EP0953053A2 - Particules virales masquees ou demasquees vis-a-vis du recepteur cellulaire - Google Patents

Particules virales masquees ou demasquees vis-a-vis du recepteur cellulaire

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Publication number
EP0953053A2
EP0953053A2 EP97925095A EP97925095A EP0953053A2 EP 0953053 A2 EP0953053 A2 EP 0953053A2 EP 97925095 A EP97925095 A EP 97925095A EP 97925095 A EP97925095 A EP 97925095A EP 0953053 A2 EP0953053 A2 EP 0953053A2
Authority
EP
European Patent Office
Prior art keywords
peptide
receptor
terminal
protein domain
retro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97925095A
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German (de)
English (en)
French (fr)
Inventor
François-Loíc COSSET
Sandrine Valsesia
Stephen J. Russell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
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Centre National de la Recherche Scientifique CNRS
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Publication of EP0953053A2 publication Critical patent/EP0953053A2/fr
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
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    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/62—DNA sequences coding for fusion proteins
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    • 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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    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
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    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
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    • C12N2810/00—Vectors comprising a targeting moiety
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    • 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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    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
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    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
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    • C12N2810/854—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from hormones

Definitions

  • Retroviruses and therefore retroviral vectors initiate their infectious cycle by recognizing specific cell surface molecules, called retroviral receptors, with envelope glycoproteins expressed on the surface of retroviral particles. This recognition then leads to the fusion between the viral and cellular membranes, a complex and poorly understood process which is also mediated by a second function of the envelope glycoprotein.
  • the present invention also relates to the construction of chimeric envelope glycoproteins using these new peptides.
  • the invention relates to the use of a peptide for the transfer of genes into a target eukaryotic cell, which peptide has from approximately 10 to approximately 200, in particular from approximately 15 to approximately 150 amino acids, and advantageously approximately 20 amino acids, in which at least 30% of the amino acids consist of proline residues, which proline residues are regularly arranged so as to induce folds of the polypeptide chain at approximately 180 ° (" ⁇ -turn” or "reverse- turn "), these folds being regularly spaced and assembling in a polyproline helix with folding type ⁇ (" polyproline ⁇ -turn helix "), in a polypeptide construction containing, on the N-terminal side (upstream) of said peptide, an N-terminal (upstream) protein domain capable of recognizing a targeted surface molecule or an antigen expressed on a cell surface, in particular an appropriate receptor (targeted receptor) located on said cell
  • this ⁇ -folded polyproline helix contains 4 ⁇ folds and therefore 4 turns, and is moreover incompatible with a secondary structure in ⁇ -helix or in ⁇ sheet.
  • the ⁇ -folding polyproline helix placed between the two domains of the chimeric protein (N-terminal domain and auxiliary domain) intrinsically has, 1) an elastomeric force, 2) the property of self-assembling with other polyproline helices, probably in relation to the trimeric nature of the envelope, 3) the property of transmitting to the auxiliary domain a deformation induced by the binding of the N-terminal domain with its receptor, causing activation of the auxiliary domain .
  • markers specifically present on various differentiated tissues (example: receptor for growth factors, peptide hormones).
  • a targeted surface molecule As an example of a targeted surface molecule, mention may be made in particular of a receptor which will hereinafter be designated as a targeted receptor.
  • substantially intact refers to a viral glycoprotein which retains all of its determinants necessary for preserving the post-translational processes, if necessary, the oligomerization, the viral incorporation and fusion properties.
  • certain alterations can be made to the glycoprotein without significantly affecting these functions and the glycoproteins containing such minor modifications are considered to be substantially intact for the purposes of the invention.
  • the glycoprotein may lack a few amino acids (e.g. about 1 to 10), especially at the N-terminus, but will generally be the same size as the wild type protein and has essentially the same biological properties than wild protein.
