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WO2000071578A2 - Nouveaux polypeptides et leur utilisation pour le sauvetage de virus ou de glycoproteines virales a defaut de fusion - Google Patents

Nouveaux polypeptides et leur utilisation pour le sauvetage de virus ou de glycoproteines virales a defaut de fusion Download PDF

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WO2000071578A2
WO2000071578A2 PCT/EP2000/004534 EP0004534W WO0071578A2 WO 2000071578 A2 WO2000071578 A2 WO 2000071578A2 EP 0004534 W EP0004534 W EP 0004534W WO 0071578 A2 WO0071578 A2 WO 0071578A2
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rbd
fusion
envelope glycoprotein
retroviruses
envelope
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WO2000071578A3 (fr
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François-Loïc Cosset
Dimitri Lavillette
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Centre National de la Recherche Scientifique CNRS
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045—Special targeting system for viral vectors
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

  • the invention relates to new polypeptides, and their use for the rescue of fusion defective virus or retrovirus glycoproteins.
  • fusion peptide a conformational change of the glycoprotein structure necessary to expose a "fusion peptide” , buried within the native envelope complex and involved in the actual membrane fusion process.
  • MLV murine leukemia virus
  • transmembrane subunit contains the "fusion machinery" composed of an amino-terminal fusion peptide associated to a coiled-coil structure that rearrange upon receptor interaction.
  • Retroviruses can be involved in two different types of membrane fusion : - a fusion between viral and cell membranes triggered by the interaction of the virion envelope glycoproteins with specific cell surface molecules, which are the viral receptors (virus-cell fusion), with said fusion being responsible for retroviral infection, - a fusion between two cell membranes triggered by the interaction of the envelope glycoproteins expressed at the surface of a cell and the viral receptors expressed on a neighbouring cell (cell-cell fusion), the visible outcome of this latter being the formation of cell syncytia containing up to 100 nuclei (also known as polykaryocytes or multinucleated giant cells); syncytium-formation results in the death of the cells which make up the syncytium.
  • a vector directing the expression on a eukaryotic cell surface of a syncytium-inducing polypeptide can be used in gene therapy of malignant diseases, as a result of selective elimination of unwanted cells, through syncytia formation (WO 98/40492).
  • the fusogenicity of envelope glycoproteins has been enhanced as described in Lavillette et al. (1998) and internal application WO 99/36561.
  • the proline-rich region (PRR) of amphotropic MLV envelope glycoprotein was shown to be an essential determinant of membrane fusion activation and specific PRR-mutated envelope glycoproteins were obtained and were characterised by an increased cell-cell or virus-cell fusogenicity.
  • H8 a particularly important residue belongs to the PHQ motif which is conserved among all MLVs and some other retroviruses, such as a gibbon ape leukemia virus SEATO and feline leukemia virus types A, B, and C.
  • the amphotropic counterpart of Del2-8, which has the first six amino acids (Met-Ala-Glu-Ser-Pro-His) deleted has been constructed. Vectors bearing the deletion mutant envelope protein could not transduce NIH 3T3 or HeLa cells (Bae et al. , Journal of Virology, March 1997, p. 2092-2099).
  • One of the aims of the invention is to enable fusion control of envelope glycoproteins with target cells.
  • the invention lies in the possibility of desactivating the inactivation of the fusogenic property of an envelope glycoprotein, said envelope glycoprotein having previously been made inactive with respect to fusion.
  • This restoration of the fusion property of the envelope glycoprotein is unexpectedly achieved by polypeptides whose common structure is the presence of the receptor binding domain (RBD) and the amino terminal part of SU in the environment of cells expressing fusion defective envelope glycoproteins and of cells expressing retroviral or viral receptors and the interaction of which will lead to the formation of syncytia, or in the environment of retroviruses incorporating fusion defective envelope glycoproteins and of cells expressing retroviral or viral receptors and the interaction of which will lead to infection.
  • RBD receptor binding domain
  • polypeptide comprising an amino acid sequence corresponding to means that the polypeptide has the same amino sequence as that of the surface subunit, or as that of an aminoacid sequence derived from the surface subunit or of a fragment and can be prepared by recombinant technique or by chemical synthesis.
  • polypeptide comprising an amino acid sequence derived from said surface subunit means that the polypeptide has an aminoacid sequence which is not strictly identical to that of the sequence of the surface subunits and may contain mutations such as deletion or substitution of aminoacids of the parental sequence or insertion of aminoacid sequence of other origins.
  • the "amino terminal part of the surface subunit” is defined as the aminoacid sequence of the first aminoacids of a length of about 10 to about 15 aminoacids of the envelope surface subunit, not including the aminoacid sequence of the signal peptide.
  • a fusion defective envelope glycoprotein derives from a parental envelope glycoprotein in which the fusogenic property has been inactivated, but which still presents the property of recognizing (attachment) target cells.
  • the expression "inactive with respect to the fusion” means that in a test for the measure of the fusogenic activity, the fusion index is either nul or significantly reduced when the fusion assay is allowed to proceed for about 20 minutes.
  • the rescue of the fusion property can be measured according to one of the following tests :
  • syncytia expressing vectors encoding the envelope glycoprotein to be tested are transfected into cells on day 0. On day 2, the transfected cells are overlaid by indicator cells which express the cell surface receptors corresponding to the tested envelope glycoprotein. Detection of syncytia (i.e. fusion of envelope-expressing cells with neighbouring indicator cells) is performed at day 3 or day 4 and fusion indexes are measured as previously described (Lavillette et al, 1998) by counting the number of syncytia and the number of nuclei outside and inside the syncytia.
  • retroviral vectors carrying a lac Z transgene are being generated with the tested envelope glycoproteins. Serial dilutions of the vectors are applied on target cells at day 0. Infected cells are stained at day 2 with X-Gal to reveal the number of cell colonies having integrated the lac Z-vectors and titers are determined according to standard procedures (Lavillette at al., 1998).
