, however, that the modification or modifications does not affect its IRES functions and / or packaging. According to an advantageous mode, the nucleotide sequence used in the context of the present invention is identical to the sequence presented in the sequence identifier SEQ ID NO: 2: (i) starting at nucleotide 1 and ending at nucleotide 578, (ii) starting at nucleotide 265 and ending at nucleotide 578, or

(iii) starting at nucleotide 452 and ending at nucleotide 578. The IRES function of said nucleotide sequence is particularly advantageous in a context poor in magnesium ion, for example in a cellular context. A high concentration of Mg ²⁺ ions can decrease the efficiency of sequence-mediated translation initiation.

A nucleotide sequence in use in the present invention is more particularly intended to be integrated into a vector for the transfer and expression of 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. In accordance with the aims pursued by the present invention, it is possible to envisage a plasmid vector or a vector derived from an animal virus and, in particular, from a poxvirus (canary pox or vaccinia virus, in particular

Coenhage or MVA), adenovirus, baculovirus, herpes virus, virus associated with an adenovirus or retrovirus. Such vectors are widely described in the literature. In particular, when it is an adenoviral vector, it can be derived from a human (preferably type 2 or 5), animal (preferably canine or bovine) adenovirus or a hybrid between various species. The general technology for adenoviruses is disclosed in Graham and Prevec (1991,

Methods in Molecular Biology, Vol 7, Gene transfer and Expression Protocols;

Ed E.J. Murray, the Human Press Inc, p 109-118).

In accordance with the aims pursued in the context of the present invention, said nucleotide sequence is preferably positioned upstream of a gene of interest to improve the translation of the expression product for which it codes. It can be implemented 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 d '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 nucleotide sequence as defined above. Particularly preferred is the use in a dicistronic cassette either upstream of the first gene of interest or upstream of the second, the latter variant being the preferred.

When a vector according to the invention comprises several expression cassettes, these can be inserted in any orientation relative to each other, either in the same orientation (promoter acting in the same direction) or in reverse orientation ( promoter acting in an opposite orientation). Furthermore, a vector according to the invention can comprise several nucleotide sequences in use according to the invention. In this case, it is preferable that they are derived from different type C retroviruses.

According to an entirely preferred embodiment, a vector according to the invention derives from a retrovirus. By way of example, mention may be made of avian 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 (FLV, FSV ....), murine retroviruses such as murine leukemia virus (MuLV), the virus Friend's (FMLV) and murine sarcoma virus (MSV) and primate retroviruses (GALV, FSV, BAEV ...). Of course, other retroviruses can be used. However, we particularly prefer to use the MoMLV virus. The numerous retroviral vectors described in the literature can be used in the context of the present invention. The retroviral vectors which can be envisaged for the purposes of the present invention comprise at least the following elements which are operatively associated: a 5 'LTR and a 3' LTR retroviral, one or more gene (s) of interest, and the nucleotide sequence use in the context of the present invention to allow or improve the packaging of said vector in a viral particle and / or as an IRES site to allow or promote the expression of a gene of interest positioned downstream of said nucleotide sequence. It goes without saying that the retroviral 5 'LTR can be used as a promoter, but an internal promoter can also be used. Furthermore, the 5 ′ and possibly 3 ′ LTR may have the same retroviral origin (for example REV) as the nucleotide sequence or a different origin. For example, a monocistronic vector will comprise from 5 'to 3' a 5 'LTR, the nucleotide sequence, a gene of interest and a 3' LTR.

Of course, a retroviral vector according to the invention can also comprise a conventional (E +) packaging region. However, the presence of the latter is not required when the nucleotide sequence in use in the present invention can alone exercise the packaging function. Such an embodiment can be more particularly envisaged when the retroviral LTR 5 ′ derives from a REV virus and, preferably from SNV, and the nucleotide sequence is substantially homologous or identical to the sequence presented in SEQ ID NO: 2, starting at nt 1 and ending at nt 578 or starting at nt 265 and ending at nt 578.

According to an advantageous embodiment, a retroviral vector according to the invention comprises at least: (a) a 5 'retroviral LTR,

(b) an encapsidation region,

(c) optionally, a first gene of interest followed by an internal promoter region of an origin different from that of said retroviral 5 ′ LTR, (d) a second gene of interest,

(e) an IRES site,

(f) a third gene of interest, and

(g) a 3 ′ retroviral LTR, at least one of the packaging region and of the IRES site being constituted by said nucleotide sequence in use according to the invention.

In the case where the retroviral vector according to the invention comprises an expression cassette directed by an internal promoter region, it is preferable to favor the gene expression, that this is in an opposite orientation with respect to the 5 'LTRs and 3 'retrovirals. Other elements can also be included, for example another IRES site and another gene of interest or another expression cassette.

A preferred retroviral vector according to the invention comprises an encapsidation region deriving from a murine retrovirus, in particular from a MoMLV, or from a VL30 type retrotransposon and an IRES site comprising a nucleotide sequence substantially homologous or identical to the sequence presented in the sequence identifier SEQ ID NO: 2:

(i) starting at nucleotide 1 and ending at nucleotide 578,

(ii) starting at nucleotide 265 and ending at nucleotide 578, or

(iii) starting at nucleotide 452 and ending at nucleotide 578.

Mention may in particular be made of the dicistronic retroviral vectors of the pREV HW-3 and HW-6 type, in which the packaging region derives from a MoMLV and the IRES site consists of a nucleotide sequence identical to the sequence presented in SEQ ID NO: 2 starting at nucleotide 265 and ending at nucleotide 578 or starting at nucleotide 452 and ending at nucleotide 578. Of course, those skilled in the art can vary the genes of interest according to the desired therapeutic effect.

For the purposes of the present invention, a gene of interest in use in the invention can be obtained from a eukaryotic or prokaryotic organism or from a virus by any conventional molecular biology technique. It can code for a polypeptide corresponding to a native protein as found in nature homologous to the host cell or not, a protein fragment, a chimeric protein originating from the fusion of polypeptides of various origins or a mutant with improved and / or modified biological properties. Such a mutant can be generated by substitution, deletion and / or addition of one or more amino acid residues. In addition, the polypeptide can be (i) intracellular (ii) membrane present on the surface of the host cell or else (iii) secreted outside the host cell and therefore comprise appropriate additional elements, such as a sequence coding for a signal of secretion or a transmembrane anchoring region.