  • non-viral recombinant gene transfer vector denotes macromolecular complexes associating the DNA containing the transferred gene, its expression regulation sequences, and molecules belonging to the class of lipids, carbohydrates, or proteins. , which have functional properties capable, 1) of targeting the deposition of DNA on the surface of the target cell, 2) of introducing this DNA into the target cell, and 3) of introducing this DNA into the nucleus of the target cell.
  • the envelope glycoprotein comprising:
  • the C-terminal (downstream) protein domain corresponds to a (retro) viral envelope glycoprotein, essentially intact, comprising the natural binding domain, the fusion and anchoring functions of the wild envelope glycoprotein from which the envelope glycoprotein carried by the recombinant (retro) viral particle is derived.
  • N-terminal protein domain upstream of the above peptide, capable of reacting with an appropriate receptor (targeted receptor) located on a eukaryotic cell, which protein domain makes it possible to specifically bind a recombinant (retro) viral particle containing said protein domain N-terminal and
  • MLV-A, GALV, FeLVB or viruses such as adenovirus, herpes virus, AAV virus (Adeno Associated Virus), or more generally viral glycoproteins derived from viruses of eukaryotic origin, in particular orthomyxovirus (such as influenza virus) or paramyxovirus (such as SV5).
  • viruses such as adenovirus, herpes virus, AAV virus (Adeno Associated Virus), or more generally viral glycoproteins derived from viruses of eukaryotic origin, in particular orthomyxovirus (such as influenza virus) or paramyxovirus (such as SV5).
  • the invention also relates to a recombinant (retro) viral particle characterized in that the 5 'end of the nucleotide sequence coding for the N-terminal (upstream) protein domain is contiguous with the 3' end of the nucleotide sequence coding for the signal peptide, the 3 'end of the nucleotide sequence coding for the N-terminal (upstream) protein domain is contiguous with the 5' end of the nucleotide sequence coding for the peptide, the 3 'end of the nucleotide sequence coding for the peptide is contiguous with the 5 ′ end of the nucleotide sequence coding for the C-terminal protein domain (downstream).
  • the invention also relates to a nucleic acid coding for a peptide or for a recombinant particle according to the invention.
  • the invention containing the nucleic acid to be transferred.
  • the invention also relates to a pharmaceutical composition containing as active substance a (retro) v ⁇ rale particle according to the invention, and also containing a gene to be transferred, in association with a physiologically appropriate pharmaceutical vehicle.
  • genes to be transferred which are important for gene therapy, these are for example IFN, IL2, p53, VEGF, TNF, CFTR, HSV-TK, lacZ, GFP, various cytokine genes, other types of suicide genes including conditional suicide genes, other genes with anti-viral activity, other genes with anti-tumor activity, other marker genes and any therapeutic gene for mono- or multi-gene disease.
  • the pathologies most specifically concerned are: most single and multi-gene diseases (mucovisidosis, myopathy, lysosomal diseases, various forms of cancer, viral diseases (AIDS), ...)
  • FIG. 1 shows the two-stage entry process of the targeting viral particle.
  • the viral particles are generated (A) with targeting envelope glycoproteins composed of an N-terminal domain (ligand, monobrin antibody, etc.), of the masking / unmasking peptide, and of a C-terminal domain (B).
  • the steps leading to the introduction of the virion into the targeted cell involve a mechanism coordinated by the masking / unmasking peptide (C).
  • FIG. 2 is a schematic representation of some of the targeting envelopes studied. The position of some functional regions is shown. Vertical arrows: proteolytic cleavage sites.
  • SU surface subunit
  • TM transmembrane subunit
  • SP signal peptide
  • PRO poly-proline region
  • T transmembrane domain
  • Ram-1 ligand binding domain for the amphotropic receptor
  • Rec-1 ligand binding domain for the ecotropic receptor
  • EGF epidermal growth factor.
  • Dark gray boxes sequence derived from the env gene of MoMLV
  • Light gray boxes sequence derived from the env gene of MLV-4070A
  • White boxes other sequences derived from MLV.