  • the activating polypeptide corresponds to the amino acid sequence of the SU of an envelope glycoprotein of a first virus or retrovirus and that the fusion defective envelope glycoprotein belongs to a second virus or retrovirus of the same type or of a type different from the first virus or retrovirus. This means that there are in fact two possibilities :
  • either the first and second viruses are of the same type, which means that the activating polypeptide and the fusion defective envelope glycoprotein are homologous,
  • the fusion defective envelope glycoprotein involved are those derived from retroviruses.
  • Retroviral envelope glycoproteins allow receptor binding of the viral particles onto which they have been incorporated during virion assembly. This preliminary attachment on the viral receptor activates a second function of the retroviral envelope glycoproteins which induces the fusion between the viral and the target cell membranes. While for type C mammalian retroviruses the determinants of receptor binding (RBD, receptor binding domain), are located at the amino-terminal end of the SU envelope subunit, the molecular determinants that are responsible of membrane fusion are located in the TM subunit of the glycoprotein.
  • RBD receptor binding domain
  • the molecular determinants or events which are involved in the conversion of binding to the receptor into a signal which activates the TM fusion dete ⁇ ninants are not precisely known although by mutagenesis analysis, they are believed to be present in all the SU subunit, in particular in the N-terminal end of the RBD itself (Bae et al. , 1997; Lavillette et al. , 2000), in the proline-rich region (Lavillette et al. , 1998) and in the carboxy-terminal domain of the SU (Nussbaum et al., 1993; Pinter et al. , 1997).
  • the control of the activation of the fusion machinery harbored by the TM subunit might be achieved by introducing specific mutations that affect the fusion-activation determinant of the SU.
  • This invention describes several mutations in the SU which can inhibit or partially inhibit the SU determinants which control activation of the TM fusion-machinery. It is important to understand that such mutations do not impair the fusogenic potential of the mutant envelope glycoproteins, but only impair the molecular determinants that control the activation of the fusion machinery. The latter one is still intact, despite the mutations introduced in the SU, but is not activated upon receptor binding.
  • the invention describes the use of soluble polypeptides that can alleviate the effect of these mutations and hence couple the fusion machinery to a device that is provided exogenously in substitution to the normal fusion control determinants which are disrupted in the mutant envelope glycoproteins.
  • the addition of the "activating" polypeptides can restore or rescue the fusogenicity of the mutant envelope glycoproteins.
  • envelope glycoprotein Several mutations or modifications of the envelope glycoprotein can be used to reversibly inhibit or reduce their fusogenicity.
  • a first method is the introduction of the delH mutation that removes an histidine from the PHQ conserved motif located at the amino-terminal end of the SU envelope subunit of type C mammalian retroviruses such as MLV-A, MoMLV and GALV (mutations H5del, H8del or H9del, respectively).
  • Such a mutation induces a drastic effect in the capacity of the receptor binding domain (RBD) of these envelope glycoproteins to triggers further events required to promote membrane fusion.
  • RBD receptor binding domain
  • a second method consists in the replacement of the SU C-terminal domain (C domain) with an heterologous C domain derived from the envelope glycoprotein of another type C mammalian retrovirus.
  • the chimeric CMO amphotropic glycoprotein (hereafter defined) can be constructed by combining the amphotropic MLV RBD and PRR (proline-rich region) with the ecotropic MoMLV C-terminal domain (example 4, Fig. 29 A and 29B).
  • RBD amphotropic origin
  • C domain ecotropic origin
  • a third method consists in the replacement of the RBD by a ligand such as a cytokine or a single chain antibody targeted to a cell surface specific receptor.
  • a ligand such as a cytokine or a single chain antibody targeted to a cell surface specific receptor.
  • a ligand such as a cytokine or a single chain antibody targeted to a cell surface specific receptor.
  • ligands do not contain the determinants that allow further activation of the Env fusogenicity.
  • Such chimeric envelope glycoproteins therefore resembles a delH envelope glycoprotein which would carry an RBD that is targeted to a specific receptor.
  • This invention relates to a method which can alleviate the inhibitory effect of these different mutations and thus restore the fusogenic properties of the mutant envelope glycoproteins.
  • This method uses preparations of polypeptides which are derived from the RBD of different type C mammalian retrovirus envelope glycoproteins. For example, the addition of an RBD which is compatible with the C domain of a fusion-defective envelope glycoprotein will fully restore its fusogenic properties and will activate the kinetic of membrane fusion. Similarly such a peptide will also restore the fusogenicity of envelope glycoproteins that harbor a delH mutation or, alternatively, which carry a cell-type specific ligand in place of the RBD.
  • This method can be applied to the activation of infectivity of retroviral vectors whose envelope glycoproteins carry one of these fusion-defective mutations. Depending on the presence of a ligand in the fusion-deficient envelope glycoprotein or, alternatively, in the activating polypeptide, control of infection to specific target cell types will therefore be obtained.
  • the invention can also be applied to the control of the hyper-fusogenic activity of retroviral envelope glycoproteins that can induce cell-cell fusion and elimination of unwanted cells by syncytia formation. Indeed while most retroviral envelope glycoproteins are not truly fusogenic when expressed at the surface of the cells that produce the retroviral particles, they acquire their fusion competency during or after assembly and release of the viral particles.
  • the fusogenicity of retroviral envelope glycoproteins can be stimulated by several ways to obtain the best effect upon activation with an exogenous activating polypeptide.
  • additional mutations will be introduced into the retroviral envelope glycoprotein, such as the truncation of the cytoplasmic tail (Rless mutation) and/or the modification of their proline-rich regions.
  • the activating polypeptide used in the invention recognizes substantially the same receptors of the target cells as the fusion defective envelope glycoprotein.
  • the activating polypeptide used in the invention recognizes receptors of the target cells which are different from the ones recognized by the fusion defective envelope glycoprotein.
  • the activating polypeptides can restore the fusogenicity of fusion defective envelope glycoproteins by interacting with retroviral receptors which are expressed at the surface of the same target cells, and which are different from the receptors recognized by the fusion defective envelope glycoprotein.