It is particularly preferred to use a gene of therapeutic interest coding for an expression product capable of inhibiting or delaying the establishment and / or the development of a genetic or acquired disease. 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, cardiovascular diseases (restenosis, arteriosclerosis, ischemia ...) and other 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: a cytokine and in particular an interleukin (IL-2, IL-7, IL-10, IL-12 ...), an interferon, a tissue necrosis factor and a growth factor and in particular hematopoietic (G-CSF, GM-CSF), 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 and in particular trypsin, a ribonuclease, alkaline phosphatase (plap) and β-galactosidase, an enzyme inhibitor such as αl-antitrypsin and inhibitors of viral proteases an expression product of a suicide gene such as thymidine kinase of the HSV virus (herpes virus) type 1, that of the fiirl gene and / or fcyl of Saccharomyces cerevisiae, ricin, an activator or an inhibitor of ion channels, 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), glucocerebrosidase and phenylhydroxylase, a protein capable of inhibiting the initiation or progression of cancers, such as the expression products of tumor suppressor genes, for example the p53, p73 and Rb genes, a protein capable of stimulating an immune response, an antibody, the antigens of the major histocompatibility complex or an immunotoxin, a protein capable of inhibiting a viral infection or its development, for example the antigenic epitopes of the virus in question or altered variants of viral proteins likely to compete with native viral proteins, a cellular or nuclear receptor or one of their ligands, a growth factor (FGF for Fibroblast Growth Factor, VEGF for Vascular Endothelial cell Growth Factor ...), and a apoptosis inducer (Bax ...), an apoptosis inhibitor (Bcl2, BclX ...), a cytostatic agent (p21, pl6, Rb ...), a nitric oxide synthase (NOS), a e apolipoprotein (apoAI, apoE ...), a catalase, a SOD, a factor acting on angiogenesis (PAI for Plasminogen Activator Inhibitor ...).

^{'Furthermore,} a use in gene of interest in the present invention may also encode a selectable marker to select or identify the transfected host cell with a vector according to the invention. We can cite the neo gene (neomycin) conferring resistance to the antibiotic G418, dhjr gene (dihydrofolate reductase), the CAT gene (Chloramphenicol Acetyl Transferase) or even the gpt gene (xanthine phosphoribosyl).

In general, use will be made, for the expression of a gene or genes of interest, of 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 may be a promoter naturally governing the expression of a gene of interest in use in the present invention or any other promoter of any origin. Furthermore, it can be constitutive or regulable in nature, in particular in response to certain tissue-specific or event-specific cellular signals. For example, it may be advantageous to target the expression of the gene of interest at the level of lymphocyte cells in the context of AIDS, of lung cells in the context of cystic fibrosis or of muscle cells in the context of myopathies.

By way of examples, the promoters suitable for the present invention can be chosen from promoters SV40 (Simian 40 virus), CMV (Cytomegalovirus), HMG (Hydroxymethyl-Glutaryl Coenzyme A), TK (Thymidine kinase), Retroviral LTRs such as that of MoMLV, RSV or MSV when a retroviral vector, the adenoviral El A and late MLP (Major Late Promotor) promoters are used, in particular in the context of an adenoviral vector, the 7.5K promoters, H5R, pKlL, p28 and pi 1 intended for poxviral vectors like the vaccinia virus, the promoter PGK (Phosphoglycero kinase), the liver-specific promoters of the genes coding for the alpha-antitrypsin, factor IX, albumin and transferrin, the promoters of the immunoglobulin genes which allow expression in lymphocytes, and finally the promoters of the genes coding for the surfactant or the CFTR protein which have a certain specificity for lung tissues. It can also be a promoter stimulating expression in a tumor or cancer cell. Muc-1 gene promoters overexpressed in breast and prostate cancers can be mentioned in particular (Chen et al., 1995, J. Clin. Invest. 96, 2775-2782), CEA (for carcinoma embryonic antigen) overexpressed in colon cancer (Schrewe et al., 1990, Mol. Cell. Biol. 10, 2738-2748), tyrosinase overexpressed in melanomas (Vile et al ., 1993, Cancer Res. 53, 3860-3864), ERB-2 overexpressed in breast and pancreatic cancers (Harris et al., 1994, Gene Therapy 1, 170-175), α-fetoprotein overexpressed in cancers liver (Kanai et al., 1997, Cancer Res. 57, 461-465), APC overexpressed in colorectal cancers, BRCA-1 and 2 (Wooster et al., 1995, Nature 378, 789-792) overexpressed in ovarian cancer and PSA (for prostate specifies antigen) overexpressed in prostate cancer. Furthermore, the gene of interest in use in the present invention may comprise other sequences improving its expression, both at the level of transcription and of translation; for example an enhancer-type transcription activating sequence, an intronic sequence, a transcription termination signal (polyA) and, as indicated above, a secretion signal or a transmembrane region.

The invention also covers the viral particles generated from a viral vector according to the invention. This is generally done by transfection of the latter into a suitable cell line. If the viral vector used is defective for replication, a complementation line will be used. In general, a person skilled in the art knows the lines that can be used to generate infectious viral particles as well as the process to be implemented according to the vector used.

For example, in the case of an adenoviral vector, use may be made of line 293 (Graham et al., 1977, J. Gen. Virol., 36, 59-72). Being a retroviral vector, one can consider using 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). However, it is particularly preferred to use 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). Generally, the infectious viral particles are recovered in the culture supernatant of the transfected complementation cells.

The invention also extends to cells comprising a vector according to the invention or infected with infectious viral particles according to the invention. The transfection methods are well known to those skilled in the art. Mention may be made of the calcium phosphate precipitation technique, that of DEAE dextran, microinjection or encapsulation in lipid vehicles. On the other hand, the vectors according to the invention can be present in the host cell in a form integrated into the cell genome or in the form of episomes both in the nucleus and in the cytoplasm. The cell according to the invention is advantageously a eukaryotic cell, in particular a mammalian cell and, preferably, a human cell. It can be a primary or tumor cell of hematopoietic origin (totipotent stem cell, leukocyte, lymphocyte, monocyte, macrophage ...), hepatic, epithelial, fibroblast, of the central nervous system and, in particular, d '' a muscle cell (myoblast, myocyte, satellite cell, smooth muscle ...), cardiac, vascular, tracheal, pulmonary or central nervous system.

The present invention also relates to the therapeutic use of a vector, a viral particle or a cell according to the invention, for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of a disease. treatable by gene therapy, including a genetic disease, an acquired disease such as cancer or an infectious disease.