  • Black boxes spacer peptides derived from the poly-proline region. All env genes are expressed from the same promoter (LTR) and polyadenylation signal (pA) from the subgenomic mRNA using the retroviral, donor (SD) and acceptor splice sites.
  • LTR promoter
  • SA SA with an identical intronic sequence of 190 nts containing the end of the pol gene ( ⁇ POL). The position of some restriction sites is shown.
  • FIG. 5 shows the expression and viral incorporation of the chimeric envelopes of the AMO series.
  • FIG. 7 shows the amino acid and nucleotide sequence of PRO (4070A).
  • FIG. 9 shows the amino acid and nucleotide sequence of PRO ⁇ (MoMLV).
  • FIG. 10 shows the amino acid and nucleotide sequence of
  • FIG. 30 shows the amino acid and nucleotide sequence of AMOEL3.
  • FIG. 35 shows the amino acid and nucleotide sequence of EL5.
  • FIG. 36 shows the amino acid and nucleotide sequence of ⁇ PR04-beta.
  • FIG. 39 shows the amino acid and nucleotide sequence of PRO-beta.
  • FIG. 40 shows the amino acid and nucleotide sequence of PRO-int.
  • Retroviruses use a number of cell surface molecules, called viral receptors, to initiate the infectious process (23). With notable exceptions, notably in the case of human immunodeficiency viruses, most of the receptors used by other retroviruses and in particular mammalian type C retroviruses are distributed on most cell types of the host organism. For example, the amphotropic murine leukemia virus (MLV-A) is capable of infecting most mammalian cells because its receptor, the phosphate transporter Ram-1, is expressed on almost all cells.
  • MLV-A amphotropic murine leukemia virus
  • polypeptides capable of binding various cell surface molecules have been inserted at the N-terminal end of the SU subunit of the envelope glycoprotein (6) (10) (13) (15) (21).
  • the study of virions generated with these various types of targeting envelopes has shown that it is possible to specifically and effectively redirect the binding of viral particles to new surface molecules.
  • Some factors that limit the effectiveness of targeting have also been identified. The first seems to depend on physiological properties of the targeted surface molecule (dimerization, internalization, intracellular transport process) (6), the second is linked to the low intrinsic fusiogenicity of the glycerol of envelope chimeras generated by insertion.
  • N terminal ligands (6) (21).
  • the natural binding domain of the support envelope is always functional.
  • this functionality of the natural binding domain does not pose problems of infection "background noise” because the ecotropic envelope of the virus is used as the support glycoprotein.
  • MoMLV which does not recognize a receptor on the cells of higher mammals.
  • N-terminal targeting glycoprotein and then an auxiliary mechanism to facilitate the entry of the virus specifically linked to the right cellular target through the natural retroviral receptor.
  • spacer masking / unmasking peptides have also been developed, inserted between the targeting domain and the glycoprotein of support envelope, and which are capable of masking the natural binding domain as long as the viral particle has not interacted with the targeted surface molecule. The realization of this interaction induces the unmasking of the natural binding domain and the interaction between the natural binding domain and the natural retroviral receptor (auxiliary mechanism) which then takes over to introduce the virus into the cell.
  • the TELCeB ⁇ cell line (7) is derived from the TELacZ line (19) by transfection and clonal selection of cells expressing the gag and pol proteins of MoMLV (Moloney Murine Leukemia Virus: Moloney murine leukemia virus).
  • TELacZ cells express the retroviral vector MFGnlslacZ capable of transducing a nuclear ⁇ -galactosidase.
  • TELCeB ⁇ cells allow the production of retroviral capsids (non-infectious because they lack envelopes) carrying the retroviral marker vector nlsLacZ.
  • the A431 (ATCC CRL1555) and TE671 (ATCC CRL8805) cells are cultured in DMEM medium (Gibco-BRL) supplemented with 10% fetal calf serum (Gibco-BRL).
  • the CHO, CERD9 (9) and CEAR13 (9) cells are cultured in DMEM medium (Gibco-BRL) supplemented with 10% fetal calf serum and proline (Gibco-BRL).