  • the activating polypeptide and/or the fusion defective envelope glycoprotein is (are) linked to a ligand, which is specific with respect to some receptors of the target cells, the ligand being preferably linked to the activating polypeptide.
  • a ligand which is specific with respect to some receptors of the target cells
  • the ligand being preferably linked to the activating polypeptide.
  • the design of a specific system to eliminate unwanted cells may consist in a fusion defective envelope glycoprotein associated to an activating polypeptide that is linked to a ligand that can specifically recognize a receptor expressed on the target cells, whereas the activating polypeptide recognizes the same receptor or a different receptor expressed on the target cell.
  • the link between the activating polypeptide and the ligand is preferably a covalent bond, whereby both the activating polypeptide and the ligand have been genetically fused and encoded by an expression vector as a single protein chain, and whereby the ligand is fused either to the amino-terminus or to the carboxy terminus of the activating polypeptide.
  • the ligand is chosen among molecules liable to recognize receptors of target cells, for instance receptors of tumoral cells, such as EGF, FGF, VEGF, or liable to recognize epitope of proteins expressed by the target cells, such as recombinant monoclonal antibody.
  • the activating polypeptide is under the form of a product as such, or is expressed by a host, such as a virus or a cell, or is expressed, in particular by the target cells, transformed by nucleotide sequences coding for said activating polypeptide.
  • soluble activating polypeptides will be sometimes used, meaning that said polypeptides are not integrated in the envelope proteins of a virus.
  • the capacity of the activating polypeptides to rescue the infectivity of fusion defective envelope glycoproteins is similar whether the target cells i) constitutively express and secrete the activating polypeptides, ii) are co-incubated with both fusion defective envelope glycoprotein and said activating polypeptides iii) are pre-incubated with said activating polypeptides before adding the fusion defective envelope glycoprotein, or iv) are pre-incubated with the fusion defective envelope glycoprotein before adding the activating polypeptides.
  • the fusion defective envelope glycoprotein is expressed by a host, such as a virus incorporating in its envelope said fusion defective envelope glycoprotein, or such as a cell, transformed by nucleotide sequences coding for said fusion defective envelope glycoprotein.
  • the activating polypeptide advantageously corresponds to a fragment of about 200 to about 250 amino acids of said surface subunit comprising the receptor binding domain (RBD) and comprising the first amino acids of the amino terminal part of the surface subunit, the number of said first aminoacids varying from about 10 to about 15, in particular comprising the RBD and the PHQ peptide, in particular the fragments corresponding to amino acid sequences derived from type C mammalian retroviruses such as: - Murine leukemia virus such as :
  • EGF-GALV-RBD - A-RBD corresponds to the activating polypeptide comprising the RBD domain and the
  • - E-RBD corresponds to the activating polypeptide comprising the RBD domain and the PHQV motif of the ecotropic MLV surface subunit.
  • - GALV-RBD corresponds to the activating polypeptide comprising the RBD domain and the PHQP motif of the GALV surface subunit.
  • - 34.1-A-RBD corresponds to the activating polypeptide A-RBD above defined, linked to a ligand which is a single chain antibody directed against MHCI.
  • - 34.1 -E-RBD corresponds to the activating polypeptide E-RBD above defined, linked to a ligand which is a single chain antibody directed against MHCI.
  • - 34.1 GALV-RBD corresponds to the activating polypeptide GALV-RBD above defined, linked to a ligand which is a single chain antibody directed against MHCI.
  • EGF-A-RBD corresponds to the activating polypeptide A-RBD above defined, linked to a ligand which is the epidermal growth factor.
  • EFG-E-RBD corresponds to the activating polypeptide E-RBD above defined, linked to a ligand which is the epidermal growth factor.
  • the fusion defective envelope glycoprotein advantageously corresponds to the amino acid sequence of a mutated form of an envelope glycoprotein of a virus or a retrovirus which, in its non mutated form, presents attachment and fusion properties with respect to target cells, said fusion defective envelope glycoprotein comprising one or several mutations in the amino terminal part of its surface subunit, particularly in the first amino acids of the amino terminal part of the surface subunit, the number of said first aminoacids varying from about 10 to about 15, said mutations inactivating the fusion property without substantially affecting the attachment property, in particular comprises one or several mutations within the PHQ amino sequence, wherein in particular the fusion defective envelope glycoprotein is derived from type C mammalian retroviruses such as:
  • - Murine leukemia virus such as :
  • fusion defective envelope glycoprotein corresponds to one of the following amino acid sequences :
  • MO del H corresponds to the ecotropic MLV envelope glycoprotein, in which the histidine residue of the PHQV motif is deleted (i.e. the 8th residue of the SU subunit is removed).
  • AdelH corresponds to the amphotropic MLV envelope glycoprotein, in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed).
  • PROMOdelH corresponds to the amphotropic MLV envelope glycoprotein, in which the proline rich region has been replaced by the proline rich region of the ecotropic MLV envelope and in which the histidine residue of the PHQV motif is deleted.
  • GALVdelH corresponds to the GALV envelope glycoprotein, (gibbon ape leukemia virus envelope glycoprotein), in which the histidine residue of the PHQP motif is deleted (i.e. the 9th residue of the SU subunit is removed).
  • C2delH corresponds to the amphotropic MLV envelope glycoprotein, in which the proline rich region has been modified by a small deletion at its carboxy terminus and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed) .
  • MO del H R less corresponds to the ecotropic MLV envelope glycoprotein, in which the histidine residue of the PHQV motif is deleted and in which the R peptide of the TM subunit has been deleted.
  • AdelHRless corresponds to the amphotropic MLV envelope glycoprotein in which the histidine residue of the PHQV motif is deleted and in which the R peptide of the TM subunit has been deleted.
  • PROMOdelHRless corresponds to the amphotropic MLV envelope glycoprotein, in which the proline rich region has been replaced by the proline rich region of the ecotropic MLV envelope, in which the histidine residue of the PHQV motif is deleted and in which the R peptide of the TM subunit has been deleted.