However, such use is not limited to an application of the somatic gene therapy type. In particular, a vector according to the invention can be used for other purposes such as the recombinant production in prokaryotic or eukaryotic cells of expression product (s) encoded by at least one of the genes of interest. . For example, it is possible to envisage the coexpression of two genes of interest in a dicistronic expression vector using a sequence nucleotide according to the invention. The coexpression of an antibiotic resistance gene as a second cistron can make it possible to increase the expression of a first cistron. It is possible to obtain a mature product by the coexpression of two genes whose expression product of one allows the maturation of the polypeptide encoded by the other (for example polypeptide precursor and a protease cleaving the precursor into mature polypeptide) . In this case, we can use prokaryotic (E. coli ...), lower eukaryotic (yeast, fungus, insect ...) or animal cells. It will then be a question of harvesting and possibly purifying said expression product of interest of the supernatant or of cell culture by conventional techniques. Another possibility of use consists in the production of transgenic animals which have integrated into their genome a cassette for the expression of one or more genes of interest and comprising a nucleotide sequence according to the invention. It can be mice, rats, rabbits, fish, primates or farm animals (cattle, sheep, pigs ...) The techniques for generating these transgenic animals are known. The polypeptide of interest can be recovered in a conventional manner, for example in the biological fluids (blood, milk, etc.) of the animal.

The invention also relates to a pharmaceutical composition comprising, as therapeutic or prophylactic agent, a vector, a viral particle or a cell according to the invention or a polypeptide of interest obtained in accordance with the use according to the invention, in combination with a pharmaceutically acceptable vehicle.

A pharmaceutical composition according to the invention can be manufactured in a conventional manner. In particular, a therapeutically effective amount of such an agent is combined with an acceptable carrier, diluent or adjuvant. It can be administered according to any route of administration and this in a single or repeated dose after a certain time interval. In particular, intravenous, intramuscular, intrapulmonary (possibly aerosolized) or intratumoral administration is preferred. The amount to be administered will be chosen according to different criteria, in particular the use as treatment or vaccine, the route of administration, the patient, the type of disease to be treated and its state of evolution, the duration of treatment, the vector retained ... etc. As an indication, a pharmaceutical composition according to the invention comprises between 10 ⁴ and 10 ¹⁴ pfu (unit forming plaques), advantageously between 10 ⁵ and 10 ¹³ pfu and, preferably, between 10 ⁶ and 10 "pfu of viral particles. A vector-based composition can be formulated as doses comprising from 0.01 to 100 mg of DNA, preferably from 0.05 to 10 mg and, most preferably, from 0.1 to 5 The formulation can also include, alone or in combination, a pharmaceutically acceptable diluent, adjuvant or excipient, as well as a solubilizing, stabilizing or preserving agent. single or in multiple doses in liquid or dry form (lyophilisate ...) capable of being reconstituted extamporanally with an appropriate diluent.

Furthermore, 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, a viral particle or a cell according to the invention is administered to a patient having need such treatment. According to a first therapeutic protocol, they can be administered directly in vivo, for example by intravenous, intramuscular, intratumoral injection or by aerosolization in the lungs. Alternatively, one can adopt an ex vivo gene therapy protocol which consists in removing the cells from a patient (stem cells from the bone marrow, peripheral blood lymphocytes, etc.), transfecting them with a vector according to the invention and cultivating them in vitro before re-implanting them in the patient.

Finally, the invention relates to the use of a vector, a viral particle or a pharmaceutical composition according to the invention for the transfection or infection of pluripotent cells, in particular pluripotent cells of the central nervous system.

The invention is illustrated below with reference to the following figures. Figure 1 is a schematic representation of the monocistronic plasmids used as templates for the in vitro synthesis of capped and non-capped RNAs. They contain the early cytomegalovirus (Po CMV) promoter usable for expression in vivo, the promoter of the gene coding for phage T7 RNA polymerase (Po T7) usable for in vitro experiments, different portions of the 5 end '' untranslated (leader) of the REV-A virus (1 to 578 for pREV CB-95, 578 to 1 for pREV CG-53, 1 to 578 deleted from nt 268 to 452 for pREV CG-54, 265 to 578 for pREV CG-55 and 452 to 578 for pREV CG-56) and the LacZ gene (ΔLacZ) coding for a truncated β-galactosidase at the C-terminal end with a molecular mass of approximately 46 kDa.

Figure 2 is a schematic representation of the dicistronic plasmids used as templates for the in vitro synthesis of capped and uncapped RNA. They contain the early cytomegalovirus promoter (Po CMV) usable for expression in vivo, the promoter of the gene coding for phage T7 RNA polymerase (Po T7) usable for in vitro experiments, the neo gene, different portions of the 5 'untranslated end (leader) of the REV-A virus (1 to 578 for pREV CB-54, 578 to 1 for pREV CG-50, 1 to 578 deleted from nt 268 to 452 for pREV CG-52, 265 to 578 for pREV CB-55 and 452 to 578 for pREV CG-58) and the LacZ gene (ΔLacZ) coding for a truncated β-galactosidase at the C-terminal end with a molecular mass of approximately 46 kDa.

Figure 3A is a schematic representation of the dicistronic retroviral vectors having two elements of different retroviral origin, as IRES and packaging region (E) and two genes of interest such as the reporter genes coding for phosphatase alkaline placenta and neo coding for neomycin phosphotransferase. B) Retroviral vectors of the pREV HW series possessing LTRs derived from MLV and placed in a plasmid context pBR322. VL30E + corresponds to the 5 'untranslated region of HaMSV and MoMLV E + corresponds to the packaging region of MoMLV. C) Reference vector pEMCV-CBTV having LTRs and the packaging region of MoMLV and 1RES of EMCV. In all cases, the sequences are numbered with respect to the cap site (position +1) of the genomic RNA. Figure 4 illustrates the effect of rapamycin on the activities A) alkaline phosphatase and B) neomycin phosphotransferase produced in GP + E-86 cells not transfected or stably transfected by the different vectors pREV HW or pEMCV-CBTV (pCBlOO ) and treated with rapamycin (full boxes) or not treated (control, dotted boxes). Figure 5 illustrates the optimization of the transduction protocol applied to Dev neuroectodermal cells. The percentage of Dev cells transduced by the pEMCV-CBTV (IRES EMCV) and pREV HW-3 viruses (IRES REV-A) is determined by flow cytometry.

EXAMPLES

The constructions described below are carried out according to the general techniques of genetic engineering and molecular cloning detailed in Sambrook et al. (1989, Molecular cloning: A Laboratory Manual, 2nd εd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) or according to the manufacturer's recommendations when using a commercial kit. 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.). Amplifications of plasmid DNA are carried out in Eschenchia coli HB101 strain 1035 (recA). Furthermore, the position of the REV-A sequences used in the constructions 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 (Darlix et al., 1992, J. Virol. 66: 7245-7252). EXAMPLE 1: Identification of an IRES site at the 5 ′ end of the REV-A RNA.

In vitro protein synthesis studies have been undertaken using a series of mono and dicistronic plasmids containing fragments of the 5 'leader of the REV-A genomic RNA in order to determine whether they allow the translation of cistrons by internal binding of ribosomes.