  • the NIH-3T3 and NIH-3T3 cell lines are cultured in DMEM medium (Gibco-BRL) supplemented with 10% newborn calf serum (Gibco-BRL). Chimerical envelopes. DNA fragments encoding polypeptides recognizing either EGFR
  • the chimeric envelope is formed by cloning of the Xhol / Notl PCR fragment and of the Notl / Clal fragment, isolated from the env EMO gene (coding for the SU and TM-P15E transmembrane proteins of MoMLV), between the Xhol / Clal sites of the env 4070A MLV gene.
  • the resulting constructs are recovered in the form of a BglII / ClaI fragment (corresponding to positions 5408 and 7676 in MoMLV) and cloned at the BamHI and ClaI sites of an expression plasmid FBMOSALF (7) in which a selection marker gene (8) fused to the polyadenylation sequences of the PGK (phospho-glycerate kinase) gene was introduced downstream of the 3 'LTR of the MLV-C57 virus.
  • a selection marker gene (8) fused to the polyadenylation sequences of the PGK (phospho-glycerate kinase) gene was introduced downstream of the 3 'LTR of the MLV-C57 virus.
  • the new recognition domain was separated from the rest of the MLV envelope by a spacer peptide consisting of three alanines, provided by the Notl cloning site (15).
  • spacer amino acids were introduced either after the domain of EGFR recognition (EGF), either after the Ram-1 recognition domain (AS208) as described below.
  • AMOl the first 208 amino acids, derived from the envelope of MLV 4070 A, were fused to amino acid 1 of the SU of MoMLV.
  • AMOlFx a site of 4 amino acids corresponding to the cleavage site of blood coagulation factor Xa (Ile-Glu-Gly-Arg) (12) was inserted after the recognition domain of Ram-1 and fused to codon + 1 of MoMLV's SU. The strategy used for these constructions is described above.
  • an oligonucleotide (5'-TCCAATTCCTTCCAAGGGGC-3 '), located just upstream of the Xhol site of the env gene of 4070A (nt 594) was used in combination with either one of the following two oligonucleotides providing Notl site:
  • PCR fragments were subjected to digestion with Xhol and NotI and cloned into the plasmid FBAMOSALF opened in Xhol / NotI, a plasmid expressing an envelope of AMO type.
  • the plasmids expressing the AMOFx, AMOl and AMOlFx envelopes were generated by cloning the NdeI / NotI fragment of FBAMOSALF (comprising the recognition domain Ram-1) in a series of plasmids (13) expressing the MoMLV envelopes modified in order to create a site Notl at codon 1 or codon 6 with (AMOlFx, AMOFx) or without (AMOl) the sequence Xa.
  • Envelopes derived from AMO and containing other types of spacer peptides were constructed. All of these spacer peptides are shown in Figure 3 A.
  • 3 'fragments were generated by PCR using the MoMLV env gene as a template, as a 5' oligonucleotide (5 '-ACTGGGGCTTACGTTTGT-3') upstream of the BamHI site, and as a 3 'oligonucleotide (5' -TATGTGCGGCCGCCGGTGGAAGTTGGGTAGGG- ') or (5' -TATGTGCGGCCGCGTCTGGCAGAACGGGGTTTGG-3 ') to build the MOAPRO and MOA ⁇ PRO envelopes, respectively.
  • These PCR fragments were digested with BamHI and NotI, and co-ligated with the 5 'fragment.
  • the sequence of spacer peptides for these two constructs is shown in Figure 3B.
  • plasmids expressing the EMOI and EMOlFx envelopes were generated.
  • the sequence of spacer peptides. for these two constructions is shown in Figure 3C.
  • the plasmids expressing the EAPRO + and EA ⁇ PRO + envelopes were generated by replacing the Sfil / Not fragment of the FBEASALF plasmid by the Sfil / Notl fragments derived from the plasmids expressing the EMOPRO + and EMO ⁇ PRO + envelopes.