  • GALVdelHRless corresponds to the GALV envelope glycoprotein, in which the histidine residue of the PHQP motif is deleted and in which the R peptide of the TM subunit has been deleted.
  • C2delHRless corresponds to the amphotropic MLV envelope glycoprotein, in which the proline rich region has been modified by a small deletion at its carboxy terminus and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed) and in which the R peptide of the TM subunit has been deleted.
  • CMO envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the SU carboxy-terminal domain, C domain, has been replaced by the C domain derived from the ecotropic MoMLV envelope glycoprotein.
  • CMOdelH envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the SU carboxy-terminal domain, C domain, has been replaced by the C domain derived from the ecotropic MoMLV envelope glycoprotein and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed).
  • TMMO envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the ectodomain of the TM subunit has been replaced by the TM ectodomain derived from the ecotropic MoMLV envelope glycoprotein.
  • TMMOdelH envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the ectodomain of the TM subunit has been replaced by the TM ectodomain derived from the ecotropic MoMLV envelope glycoprotein and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed).
  • CTMMO envelope corresponds to the amphotropic MLV envelope glycoprotein, in which both the SU carboxy-terminal domain, C domain, and the ectodomain of the TM subunit have been replaced by the C domain and TM ectodomain derived from the ecotropic MoMLV envelope glycoprotein.
  • CTMMOdelH envelope corresponds to the amphotropic MLV envelope glycoprotein, in which both the SU carboxy-terminal domain, C domain, and the ectodomain of the TM subunit have been replaced by the C domain and TM ectodomain derived from the ecotropic MoMLV envelope glycoprotein and in which the histidine residue of the PHQV motif is deleted (i.e. the 5th residue of the SU subunit is removed).
  • * 34.1-A envelope corresponds to the amphotropic MLV envelope glycoprotein, in which the receptor binding domain has been removed and replaced by an single chain antibody against human class I MHC molecules.
  • the invention also relates to the use of at least one activating polypeptide and of at least one fusion defective envelope glycoprotein for the preparation of a drug useful for the treatment of cancer and infectious diseases.
  • the invention also relates to products containing an activating polypeptide and a fusion defective envelope glycoprotein as defined above, as a combined preparation for simultaneous, separate, or sequential use for the rescue of the fusion defective property of said fusion defective envelope glycoprotein.
  • the invention also relates to products containing an activating polypeptide and a fusion defective envelope glycoprotein as defined above, as a combined preparation for simultaneous, separate or sequential use for the treatment of cancer pathologies.
  • the invention also relates, as new products, to activating polypeptides corresponding to a fragment of about 200 to about 250 amino acids of said surface subunit comprising the RBD and the PHQV peptide, in particular the fragments corresponding to amino acid sequences derived from type C mammalian retroviruses such as: - Murine leukemia virus such as : . amphotropic
  • the invention also relates, as new products, to fusion defective envelope glycoprotein to the amino sequence of an envelope glycoprotein of a virus or a retrovirus which in its native state presents attachment and fusion properties with respect to target cells, and comprises one or several mutations in the .imino terminal part of its surface subunit, inactivating the fusion property without substantially affecting the attachment property, in particular comprises one or several mutations within the PHQ amino sequence of the amino terminal part of its surface subunit, wherein in particular the fusion defective envelope glycoprotein is derived
  • - Murine leukemia virus such as :
  • fusion defective envelope glycoprotein corresponds to one of the following amino acid sequences :
  • the invention also relates to the use of at least one activating polypeptide to the rescue of the fusion property of a fusion defective envelope glycoprotein wherein the fusion defective envelope glycoprotein corresponds to the amino acid sequence of a mutated form of an envelope glycoprotein of a virus or a retrovirus which, in its non mutated form, presents attachment and fusion properties with respect to target cells, said fusion defective envelope glycoprotein being advantagously derived from type C mammalian retroviruses such as: - Murine leukemia virus such as :
  • retroviruses and related retrovirus such as SSAV, FELV-A, FELV- B, FELV-C and comprising :
  • the fusion defective envelope glycoprotein corresponds to the following amino-acid sequence :
  • Fig 1A, IB and 1C represent titres of AdelH retroviruses in the presence of soluble envelope fragments of the invention.
  • Retroviral vectors carrying VSV-G or/and wild-type amphotropic, A, envelope glycoproteins were used as controls.
  • the titres are on the y-axis.
  • Fig 1A Different target cell types (XC, NIH3T3, CHO-PIT-2, TE 671, from left to right) were incubated with both lacZ retroviruses and conditioned media containing, or not (control), the indicated polypeptides during the 3 hrs of infection.
  • the black columns correspond to the control
  • the grey columns correspond to A-RBD
  • the hatched columns correspond to SU polypeptide.
  • Fig IB XC target cells were incubated with dilutions of A-RBD (x axis) and infected with LacZ retroviruses carrying the AdelH envelope glycoproteins (curve with black circles), A envelope glycoproteins (curve with black squares) or VSV-G envelope glycoproteins (curve with hollow squares).
  • Retroviral vectors carrying A envelope glycoproteins (curves with black squares) or AdelH envelope glycoproteins (curves with black circles) were incubated with XC target cells for 1 hr at 4°C to allow virion binding while preventing cell entry. After PBS washing to remove unbound retroviruses, cells were incubated at 37 °C with undiluted A-RBD- containing supernatants for the indicated times of dilution represented on the x axis, the titres being expressed on the y axis. A-RBD was then eliminated from the cell supernatant by washing the cells four times with 1 ml PBS (resulting in dilution of unbound A-RBD by more than 100,000 times). Cells were then grown in regular medium for 2 days before X- Gal staining.
  • Fig 2. represents the titers of A-RBD-mediated activation of AdelH retroviruses.
  • RBD (used undiluted) was bound to XC target cells at 37 °C for 30 min. Unbound A-RBD was removed and cells were washed two times with PBS and incubated at 37°C. At the indicated times, cells were further washed two times with PBS and incubated at 37 °C for 3 hrs with retroviruses carrying A envelope glycoproteins or AdelH envelope glycoproteins. Titers, determined 2 days later by X-Gal staining, are expressed as lacZ i.u./ml (y-axis), as a function of time before infection (x-axis). The curve with black circles corresponds to retrovirus carrying A envelope glycoproteins and the curve with black squares corresponds to retrovirus carrying AdelH envelope glycoproteins.