1. Construction of mono and dicistronic plasmids.

The DNA fragments corresponding to sequences 1 to 578, 265 to 578 and 452 to 578 of the REV-A RNA are isolated by PCR from the pREVSC-1 template (Darlix et al., 1992, J. Virol. 66, 7245-7252). Appropriate primers are used that a person skilled in the art can design, provided at their ends with a Mzel site. After digestion with this enzyme, the PCR fragments are inserted upstream of the LacZ gene in the vector pEMCV-M260-837 (Berlioz et al., 1995, J. Virol. 69, 6400-6407) previously cleaved by Nhel. The LacZ gene used codes for a truncated β-galactosidase product at the C-terminal end. The monocistronic plasmids pREV CB-95 (1-578), pREV CG-55 (265-578) and pREV CG-56 (452-578) are obtained, illustrated in Figure 1. The dicistronic plasmids pREV CB-54 (1- 578), pREV CB-55 (265-578) and pREV CG-58 (452-578) are shown in Figure 2 and result from the insertion of the previous PCR fragments between the neo and LacZ genes of pEMCV-D260-837 (pCBlOl) (Berlioz et al., 1995, J. Virol. 69, 6400-6407) also subjected to digestion with Nhel. Amplification of nt 1 to 578 deleted from sequences 268 to 452 is carried out using the vector pREVSC-1 previously digested with Kpnl and Sα, treated with the Klenow fragment of AD de polymerase from E. coli and religious. The amplified fragment digested by Mzel is cloned in pEMCV-M260-837 upstream of the LacZ gene or between the neo and LacZ genes of pEMCV-D260-837, the two vectors having been digested with Mzel, to give respectively pREV CG-54 (Fig 1) and pREV CG-52 (Fig 2). Finally, monistristrum control plasmids pREV CG-53 (Fig 1) and dicistronic pREV CG-50 (Fig 2) were constructed by introduction of the PCR fragment carrying the sequences REV-A l to 578 in the previous vectors in reverse orientation (578- 1). In all of the constructions containing the REV-A sequences in sense orientation, the initiation of the translation of β-galactosidase is under the control of the AUG codon of the gag gene of REV-A located at position 574-576, while in the control plasmids, the synthesis of β-galactosidase depends on an AUG placed in a favorable Kozak context introduced by PCR.

2. Synthesis of RNA and in vitro translation.

The capped and uncapped RNAs are synthesized from 1 μg of plasmid DNA linearized by Sspl (position 1240 in the LacZ gene) using T7 RNA polymerase (mMessage mMachine or MAXIscript, Ambion) in a reaction volume 20 μl according to the protocol indicated by the supplier. Transcription is stopped by treatment of the DNA matrix with the enzyme DNasel followed by precipitation of the RNAs in the presence of lithium chloride. The RNAs are taken up in 50 μl of TE buffer (10 mM Tris-HCl pH7.5, EDTA ImM) before being purified and desalted by passage through an S-300 MicroSpin ™ column (Pharmacia BioTech) according to the supplier's instructions. The integrity of the transcribed RNAs is verified by electrophoresis on 0.7% agarose gel containing ethidium bromide (0.5 μg / ml). The final RNA concentration is determined spectrophotometrically. The capped and uncapped RNAs (10 μg / ml) are translated into the RRL cellular system (Promega) used at 50% of its initial concentration in the presence of 1 mCi of [ ³⁵ S] methionine per ml (Amersham) at 31 ° C. for 1 hour. The reaction mixture is supplemented with potassium acetate to a final concentration of 120 mM. Furthermore, RNA luciferase is tested in same reaction conditions (positive control). A test carried out in the absence of RNA constitutes the negative control. In parallel, we tested the effect of the L and FMDV (Foot and mouth disease virus) protease on the translation of dicistronic RNAs. This protease cleaves the eIF4G translation initiation factor between glycine at position 479 and arginine at position 480 to generate two peptide fragments lacking initiating activity (Kirchweger et al., 1995, J. Virol. 68, 5677 -5684). In addition, Ohlmann et al. (1995, Nucleic Acid Res. 23, 334-340; 1996, EMBO J. 15, 1371-1382) has shown that the treatment of reticulocyte lysates by protease L inhibits the in vitro translation of capped cellular RNAs while the internal initiation directed by 1TRE of the cardiovirus is not affected. Thus if an IRES element exists in the 5 ′ leader REV-A, the presence of protease L should not affect the expression of the cistron whose translation is dependent on it. In this case, the tests use the RRL Flexi system (Promega) at 50% of its initial concentration, 8 μg / ml of RNA, 1 mCi of [ ³⁵ S] methionine per ml (Amersham) and 30 ng of protease L recombinant and purified by conventional methods. The reaction mixture is supplemented with potassium chloride to a final concentration of 80 mM. The reaction is continued at 31 ° C for 1 h.

The samples are heat denatured in 62.5 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulphate (SDS), 10% glycerol, 5% β-mercaptoethanol and 0.02% bromophenol blue and the labeled proteins analyzed by electrophoresis on polyacrylamide gel 12% (weight / vol), 0.2% SDS. The neo gene product and β-galactosidase migrate at a molecular mass of approximately 28 and 46 kDa respectively. The efficiency of the cap-dependent and independent translation is quantified by CT scan (Phospho-Imageύr Storm 840, version 4.00, Molecular Dynamics; Image Quant ™ version 1.1, Molecular Dynamics). In the case of dicistronic vectors, the intensity of the labeling of the expression product of the second cistron (β-galactosidase) whose translation is mediated by PIRES is evaluated after standardization of the level of expression of the neo product.

It is observed that the translation of the non-capped RNAs obtained from the monocistronic plasmids pREV CB-95, pREV CG-53, pREV CG-54, pREV CG-55 and pREV CG-56 is as efficient as that of the capped RNAs. However, as expected, the amount of β-galactosidase generated from the plasmid pREV CG-53 in which the REV-A sequences (1 to 578) are in antisense orientation is much lower than that obtained with the constructions using a REV sequence -A in sense orientation. When the REV-A fragments are implemented in a dicistronic manner between the neo and LacZ genes, the expression of β-galactosidase only appears in the presence of a functional IRES (pREV CB-54, pREV CB -55, and pREV CG-52) while that of the first cistron (neo) is effective in all cases (including pREV CG-50). Compative in vitro translation tests carried out with the preceding vectors and the plasmid pEMCV-D260-837 comprising TIRES EMCV reference show that the fragments REV-A 1-578, 265-578 and 452-578 are capable of initiating translation of the second cistron in a more efficient way than that directed by TIRES EMCV. Furthermore, the treatment of reticulocyte lysates with protease L is accompanied by an inhibition of the cap-dependent expression of the neo gene, while the expression of TIRES-dependent β-galactosidase is significantly increased. For the pREV CG-50 control, the inhibition of neo expression is also observed while the expression of β-galactosidase is barely detectable whether or not the treatment with protease L takes place. As an indication, the effect of the protease on the expression of the two reporter genes is illustrated below.