  • the virus-producing cells are lysed for 10 min at 4 ° C. in 20 mM Tris-HCL buffer (pH 7.5), containing 1% tritonXIOO, 0.05% SDS, 5 mg / ml deoxycholate, 150 mM NaCl and PMSF
  • Target cells were washed with PBS and detached by incubation
  • the target cells are incubated for 30 min at 37 ° C. in a medium containing 10 ⁇ 6 M of recombinant human EGF (236-EG, R&D Systems, UK). The cells are then rinsed and the infections carried out as previously described. In order to block the acidification of the endosomes, 100 mM of chloroquine phosphate (Sigma, UK) is added to the medium. Six hours after infection, the cells are rinsed and incubated in a normal medium. Results and discussion.
  • a first envelope targeting Ram-1, AMO was constructed by N-terminal insertion of the MoMLV envelope (by fusion with codon 7) of a polypeptide recognizing Ram-1 (AS208, FIG. 3 A) and corresponding to the first 208 amino acids of the SU of MLV-A (1).
  • the sequence coding for EGF was inserted into the env gene of MLV at position +6 of the SU of
  • the new binding domains were separated from the recognition domain of the retroviral receptor by a spacer peptide corresponding to three alanines.
  • various constructions were then generated by insertion of spacers of different sizes and structures. The protein sequences of these different spacers are reported in Figure 3 A for the envelopes targeting Ram-1 and in Figure 3C for the envelopes targeting EGFR.
  • the plasmids expressing the various envelopes were transfected into the TELCeB ⁇ cell line which expresses the proteins coded by the gag and pol genes, as well as a retroviral vector nlsLacZ ( 7).
  • Cell surface expression was determined by FACS analysis of producer cells, using antibodies against SU, or using anti-EGF monoclonal antibody.
  • the cells transfected with the different envelopes can be labeled with the anti-SU antibody (not shown).
  • Only cells expressing EGF fusion envelopes can be labeled using anti-EGF monoclonal antibodies ( Figure 4). This demonstrates the expression of the chimeric envelopes on the cell surface and the correct folding of the EGF on the chimeric glycoproteins.
  • Envelopes targeting EGFR are also capable of binding to A431 cells, over expressing EGFR ( Figure 6A). This connection seems specific since a pre-incubation of A431 cells in the presence of EGF (inducing the endocytosis of EGFR) inhibits this binding (not shown). Ram-1 and Rec-1 cooperation in infection.
  • the transduction of the pseudotyped retroviral vectors by the various targeting envelopes was measured on cells expressing different types of receptors: human TE671 cells expressing the EGF receptors and
  • Viruses with AMO chimeric envelopes are capable of infecting TE671 cells in a titer of 4.10 ⁇ lacZ i.u./ml (Table 1). In comparison; despite a similar receptor binding efficiency ( Figure 6B), the titers obtained with wild envelopes are 10,000 times higher. Su ⁇ renantly, the viruses expressing AMOPRO envelopes, despite good binding efficiency, have been found incapable of infecting human TE671 cells.
  • Viruses carrying the chimeric envelopes in which the Ram-1 binding domain has been separated from the SU of MoMLV by different spacers prove to be very infectious on 3T3 cells.
  • an increase from 200 (for AMOPRO) to more than 1000 times (for AMOlFx) of the viral titers was measured (Table 1).
  • the virions containing the AMO ⁇ PRO envelope can effectively infect cells when Ram-1 is expressed there alone.
  • the infectivity is increased by approximately 10 times when Rec-1 is also present on the cell surface. This difference is not due to the simple fact that AMO ⁇ PRO virions preferentially use Rec-1 for infection.
  • infection of cells on which only Rec-1 is available is extremely low (Table 1) or even undetectable (Table 2) compared to when Ram-1 and Rec-1 are co-expressed.
  • the RecID index is less than 10 ⁇ 5 (Table 2). This also demonstrates that the two receptors can synergize the infection.
  • the AMO ⁇ PRO virions are compared to the virions containing the envelopes with the flexible spacers AMOlFx, AMOGIFx and AMOG2.