  • Fig 3. represents titers of AdelH retroviruses after ultrafiltration. Titers expressed on the y-axis (as lacZ i.uJml) of retroviruses carrying wild-type, A, or AdelH envelope glycoproteins before (1) or after incubation with A-RBD (2), and following A-RBD elution on two successive 700 KD-cut off ultrafiltration cartridges (3). After elution AdelH retroviruses were subjected to re-stimulation with fresh A-RBD (4).
  • Fig. 4 and 4B represent infection assays of AdelH and MOdelH retroviruses in the presence of RBD or RBDdelH polypeptides.
  • FIG. 4A Retroviruses carrying A (upper left) or AdelH (upper right) envelope glycoproteins were mixed with A-RBD (hatched column) or A-RBDdelH (grey column) during infection of XC cells. Retroviruses carrying MO (lower left) or MOdelH (lower right) envelope glycoproteins were mixed with E-RBD (hatched column) or E-RBDdelH
  • Fig. 4B represents binding assays of A-RBD (solid lines) and A-RBDdelH fragments (broken lines) on Cearl3 (top), and binding assays of E-RBD (solid lines) and E-RBDdelH fragments (broken lines) on Cearl3 cells (bottom).
  • the background of fluorescence was provided by incubating the cells with supernatants devoid of envelope fragments.
  • Fig 5A and 5B represent the cross-activation of AdelH and MOdelH retroviruses by MLV RBDs.
  • Fig. 5A Titers of retroviruses carrying AdelH or MOdelH envelope glycoproteins in the presence of the indicated RBDs. Titers are determined (y-axis) on XC target cells as lacZ i.uJml. On figure 5A, the black column corresponds to control, the hatched column corresponds to A-RBD and the grey column corresponds to E-RBD.
  • Fig. 5B Titers of AdelH retroviruses (y-axis) on CHO cells expressing, or not (control),
  • PiT-2 and/or mCAT-1 MLV receptors in the presence of the indicated RBDs.
  • the hatched column corresponds to E-RBD
  • the grey column corresponds to A-RBD
  • the black column corresponds to control.
  • Fig. 6 represents the A-RBD sequence
  • Fig. 7 represents the E-RBD sequence
  • Fig. 8 represents the GALV-RBD sequence
  • Fig. 9 represents the EGF-A-RBD sequence
  • Fig. 10 represents the EGF-E-RBD sequence
  • Fig. 11 represents the EGF-GALV-RBD sequence
  • Fig. 12 represents the 34.1-A-RBD sequence
  • Fig. 13 represents the 34.1-E-RBD sequence
  • Fig. 14 represents the 34.1-GALV-RBD sequence
  • Fig. 15 represents the AdelH sequence
  • Fig. 16 represents the PROMOdelH sequence
  • Fig. 17 represents the GALVdelH sequence
  • Fig. 18 represents the C2delH sequence
  • Fig. 19 represents the AdelHRless sequence
  • Fig. 20 represents the PROMOdelHRless sequence
  • Fig. 21 represents the GALVdelHRless sequence
  • Fig. 22 represents the C2delHRless sequence
  • Fig. 23 represents the C MO sequence
  • Fig. 24 represents the C MO delH sequence
  • Fig. 25 represents the TM MO sequence
  • Fig. 26 represents the TM MOdelH sequence
  • Fig. 27 represents the C TM MO sequence
  • Fig. 28 represents the C TM MO delH sequence
  • Fig. 29A represents the schematic structure of chimeric amphotropic envelope glycoproteins in which the C domain (CMO chimera), the TM ectodomain (TMMO chimera) or both C and TM domains (CTMMO chimera) are replaced by the ecotropic counterpart derived from MoMLV envelope glycoprotein.
  • CMO chimera C domain
  • TMMO chimera TM ectodomain
  • CTMMO chimera both C and TM domains
  • Figure 29B represents the kinetic of infection of retroviral vectors carrying the wild-type amphotropic envelope glycoprotein (A) or the CTMMO amphotropic chimera on XC cells in the presence, or not, of soluble polypeptides encompassing the amphotropic RBD (A- RBD) or the ecotropic RBD (E-RBD), as indicated.
  • A- RBD amphotropic envelope glycoprotein
  • E-RBD ecotropic RBD
  • the curve with points and black circles corresponds CTMMO on XC cells + E-RBD
  • the curve with points and hollow circles corresponds to A on XC cells + E-RBD
  • the curve with continuous line and hollow circles corresponds to A on XC cells
  • the curve with dotted line and hollow circles corresponds to A on XC cells + A-RBD
  • the curve with continuous line and black circles corresponds to CTMMO on XC cells
  • the curve with dotted line and black circles corresponds to CTMMO on XC cells + A- RBD
  • the X axis corresponds to the time expressed in seconds.
  • the Y axis corresponds to the number of infected cells.
  • Fig. 30 represents the 34.1-A Sequence
  • the TELCeB ⁇ cell line (Cosset et al., 1995b) has been derived from the TELac2 line after transfection and clonal selection of a Moloney murine leukemia virus (MoMLV)- based expression plasmid to produce Gag and Pol proteins.
  • MoMLV Moloney murine leukemia virus
  • the TELac2 cells were originally derived from the TE671 human rhabdomyosarcoma cells (ATCC CRL8805) to express the nlsLacZ reporter retroviral vector (Takeuchi et al., 1994). Production of infectious retroviral particles by TELCeB ⁇ cells depends on newly introduced envelope expression vectors.
  • Cerd9 and Cearl3 cells were derived from CHO (Chinese hamster ovary) cells (ATCC CCL-61) and express ecotropic MLV receptors alone or both ecotropic and amphotropic receptors or amphotropic MLV receptors alone, respectively.