Table 1: Report of gene expression in the presence and absence of FMDV protease L.

EXAMPLE 2 Retroviral vectors comprising an IRES REV-A sequence

A series of retroviral vectors has been constructed using the REV-A sequences as IRES sites or as elements that increase packaging. FIG. 3 illustrates the vectors of the pREV HW series having LTRs of MoMLV type and the control vectors used in the experiments described below. Although these are not represented, vectors designated pMC have also been constructed which differ from pREV HW only in that their LTRs are of SNV origin and negative controls in which the REV-A sequences are positioned in orientation reverse (3 '-> 5') compared to LTRs. For all the molecular biology stages, these vectors are introduced into the plasmid pBR322.

1. Construction of retroviral vectors.

The control vector pEMCV-CBTV (pBC100) is a dicistronic vector comprising, in addition to the LTRs and the packaging region derived from MoMLV, the gene coding for placental alkaline phosphatase (plap), the translation of which is cap-dependent and the gene neo. whose translation is site dependent

IRES EMCV (Torrent et al., 1996, Human Gène Therapy 7, 603-611).

The pREV HW vectors were obtained in the following way: pREV HW-1: the REV-A fragment extending from nt 265 to 578 generated by PCR and digested with zel is cloned between the plap and neo genes of pMLV-CB71 (Berlioz and Darlix, 1995, J. Virol. 69, 2214-2222). pREV HW-2: the EcoRI fragment of pVL CBT5 (Torrent et al., 1996, Human Gene Therapy 7, 603-611) carrying the LTR 5 ′ MoMLV and the packaging sequences of VL30 is introduced into the vector pRΕV HW- 1 linearized by EcoRI. pRΕV HW-3: the EcoRI fragment from pΕMCV-CBTV containing the 5 'LTR and the packaging sequences of MoMLV is inserted into the vector pRΕV HW-1 linearized by EcoRI. pRΕV HW-4: the RΕV-A fragment extending from nt 452 to 578 generated by PCR and digested with Nftel is cloned between the plap and neo genes of pMLV-CB71. pRΕV HW-5: the EcoRI fragment of pVL CBT5 carrying the LTR 5 ′ MoMLV and the packaging sequences of VL30 is introduced into the vector pRΕV HW-4 linearized by EcoRI. pRΕV HW-6: the EcoRI fragment from pΕMCV-CBTV containing the 5 'LTR and the MoMLV packaging sequences is inserted into the vector pRΕV HW-4 linearized by EcoRI.

The pMC series vectors are obtained according to the following construction scheme: The SΝV LTRs are generated by PCR from the plasmid RΕV-A 2-20-6 (O'Rear and Temin, 1982, Proc. Νatl. Acad. Sci USA 79, 1230-1234; Darlix et al., 1992, J. Virol. 66, 7245-7252). The neo gene is isolated from pMLV-CB71 by digestion with Sali and BamHI then introduced between the same sites of the vector pUC19 (Gibco BRL). The 5 'LTR of SΝV (nt 1 to 861) is digested with H diπ and Sali, and inserted into pUC19-neo previously cleaved by these same enzymes. The LTR 3 'SΝV (nt 7230-8300) digested with Smαl and EcoRI is cloned in the preceding vector to give pCG-61 containing LTR 5' SΝV-neo-LTR 3 'SΝV. In parallel, a vector designated pCG-62 is generated which differs from the previous one by deleting the env sequences (nt 7230-7691) obtained by BglR-AvrTl, Klenow treatment and religation. The plap gene isolated from the clone Cla-12AP (DGoff) is introduced between the EcoRI and Xbal sites of a bluescript plasmid previously deleted from the Sali site (EcoRI-.X7zoI digestion) before being re-isolated in the form of a Kpnl-SaR fragment and cloned between the same pCG-61 sites and pCG-62, to give pCG-63 and pCG-64 respectively. The LacZ gene is obtained by partial digestion of pRΕV CB-95 with the enzymes Sali and BamΑl. Its insertion between the SalI and BamUl sites of pCG-61 and pCG-62 gives rise to pCG-65 and pCG-66 respectively. Finally, the LTR-Gene-LTR block is isolated from each plasmid pCG-62, pCG-64 and pCG-66 by H dlII-EcoRI digestion to be inserted into the vector pBR322 cleaved by these same enzymes. PMCl, pMC2 and pMC3 are generated.

2. Generation of infectious viral particles and determination of the viral titer and of the expression of the plap and neo reporter genes.

The GP + Ε-86 ecotropic complementation line (Markowitz et al., 1988, J. Virol., 62, 1120-1124) and the NIΗ3T3 target cells (mouse fibroblastic cells) available at TATCC, are cultured at 37 ° C. in the presence of 5% CO ₂ in DMΕM medium (Dulbecco's Modified Eagle's Medium, Gibco BRL) supplemented with 10% newborn calf serum. GP + Ε-86 helper cells and NIH3T3 target cells are cultured the day before transfection and infection. Viral infections are carried out according to the conventional protocol described in the literature.

20 μg of pRΕV HW-1 to 6 vectors as well as of reference vector pΕMCV-CBTV are transfected in parallel in GP + Ε-86 cells ( ⁵ × 10 ⁵ cells per 10 cm dish) according to the method of Chen and Okyama (1987, Mol. Cell. Biol., 7, 2745-2753; 1988, Bio / Techniques 6, 632-637). Different dilutions of supernatants from stable or transient GP + Ε-86 cultures are used to infect the target NIH3T3 cells seeded the day before infection at the rate of 2x10 * cells per well. Beforehand, the viral supernatants were filtered (on 0.45 μm filters) and placed in the presence of polybrene to a final concentration of 8 μg / ml. The infection is continued overnight at 37 ° C. and the following day, the cells are washed and cultured in fresh medium. After 48 h, the cells are placed in a selective medium (1 mg / ml of G418) or stained to determine the number of cells expressing the alkaline phosphatase plap. First of all, fixing is carried out in PBSxl buffer containing 2% formaldehyde and 0.2% glutaraldehyde. After two rinses in PBSxl followed by a 30 min incubation at 65 ° C in PBSxl, the cells are washed twice in AP buffer (0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 50 mM MgCl ₂ in PBSxl) and placed for 5 h in the staining solution (0.1 mg / ml of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 1 mg / ml of a terazolium salt of Nitroblue (NBT) and 1 mM Levamisol in buffer AP). These histochemical staining experiments confirm the expression of alkaline phosphatase in the GP + E-86 helper cells and in the NIH3T3 target cells. The titer of the recombinant viruses is determined after transfection of the GP + E-86 ecotropic cells. After two days of incubation, the viral supernatant is harvested and used to determine the viral titer (transient expression). Then, the transfected cells are selected with G418 for one month. After this selection, the viral titer is determined on the harvested supernatant (stable expression). This corresponds to the number of infectious particles per ml of supernatant. By the limit dilution method, the NIH3T3 target cells are infected with serial dilutions of viral supernatant and, after two days of incubation, the cells are stained histochemically and counted. The following results are obtained (Table 2):