  • the ⁇ PRO peptide is contains 5 prolines probably arranged in a polyproline type II helix, while the AMOGIFx and AMOG2 envelopes essentially contain glycines.
  • virions containing AMOPRO envelopes require the simultaneous presence of both types of receptors to infect cells.
  • the infectious titer in cell types co-expressing the two receptors is however lower than that which is observed with the AMO ⁇ PRO virions, without however excluding the hypothesis that the slightest inco deoration of these envelopes is responsible for this result.
  • AMOPRO viruses cannot infect cells when either of the two receptors is expressed alone (Table 2).
  • the two indices RamID and RecID are indeed lower than A.
  • MOAPRO envelopes include the binding domain of the ecotropic envelope followed by the proline region rich in this same envelope, all fused to the N-terminal end of the amphotropic envelope ( Figure 2).
  • Table 2 The results, shown in Table 2, indicate that, similarly to the virions containing the AMOPRO envelopes, the MOAPRO virions can hardly, if at all, infect cells expressing only one or the other of the Rec-1 receptors or Ram-1.
  • the Ram-1 field in the MOAPRO envelope is even less accessible (Ramid less than 7xl0 "5) than the field is the Rec-1 in AMOPRO envelope (RECID less than 5, 6x10-4
  • the MOAPRO envelopes can effectively infect cells expressing the two types of receptors, with titers of the order of 10 ⁇ lacZ iu / ml, suggesting that, in the case of this envelope too, the presence of the two receptors synergizes the infectious process.
  • the spacer peptide inserted between the targeting domain and the rest of the retroviral envelope exerts a control on the accessibility of the domain located downstream of said peptide and on the activation of the fusion. This control depends on the peptide itself and is influenced by its length and by its biochemical composition.
  • the hypothesis formulated is that the spacer peptide PRO would ultimately play the same role as the proline-rich region from which it originates and which is located, in the unmodified glycoprotein, between the receptor binding domain and the fusion domain. This role would be the masking of the downstream domain (fusion domain for the wild envelope or binding domain for the chimeric envelope) and the unmasking following the interaction of the upstream domain with its receptor.
  • this unmasking would lead to activation of the fusion, while in the case of chimeric envelopes, the unmasking would lead to the accessibility of the binding domain to the viral receptor. If the receptor is expressed on the cell surface, there can then be interaction, this then triggering the activation of the fusion domain explaining why the simultaneous presence of the two receptors synergizes the infection.
  • a targeting envelope in two stages for which a targeting envelope is constructed with different domains whose functions are activated and coordinated by means of specific spacer peptides containing sequences rich in proline.
  • These chimeric envelope glycoproteins can be designed in the following way, with from N-terminal to C-terminal, a "targeting" domain capable of recognizing a cell surface molecule specifically expressed on the targeted tissue or cell (for example a single chain antico ⁇ s or a ligand for a surface receptor); a spacer peptide capable of masking an auxiliary domain itself capable of facilitating the penetration of the virus when it is activated.
  • auxiliary domain can be an entire retroviral envelope, i.e.
  • the helper domain should be masked until the virus particle has specifically interacted with the targeted surface molecule.
  • the targeted surface molecule is Ram-1 while the auxiliary domain is the ecotropic envelope. EGFR and Rec-1 cooperation in infection.
  • the targeting domain is EGF
  • the auxiliary domain is the ecotropic envelope.
  • the infection tests were carried out with cells expressing Rec-1 only (Cerd9 cells) or with cells co-expressing Rec-1 and EGFR (3T3 cells). The results of a typical experiment are reported in Table 3. As expected by the results obtained with the AMO envelopes, viruses containing the EMO envelopes can effectively infect Cerd9 and 3T3 cells, indicating that the Rec- binding domain 1 in these envelopes is not hidden. Compared to EMO viruses, viral particles containing the EMOPRO + and EMO ⁇ PRO + envelopes can only with difficulty infect Cerd9 cells (between 1,000 and 10,000 times worse than EMO viruses).