  • XC-A-RBD cells were derived from XC rat sarcoma cells (ATCC CCL-165) after transfection with the pA-ST plasmid expressing the amino terminal receptor binding domain of the amphotropic envelope glycoprotein (Battini et al., 1996).
  • TE671 and TELCeB ⁇ cells were grown in DMEM (Life-Technologies) supplemented with 10% fetal bovine serum.
  • NIH-3T3 mouse fibroblasts were grown in DMEM supplemented with 10% new-born calf serum.
  • Cerd9, Cearl3, CHO-PiT-2 and CHO cells were grown in DMEM supplemented with 10% fetal bovine serum and with proline (Life-Technologies).
  • Plasmids FBASALF and FBMOSALF carrying a phleomycin-resistance gene and encoding the MLV-4070A amphotropic (noted as A) and MoMLV ecotropic (noted as MO) envelope glycoproteins, respectively, have been described elsewhere (Cosset et al. , 1995a) and were used as backbones for construction and expression of envelope mutants. All constructs were generated by PCR-mediated and ohgonucleotide site-directed mutagenesis (details and sequences available upon request) .
  • the FBASALF plasmid was modified to produce a cell entry-defective form of the amphotropic glycoprotein, designated AdelH envelope, by deleting the 36th codon of the 4070A env gene (Ott et al. , 1990).
  • Plasmids encoding soluble receptor binding domains were derived from FBASALF and FBMOSALF expression vectors.
  • Residues are numbered starting from the initiation methionine deduced from the aminoacid sequences of the 4070A amphotropic MLV (Ott et al. , 1990) or the Moloney MLV (Shinnick et al. , 1981).
  • Expression vectors encoding either A-RBDdelH or E-RBDdelH were generated similarly by using the plasmids expressing the AdelH or MOdelH envelope glycoproteins.
  • plasmids encoding A-RBD, E-RBD, A-RBDdelH or E- RBDdelH polypeptides the 11 aminoacid-long VSV tag (YTDIEMNRLGK) (Kreis and Lodish, 1986) was inserted after either RBDs.
  • Envelope glycoproteins expression plasmids were transfected into TELCeB ⁇ as reported elsewhere (Cosset et al. , 1995a).
  • RBD expression vectors were transfected by calcium phosphate precipitation in NIH-3T3, XC or TE671 cells. Transfected cells were selected with phleomycin (50 ⁇ g/ml) and phleomycin-resistant colonies were pooled.
  • RBD-containing supernatants were collected after an overnight production from confluent RBD-transfected cells, filtrated trough 0.45 micron pore-sized membranes and stored at 4°C.
  • Virus-containing supernatants were collected after an overnight production from freshly confluent env-transfected TELCeB ⁇ cells in regular medium.
  • the supernatant were filtered trough 0.45 micron pore-sized membranes and diluted in DMEM for titration assays.
  • Target cells were seeded in 24 multi-well plates at a density of 5xl0 4 cells per well and incubated overnight at 37°C. Unless otherwise indicated in figure legends, 200 ⁇ l of conditioned cell culture media containing the RBDs were added to the cells after removing their supernatants. Then 200 ⁇ l of viral supernatant dilutions containing 5 ⁇ g/ml polybrene were added and cells were incubated for 3 hrs at 37 °C. Cell supernatants were then removed and cells were incubated in regular medium for 48 hrs. X-Gal staining and viral titer determination were performed as previously described (Cosset et al. , 1995a) and expressed as lacZ i.u. (infectious units)/ml.
  • Antibodies a rat monoclonal antibody 83A25 (Evans et al. , 1990) cell culture supernatant against MLV SU used undiluted for FACS analysis.
  • Anti- VSV tag a purified mouse monoclonal antibody P5D4 (SIGMA-Aldrich) used diluted 1/100 for FACS analysis.
  • Binding assays and FACS analysis For binding assays, target cells were washed in PBS and detached by a 10 min incubation at 37°C with versene 0.02% in PBS. Cells were washed in PBA (PBS with 2% FCS and 0.1 % sodium azide). 5xl0 5 cells were incubated with 1 ml of conditioned supernatants containing the VSV tagged RBDs for 45 min at 37 °C. Cells were then washed with PBA and were incubated with the P5D4 antibody, for 45 min at 4°C. Cells were washed twice with PBA and incubated with anti-mouse FITC-conjugated antibodies (DAKO, U.K.), respectively.
  • DAKO anti-mouse FITC-conjugated antibodies
  • H5del phenotype can be efficiently compensated by soluble A-RBD.
  • Viral particles were generated with either wild-type amphotropic A or AdelH envelope glycoproteins and used to infect target cells in the presence or in the absence of soluble SU or soluble A-RBD, an envelope fragment encompassing the amphotropic receptor binding domain.
  • SU or A- RBD were provided during infection the infectivity of retroviruses carrying wild-type amphotropic envelope glycoproteins was decreased by approximately 5-100 times (Fig. 1A), most likely owing to the partial blocking of PiT-2 receptors on the target cell surface.
  • the capacity of the soluble envelope fragments to rescue the infectivity of AdelH retroviruses was similar whether the target cells i) constitutively expressed and secreted the envelope fragments, ii) were co-incubated with both AdelH retroviruses and A-RBD, iii) were pre-incubated with A-RBD prior to infection, or iv) were pre-incubated with the AdelH retroviruses before adding A-RBD (Table 1). Using this latter experimental condition, the kinetic of activation of infection was found very rapid. Indeed a brief incubation of target cells with A-RBD, by no more than 30 sec, was found sufficient to fully stimulate the infectivity of pre-bound AdelH retroviruses (Fig. 1C).
  • A-RBD stimulates the target cell membrane rather than the AdelH envelope.
  • the envelope fragments might rescue the infectivity of AdelH retroviruses either by interacting with the fusion-defective viral particles or by activating the target cell membrane upon receptor binding.
  • the SU subunits are not tightly attached to either the TM subunits or to the other SU units and can easily shed off the viral particles (Hunter and Swanstrom, 1990).