The pREV HW-1 and pREV HW-4 vectors lacking a conventional packaging region are incapable of producing infectious viral particles after transfection into the MLV helper line (GP + E-86). However, it is indicated that the vector pMC1 can be packaged in SNV viral particles after transfection of the helper line SNV D17-C3A2 (for example ATCC CRL8468), indicating that the REV-A sequences extending from nt 265 to 578 can be used in this context as an encapsidation region. Retroviral vectors comprising both a REV-A sequence (265-578 or 452-578) and a conventional packaging region produce high titer viral particles (pREV HW-2, 3, 5 and 6). However, the association with the packaging region of MLV proves to be particularly advantageous since it gives viral titers 2 (pREV HW-6) to 5 times (pREV HW-3) higher than the reference vector pEMCV-CBTV combining this same encapsidation region and TIRES EMCV. Furthermore, the comparison of the data obtained with the identical vectors varying only at the level of the REV-A segment used (pREV HW-2 and pREV HW-5 oμ pREV HW-3 and pREV HW-6) suggests that the sequence ranging from nt 265 to 578 is capable of cooperating with the packaging region and thus improving the packaging of viral RNAs and consequently the viral titers. An element interacting positively with packaging could be present between nt 452 and 265 in the REV-A genome.

3. Analysis of recombinant viruses by electron microscopy.

The morphology of the recombinant pREV HW virions produced after transfection of the GP + E-86 line with the corresponding vectors is analyzed by electron microcopying. The viruses obtained from pEMCV-CBTV under the same conditions are used as controls and the wild retroviruses obtained after infection of the NIH3T3 cells with the FMLV-29 strain (Friend murine leukemia virus strain 29). Microscopy results indicate that the RNA content does not affect the morphology of the recombinant viruses.

4. Effect of rapamycin on the expression of plap and neo cistrons.

GP + E-86 cells are stably transfected with 20 μg of vectors of the pREV HW or pEMCV-CBTV series and cultured under selective conditions (G418) for 15 days. At 70 to 80% confluence, they are brought into the presence of rapamycin at a final concentration of 20 ng / ml. The latter has an inhibitory effect on cap-dependent translation varying from 15 to 40% depending on the cell line (Beretta et al., 1996, EMBO J. 15, 658-664) but does not affect cap-independent translation. Cell extracts are conventionally prepared after 20 h of incubation. Briefly, the cells are washed twice in PBSxl, placed in 1 ml of TEN (40 mM Tris-HCl pH7.5, 1 mM EDTA, 50 mM NaCl) for a 10 cm dish then recovered by scraping and centrifuged on low speed. The pellet is taken up in 100 μl of 0.25 M Tris-HCl, pH8 and subjected to cell lysis by 3 freeze-thaw cycles. After centrifugation for 10 min at 14,000 g, the supernatant is recovered and can be stored at -70 ° C while waiting for the enzymatic tests. The final protein concentration is determined by the Micro BCA test (Pierce). The plap enzymatic activity of the cell extracts is evaluated spectrophotometrically (alkaline phosphatase kit, BIORAD). The plap units are determined in relation to a standard consisting of alkaline phosphatase from calf intestine (Boehringer Mannheim). The neo activities are measured by the transfer of phosphate labeled with [γ- ³² P] on neomycin (Ramesh and Osborne, 1991, Anal. Biochem. 193, 316-318). The results of the expression of the plap and neo genes measured in the absence and in the presence of rapamycin are illustrated in FIG. 4 and presented in Table 3.

Table 3: effect of rapamycin on the expression of plap and neo cistrons expressed in% relative to untreated cells.

In the vectors pREV HW-1 and pREV HW-4, the presence of rapamycin reduces the cap-dependent translation and increases that dependent on TIRES. This stimulation can be explained by less competition for the translational machinery in the presence of rapamycin. When two IRES elements are present, the addition of rapamycin is accompanied by an increase in the expression of the two genes. However, quantitative expression data indicate that the relative activity of IRES is different, suggesting a competition between them for ribosomes.

In summary, all the data show the presence of an IRES site effective in the 5 'leader of REV-A virus RNA and probably of a positive interacting element with packaging. The minimum IRES element is contained within a sequence of 129 nt (positions 452 to 578).

EXAMPLE 3 Transduction of Pluripotent Human Neuroectodermal Cells

This study is carried out in the human Dev cell line, which constitutes a cellular model of the central nervous system, a potential target for human gene therapy. Dev cells are derived from a human primary tumor of neuroectodermal origin (PNET) and behave like pluripotent stem cells (Derrington et al., 1997, Oncogene, in press). Moreover, it is possible to induce their differentiation either into neurons or into glial cells (Derrington et al., 1997, supra; Dufay et al., 1994, Eur. J. Neurosci. 6, 1633-1640; Giraudon et al ., 1993, Neurosci. 52, 1069-1079). In this study, the dicistronic vector pREV HW-3 (IRES REV-A 265-578) is compared with the control vector pEMCV CBTV (IRES EMCV).

The Dev cells are infected with a 1/100 dilution of viral supernatant for 2 days then fixed, histochemically stained according to the above protocol and counted. The viral titer is similar for the two vectors and of the order of ³ × 10 ³ TU / ml. The transduction units (TU / ml) correspond to the ratio of the number of colonies x dilution of the infecting retrovirus by the total volume (ml) of the diluted vector placed on the cells.

We also check the expression of the two neo and plap cistrons. To do this, the target cells are as previously transduced with a 1/100 dilution of viral supernatant and selected in the presence of G418 for 3 weeks. The intensity of the fluorescence is determined by flow cytometry with an antibody specific for the plap (DAKO). It is indicated that a polyclonal antibody is suitable. The results show the production of the enzyme plap by cells transduced and selected with G418. In parallel, the synthesis of the enzyme plap is confirmed by histochemical staining of the resistant clones. These data show that the dicistronic vector pREV HW-3 can efficiently transduce human cells derived from the central nervous system and transfer genes of interest therein and confirm the functionality of the IRES REV-A site to mediate the production of the product. expression of the second cistron.