  • the viral particles containing the EMOPRO + and EMO ⁇ PRO + envelopes are 20 and 60 times more infectious, respectively, compared to when Rec-1 is expressed alone.
  • the masking is apparently less successful, and this results in significant infectivity on Cerd9 cells. This may be due to the fact that the spacer peptides PRO + and ⁇ PRO + are not optimized for their function, but perhaps also to the fact that the Cerd9 cells express a little EGF receptor which would help activate the EMOPRO + and EMO ⁇ envelopes PRO +.
  • a percentages calculated taking as values 100 the titers obtained on 3T3 b: infection on 3T3 chronically infected with MLV-A (3T3-MLV-A) or with MoMLV (3T3-MoMLV) c: receptor available on the surface of the cell considered
  • EXAMPLE 2 In order to characterize the cooperation between the Rec-1 and Ram-1 receptors, as well as the peptides capable of regulating this cooperation of receptors, a new series of AMO-type chimeric envelope glycoproteins (see previous example) a been built - in order to check whether the infection obtained with the AMOPRO and AMODeltaPRO envelopes passes, in a second step, through an interaction with Rec-1, the binding site with Rec-1 was inactivated by point mutagenesis (D84K mutation) (MacKrell et al.,]. Virology, 70: 1768-1774.
  • the AMOEL3 and AMOEL5 envelopes were constructed. These envelopes respectively carry 3 and 5 turns of a type II polyproline helix characterized in the literature (Urry, Journal of Protein Chemistry 7: 1-34. (1988)).
  • Retroviruses were generated with these chimeric envelopes and were characterized by infection of cells expressing either Rec-1 alone, or Ram-1 alone, or the two molecules Ram-1 and Rec-1.
  • oligonucleotides elast3U (5'-TTT ATG GTC ACC GCG GCC GCA CCT GGG GTA GGG GCT CCG GGG GTA GGG GCT CCT GGG GTG GCC ATA TAA) and elast3L (5'-TTA TAT GGC CAC CCC AGG AGC CCC TAC
  • the oligonucleotides UpE15 (5 * -G AT GTA CCT GGG GTA GGC GCC CCT GGA GTC GGG GCT CCT GGG GTA GGA TTC AT) and LowE15: (5 '-ATG AAT CCT ACC CCA GGA GCC CCG ACT CCA GGG GCG CCT
  • the DELASTIN3-V Upper oligonucleotides (5'-GTC ACC GCG GCC GTC CCT GGG GTA GGG GTG CCG GGG GTA GGG GTG CCT GGG GTG GCC ATA TA A) and DELASTIN3-V Lower (5'-TTA TAT GGC CAC CCC AGG CAC CCC TAC CCC CGG CAC CCC TAC CCC AGG GAC GGC CGC GGT GAC) have been hybridized.
  • the resulting double-stranded DNA fragment was digested with the restriction enzyme Eael and cloned into the expression plasmid FBAMOSALF previously opened in NotI. This results in the plasmid
  • FBAMOEL3-VSALF (see sequence of the AMOEL3-V gene in FIG. 34) comprising the peptide EL3-V whose peptide sequence is represented in table 4 (see nucleotide sequence in FIG. 35).
  • FBAMOEL3-ISALF comprising the peptide EL3-I whose peptide sequence is represented in table 4.
  • Expression and viral inco ⁇ oration of chimeric envelopes Expression plasmids for the envelopes AMO, AMODeltaPRO, AMOPRO, AMOEL3, AMOEL5, AMOEL3-V, AMOEL3-I, AMOIFX, AMOG1X, AMOD84K, AMODeltaPROD84K, AMOPROD84K, AMOG1XD84K, AMODeltaPR02
  • the corresponding DNA fragments were digested with the enzyme EagI and separately inserted into the plasmid FBEASALF (expressing the chimeric envelope glycoproteins EA) (Cosset et al., Journal of Virology 69: 6314-6322.

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