  • the reassociation of shed SU with the viral particle is unlikely since they are incorporated on virions by virtue of their association with the TM subunit that results from their synthesis as a common SU-TM polypeptide precursor (Hunter and Swanstrom, 1990). Therefore it is unlikely that soluble SU or A-RBD mixed with AdelH retroviruses may interact with the envelope complex of the latter retroviruses and thus rescue their fusion defect. Nevertheless the following experiments were performed to address this possibility.
  • A-RBD was bound to target cells at 37°C for 30 min. After removal of unbound fragments by washing target cells, binding of A-RBD was verified by FACS analysis. Cells were then incubated at 37 °C for 0 (TO) to 28 hrs (T28) to allow internalisation of receptor/A-RBD complexes (Rodrigues and Heard, 1999) and A-RBD disappearance from the cell surface, as checked by FACS analysis (Data not shown). Cells were then washed again to remove A- RBD that may have been released by the cells and the AdelH retroviruses were then added at the different time points.
  • A-RBD could not associate with the AdelH retroviruses. Indeed retroviruses generated with either A or AdelH envelopes and mixed or co-expressed with A-RBD were not found to incorporate A-RBD as shown by western-blotting of purified viral particles with antibodies recognising A-RBD (data not shown). However these experiments could not exclude that undetected quantities of A-RBD might be associated with the virions. Therefore to rule out this possibility, a mixture of A-RBD and retroviruses carrying either AdelH or wild-type A envelope glycoproteins were separated on two consecutive 700 KD-pore sized ultrafiltration columns in order to elute out A-RBD.
  • the histidine residue deleted in the H5del mutant glycoprotein belongs to a peptide motif, PHQ, found at the amino-termini of the SUs of all type C mammalian retroviruses, suggesting a conserved function of this motif.
  • PHQ peptide motif
  • the H8del mutant ecotropic envelope glycoprotein (MOdelH) harboring the deletion of the histidine in the PHQV peptide motif of the MoMLV SU, is impaired for both cell-cell and virus-cell fusion (Bae et al. , 1997).
  • the H9del mutation of the GALV envelope glycoprotein GAdelH mutant results in impairement of both cell-cell and virus-cell fusion.
  • delH mutations H5del, H8del, were introduced in the A- RBD or in the E-RBD fragments, respectively.
  • the resultant A-RBDdelH and E-RBDdelH could bind their respective cell surface receptors as efficiently as the parental envelope fragments (Fig. 4B). They could also decrease the infectivity of retroviruses bearing wild- type amphotropic or ecotropic glycoproteins, respectively, by up to 10-fold (Fig. 4A). This effect was most likely a consequence of partial receptor blocking.
  • receptor binding domain located in the amino-terminal half of the MLV SU may in fact be composed of two different entities, one involved in specific receptor binding and a second, likely to be non functional in AdelH or MOdelH envelopes, involved in transmission of a signal that may activate cell entry upon receptor binding.
  • This fragment could activate neither AdelH nor MOdelH retroviruses despite the presence of both amphotropic and ecotropic receptors on target cells (Fig. 5A).
  • Cross-activation of AdelH retroviruses could also be obtained by using soluble envelope glycoproteins from Gibbon Ape leukemia virus (data not shown), thus indicating that infection by delH retroviruses could be activated by a pathway common to type C mammalian retrovirus.
  • Target cells were pre-incubated for one hr at 4°C with conditioned supernatants containing A-RBD (used undiluted) or with lacZ retroviral vectors carrying AdelH envelope glycoproteins, as indicated.
  • b Target cells were incubated for 3 hrs at 37 °C.
  • c Titres as lacZ i.uJml determined 48 hrs post-infection.
  • d Both A-RBD (used undiluted) and AdelH retroviruses were added at the same time.
  • e XC cells constitutively expressing A-RBD polypeptides.
  • the transfer of therapeutic genes into tumour cells should lead both to direct local cell destruction and to activation of anti-tumour immunity to clear tumour deposits to which the genes cannot be delivered.
  • the most commonly used genes for the control of local tumour growth have been the suicide gene/prodrug systems such as Herpes Simplex Virus thymidine kinase (HSVtk)/Ganciclovir (GCV) or cytosine deaminase (CD)/5FC systems.
  • HSVtk Herpes Simplex Virus thymidine kinase
  • Ganciclovir Ganciclovir
  • CD cytosine deaminase
  • Table 2 shows the results of four FMGs derived from the non fusogenic ecotropic or amphotropic murine leukemia virus glycoproteins, whose fusogenicity were increased by deleting the p2R peptide (naturally removed during retrovirus egress) for the ARless and MoRless FMG and also by modifying the proline-riche region (Lavillette et al., 1998) for the C2Rless and PROMORless FMGs.
  • virus-cell fusion activity of delH MLV envelopes bearing the delH mutation can be efficiently rescued by using polypeptides encompassing the first 213 amino-acids of amphotropic MLV SU (A-RBD) or the first 236 amino-acids of ecotropic MoMLV SU (E-RBD) (example 1 and Lavillette et al. , 2000).
  • Example 2 the data in Table 2 demonstrated that the cell-cell fusion activity of the four FMGs engineered to harbor the delH mutation, AdelRless, C2delHRless, and PROMOdelHRless and MOdelHRless, could be rescued by providing either the A-RBD or E-RBD polypeptides thus leading to syncytia formation and elimination of target cells that express the relevant viral receptors.
  • formation of syncytia by delH FMGs could be achieved by using an heterologous polypeptide (E-RBD) that interacted with a receptor different of that recognised by the delH FMGs.
  • E-RBD heterologous polypeptide
  • fusion indexes (calculated according to Lavillette et al. , 1998) -: less than 100, +/-: 100-304, + : 300-1000, + + : 1000-10 000, + + + : 10 000-15 000, + + + + : > 15000
  • similar results of fusion activation with RBD polypeptides were obtained with the GALVdelHRless envelope glycoprotein, indicating that the delH mutation of the potent GALVRless FMG (Bateman et al. , 2000) can be controlled by adding a soluble Env- derived polypeptide (Lavillette and Cosset, unpublished results).