In addition, different transduction protocols have been studied for the purpose of optimization. Variants are the cell culture conditions in the presence or absence of serum and the presence or absence of growth factors (FGF-2) and the production of viruses in the presence or absence of serum.

The following six protocols were compared with the pEMCV CBTV and pREV HW-3 viruses possessing the GaLV polytrope envelope (production on cells

PG13, ATCC CRL-10686). Protocol 1: cells cultured in medium with serum (10%), virus produced in the presence of serum (10%).

Protocol 2: cells cultured in medium with serum (10%), virus produced in the absence of serum. Protocol 3: cells cultured in serum-free medium, virus produced in the presence of serum (10%).

Protocol 4: cells cultured in serum-free medium, virus produced in the absence of serum. Protocol 5: cells cultured in serum-free medium, virus produced in the presence of serum (10%), presence of FGF-2. Protocol 6: cells cultured in serum-free medium, virus produced in the absence of serum, presence of FGF-2. The percentage of Dev cells transduced is determined by flow cytometry (Figure 5). The percentage of cells transduced by pREV HW-3 exceeds 30% when the culture of Dev cells is carried out in the absence of serum. Such a% is not reached with the conventional virus pEMCV CBTV. The addition of growth factors is also advantageous. Among all the protocols used, protocol 5 allows more than 50% of Dev cells to be transduced. These results show that the polycistronic vectors of the invention carrying TIRES REV-A are functional for transducing pluripotent neuroectodermal cells. The development of protocols for the efficient transduction of human cell lines is an essential point in the development of gene transfer strategies.

The expression of plap and neo cistrons is evaluated after neuronal and glial differentiation (Derrington et al., 1997, supra). Briefly, the Dev cells adopt a differentiated phenotype in the presence of serum and FGF-2 whereas when the culture is carried out in the absence of serum, the phenotype is pluripotent. Immunofluorescence results with a specific anti-plap antibody on transduced Dev cells, selected with G418 and differentiated show expression of the plap in neuronal and glial cells. These data suggest that the state of differentiation does not inhibit TIRES REV-A mediated translation.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: INSERM

(B) STREET: 101 rue de Tolbiac

(C) CITY: Paris cedex 13

(E) COUNTRY: France

(F) POSTAL CODE: 75654

(G) TELEPHONE: 01 44 23 60 00 (H) FAX: 01 45 85 68 56

(ii) TITLE OF THE INVENTION: New internal ribosome entry site and vector containing it.

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER-READABLE FORM:

(A) TYPE OF SUPPORT: Tape

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentln Release # 1.0, Version # 1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 940 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: RNA (genomics) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO

(vi) ORIGIN:

(A) ORGANISM: Reticuloendotheliosis virus

(B) STRAIN: type A (REV-A)

(C) INDIVIDUAL ISOLATED: leader 5 'of the genomic RNA REV-A

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

AAUGUGGGAG GGAGCUCCGG GGGGAAUAGC GCUGGCUCGC UAACUGCCAU AUUAGCUUCU 60

GUAAUCAUGC UUGCUUGCCU UAGCCGCCAU UGUACUUGAU AUAUUUCGCU GAUAUCAUUU 120

CUCGGAAUCG ^' GCAUCAUUUC UCGGAAUCGG CAUCAAGAGC AGGCUCAUAG ACCAUAAAAG 180

GAAAUGUUCG ^' UUGGAGGCGA GCAUCAGACC ACUUGCGCCA UCCAAUCACG AGCAAACACG 240

AGAUCGAACU AUCAUACUGA GCCAAUGGUU GUAAAGGGCA GAUGCUAUCC UCCAAUGAGG 300

GAAAAUGUCA UGCAACAUCC UGUCCUGUAA GCGGCUAUAU AAGCCAGGUG CAUCUCUUGC 360 UCGGGGUCGC CGUCCUACAC AUUGUUGUGA CGCGCGGCCC AGAUUCGAAU CUGUAAUAAA 420

AGUUUUUUUC UUCUAUAUCC UCAGAUUGGC AGUGAGAGGA GAUUUUGUUC GUGGUGUAGG 480

CUGGCCUACU GGGUGGGGUA GGGGUCCGGA CUGAAUCCGU AGUAUUUCGA UACAACAUUU 540

GGGGGCUCGU CCGGGAUUCC UCCCCAUCGG CAGAAGUGCC UACUGUUUCU UCGAACUCCG 600

GCGCCGGUAA GUAAGUACUU GAUUUUGGUA CCUCGCGAGG GUUUGGGAGG AUCGGAGUGG 660

CGGGACGCUG CCGGGAAGCU CCACCUCCGC UCAGCAGGGG ACGCCCUGAU CUGAGCUCUG 720

UGGUAUCUGA UUGUUGUUGG ACCGUCUCCA AGACGGUGAU AAUAUAAGUC GUGGUUUGUG 780

UGUUUGUUUG UUACCUUGUG UUUGUUCGUC ACUUGUCGAC AGCGCCCUGC GAAUUGGUGU 840

GCCCACACCG CGCGGCUUGC GAAUAAUACU UUGGAGAGUC UUUUGCCUCC AGUGUCUUCC 900

GUUUGUACUC GUCCUCCUCU CCCUCUCCGG CCGGGAUGGG 940 (2) INFORMATION FOR SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 578 base pairs

(B) TYPE: nucleic acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: linear

(ii) TYPE OF MOLECULE: RNA (genomics) (iii) HYPOTHETIC: NO (iii) ANTI-SENSE: NO

(vi) ORIGIN:

(A) ORGANISM: Reticuloendotheliosis virus

(B) STRAIN: type A (REV-A)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

GGGGUCGCCG UCCUACACAU UGUUGUGACG CGCGGCCCAG AUUCGAAUCU GUAAUAAAAG 60

UUUUUUUCUU CUAUAUCCUC AGAUUGGCAG UGAGAGGAGA UUUUGUUCGU GGUGUAGGCU 120

GGCCUACUGG GUGGGGUAGG GGUCCGGACU GAAUCCGUAG UAUUUCGAUA CAACAUUUGG 180

GGGCUCGUCC GGGAUUCCUC CCCAUCGGCA GAAGUGCCUA CUGUUUCUUC GAACUCCGGC 240

GCCGGUAAGU AAGUACUUGA UUUUGGUACC UCGCGAGGGU UUGGGAGGAU CGGAGUGGCG 300

GGACGCUGCC GGGAAGCUCC ACCUCCGCUC AGCAGGGGAC GCCCUGAUCU GAGCUCUGUG 360

GUAUCUGAUU GUUGUUGGAC CGUCUCCAAG ACGGUGAUAA UAUAAGUCGU GGUUUGUGUG 420

UUUGUUUGUU ACCUUGUGUU UGUUCGUCAC UUGUCGACAG CGCCCUGCGA AUUGGUGUGC 480

CCACACCGCG CGGCUUGCGA AUAAUACUUU GGAGAGUCUU UUGCCUCCAG UGUCUUCCGU 540

UUGUACUCGU CCUCCUCUCC CUCUCCGGCC GGGAUGGG 578

Claims

claims

1. Use of a nucleotide sequence derived from all or part of the 5 'end of the genomic RNA of a type C retrovirus with the exception of the murine leukemia viruses of Friend (FMLV) and Moloney

(MoMLV), as an internal ribosome entry site (IRES) in a vector and / or to allow or improve the encapsidation of a retroviral vector.

2. Use according to claim 1, according to which the type C retrovirus is selected from the REV, MSV, MHV, MEV, FMOV, AMLV, MEELV, SFFV, RASV, FLV, FSV, EFLV, SSV, GALV and BAEV viruses.

3. Use according to claim 2, according to which said nucleotide sequence derives from all or part of the 5 ′ end of genomic RNA of a reticuloendoteliosis virus.

4. Use according to claim 3, according to which said nucleotide sequence derives from all or part of the 5 'end of genomic RNA of an avian reticuloendoteliosis virus, and in particular of type A.

5. Use according to one of claims 1 to 4, according to which said nucleotide sequence is substantially homologous or identical to all or part of the sequence presented in the sequence identifier SEQ ID NO: 1.

6. Use according to claim 5, according to which said nucleotide sequence is substantially homologous or identical to the sequence presented in the sequence identifier SEQ ID NO: 2: (i) starting at nucleotide 1 and ending at nucleotide 578, (ii) starting at nucleotide 265 and ending at nucleotide 578, or (iii) starting at nucleotide 452 and ending at nucleotide 578.

7. Use according to claim 6, according to which said nucleotide sequence is identical to the sequence presented in the sequence identifier SEQ ID NO: 2:

(i) starting at nucleotide 1 and ending at nucleotide 578, (ii) starting at nucleotide 265 and ending at nucleotide 578, or (iii) starting at nucleotide 452 and ending at nucleotide 578.

8. Vector for the expression of one or more gene (s) of interest comprising said nucleotide sequence in use according to one of claims 1 to 7.

9. Vector according to claim 8, characterized in that it is a plasmid vector or a viral vector derived from a virus selected from the group of poxviruses, adenoviruses, baculoviruses, herpes viruses, virus associated with an adenovirus and retrovirus.

10. Vector according to claim 8 or 9, derived from a retrovirus and comprising at least the following elements associated in a functional manner: a 5 'LTR and a 3' LTR retroviral, one or more gene (s) of interest , and said nucleotide sequence as defined in one of claims 1 to 7 to allow or improve the encapsidation of said vector in a viral particle and / or as an IRES site to allow or improve the expression of a gene of interest positioned downstream of said nucleotide sequence.

1 1. The retroviral vector according to claim 10, wherein said nucleotide sequence is as IRES site and further comprising a packaging region heterologous to said nucleotide sequence.

12. The retroviral vector according to claim 10 or 1 1, comprising at least:

(a) a retroviral 5 'LTR,

(b) an encapsidation region,

(c) optionally, a first gene of interest followed by an internal promoter region of an origin different from that of said retroviral 5 ′ LTR,

(d) a second gene of interest,

(e) a 1RES site ,,

(f) a third gene of interest, and

(g) a 3 ′ retroviral LTR, at least one of the packaging region and of the IRES site consisting of said nucleotide sequence in use according to one of claims 1 to 7.

13. The retroviral vector according to claim 12, in which the internal promoter region, the second gene of interest, the IRES site and the third gene of interest are in a reverse orientation with respect to the retroviral 5 'and 3' LTRs.

14. The retroviral vector according to claim 12 or 13, in which the packaging region derives from a murine retrovirus, in particular from a MoMLV, or from a VL30 type retrotransposon and the IRES site comprises a nucleotide sequence as defined. to claim 6.

15. The retroviral vector of claim 14, wherein the region packaging derived from a MoMLV and the IRES site includes a nucleotide sequence identical to the sequence presented in the sequence identifier SEQ ID NO: 2 starting at nucleotide 265 and ending at nucleotide 578 or starting at nucleotide 452 and ending at nucleotide 578.

16. The retroviral vector according to claim 10, comprising a 5 ′ retroviral LTR derived from a REV virus, in particular SNV, a 3 ′ retroviral LTR of any origin, one or more gene (s) of interest, and a sequence nucleotide substantially homologous or identical to the sequence presented in the sequence identifier SEQ ID NO: 2 starting at nucleotide 1 and ending at nucleotide 578, or starting at nucleotide 265 and ending at nucleotide 578, as an encapsidation region .

17. A vector according to one of claims 8 to 16, comprising a gene of interest encoding an expression product selected from factor VIII, factor IX, the CFTR protein, dystrophin, insulin, the interferon alpha, beta, gamma, an interleukin (IL) including TIL-2 and a selection marker.

18. A viral particle generated from a viral vector according to one of claims 8 to 17.

19. A cell comprising a vector according to one of claims 8 to 17 or infected with a viral particle according to claim 18.

20. Use of a vector according to one of claims 8 to 17, of a viral particle according to claim 18 or of a cell according to claim 19 for the preparation of a pharmaceutical composition intended for the treatment and / or prevention of a disease treatable by gene therapy.

21. Use of a vector according to one of claims 8 to 17, of a viral particle according to claim 18 or of a cell according to claim 19 for the preparation of one or more polypeptides of interest by recombinant route or for the production of a transgenic animal.

22. A pharmaceutical composition comprising, as therapeutic or prophylactic agent, a vector according to one of claims 8 to 17, a viral particle according to claim 18, a cell according to claim 19 or a polypeptide of interest obtained according to l use according to claim 21, in combination with a pharmaceutically acceptable vehicle.

23. A pharmaceutical composition according to claim 22, characterized in that it comprises between 10 ⁴ and 10 ′ ⁴ pfu, and preferably between 10 ⁶ and 10 ″ pfu viral particles according to claim 18.

24. Use of a vector according to one of claims 8 to 17, of a viral particle according to claim 18 or of a pharmaceutical composition according to claim 22 or 23, for the transfection or infection of pluripotent cells, in particular pluripotent cells of the central nervous system.