  • delH FMGs in cell elimination stategies.
  • viral vectors such as adenovirus vectors can be used to transduce both the delH FMG and the gene encoding the activating RBD polypeptide in cancer cells in vivo.
  • cells transduced in vitro or ex vivo by a vector expressing a delH fusion-defective FMG can be induced to form syncytia after inoculation of an RBD presented as a protein preparation.
  • delH FMG and RBD can be effectively delivered through cell-mediated mechanisms, which will also be useful as an option for in vivo delivery of FMG into tumours for both local tumour eradication as well as enhancement of anti tumour vaccination strategies.
  • a ligand was inserted at the amino-terminal end of the RBD.
  • the anti-MHC-I 34.1 scFv was chosen as we previously demonstrated that it could redirect the host-range of retroviral vectors carrying 34.1 -ecotropic MLV chimeric envelope glycoproteins and allow specific infection of human cells (Marin et al. 1996). Therefore the 34.1 scFV was fused to A-RBD or to E- RBD and preparation of 34.1-A-RBD or 34.1-E-RBD polypeptides were obtained.
  • Retroviral vectors carrying wild-type envelope glycoproteins, A, or AdelH fusion-defective amphotropic envelope glycoproteins were used to infect target cells in the presence of A- RBD, E-RBD, 34.1-E-RBD or 34.1-A-RBD polypeptides (Table 3). While either polypeptides could only weakly decrease the infectivity of parental retroviruses, they could strongly stimulate the infectivity of AdelH retrovirus. No significant differences could be detected when comparing the A-RBD, the E-RBD, the 34.1-E-RBD or the 34.1-A-RBD polypeptides in activation of AdelH retrovirus infectivity (Table 3). Table 3. Modulation of virus-cell fusion by ligand-displaying envelope fragments
  • the H5del mutation was introduced into the CMO, the TMO and the CTMMO MLV amphotropic envelope glycoprotein (Lavillette et al., in preparation), derived from the MLV 4070A envelope glycoprotein by substitution of the SU carboxy-terminal domain (C domain) and/or TM ectodomain with those derived from the MLV ecotropic envelope glycoprotein (Figure 29A).
  • C domain carboxy-terminal domain
  • TM ectodomain those derived from the MLV ecotropic envelope glycoprotein
  • Figure 29A Recent evidence from the literature suggest that the C domain of MLV envelope glycoproteins is involved in activation and/or control of the fusion machinery contained in the TM subunit of the envelope glycoprotein (Nussbaum et al., 1993; Pinter et al. , 1997).
  • Retroviruses carrying either the parental or the delH-mutated CMOdelH, TMOdelH and CTMMOdelH chimeric envelope glycoproteins were prepared and their infectivity was assayed in the presence or the absence of A-RBD or E-RBD soluble polypeptides (Table 5). While the infectivity of AdelH control retroviruses could easily be rescued when either A-RBD or E-RBD polypeptides were present in the infection mixture (Lavillette et al. , 2000), the infectivity of CMOdelH, TMMOdelH and CTMMOdelH retroviruses could not be rescued with the A-RBD polypeptide although it was efficiently rescued with the E-RBD polypeptide (Table 5).
  • Example 5 Retargeted gene delivery using RBD fragments.
  • the RBD of a retroviral envelope glycoprotein is not only a domain responsible of attaching the viral particle to a cell surface receptor but is also a domain that is responsible, upon receptor binding, of fusion activation most likely via an interaction with the carboxy-terminal domain of the SU Env subunit (C domain).
  • the RBD determinant responsible of this activation appears to be located at its amino-terminal end and can be disrupted by the delH mutation. Thus receptor binding of RBD most probably promotes a conformational change of this amino-terminal end which, in turn, is rendered competent for inducing further conformational changes in the Env C domain that control the fusion machinery located in the TM Env subunit.
  • LacZ retroviral vectors were generated with the indicated envelope glycoproteins.
  • A wild-type amphotropic Env; 34.1-A, a chimeric envelope glycoprotein derived from the MLV amphotropic Env in which the receptor binding domain was replaced by a single chain antibody (scFv) against human class I MHC molecule (Marin et al, 1995).
  • the vectors were used to infect class I positive human cells. Titers as lacZ i.uJml.
  • a preparation of A-RBD polypeptide was added during infection. Similar results were obtained when the A-RBD polypeptide was added before or after infection with the 34.1-A retroviruses .
  • Anti-MHC-I IgGs corresponding to the Mab which was used to derive the anti-MHC-I scFv inserted into the 34.1-A chimera, were used as competitor in this infection assay.
  • the anti-MHC-I IgGs were used to block the targeted antigens before infection with the 34.1-A retroviruses .

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Abstract

L'invention concerne l'utilisation d'au moins un polypeptide d'activation comprenant une séquence d'acides aminés correspondant, par exemple, à : la séquence d'acides aminés de la sous-unité de surface (SU) d'une glycoprotéine d'enveloppe d'un premier virus ou d'un premier rétrovirus, pour le rétablissement de la propriété de fusion d'une glycoprotéine d'enveloppe à défaut de fusion, possédant la propriété de se fixer à des cellules cibles et inactive par rapport à la fusion auxdites cellules cibles. Ladite glycoprotéine d'enveloppe à défaut de fusion appartient à un second virus ou à un second rétrovirus d'un type similaire à ou différent du « premier virus ou premier rétrovirus » susmentionné.
PCT/EP2000/004534 1999-05-20 2000-05-19 Nouveaux polypeptides et leur utilisation pour le sauvetage de virus ou de glycoproteines virales a defaut de fusion Ceased WO2000071578A2 (fr)

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FR2832424A1 (fr) * 2001-11-20 2003-05-23 Genethon Iii Plasmide chimere comprenant un genome retroviral replicatif et utilisations
WO2003044202A1 (fr) * 2001-11-20 2003-05-30 Genethon Plasmide chimere comprenant un genome retroviral replicatif et utilisations

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