AU725632B2 - Viral particles which are masked or unmasked with respect to a cell receptor - Google Patents

Viral particles which are masked or unmasked with respect to a cell receptor Download PDF

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AU725632B2
AU725632B2 AU30356/97A AU3035697A AU725632B2 AU 725632 B2 AU725632 B2 AU 725632B2 AU 30356/97 A AU30356/97 A AU 30356/97A AU 3035697 A AU3035697 A AU 3035697A AU 725632 B2 AU725632 B2 AU 725632B2
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peptide
retro
receptor
terminal
viral
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Francois-Loic Cosset
Stephen J. Russell
Sandrine Valsesia
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Centre National de la Recherche Scientifique CNRS
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Description

VIRAL PARTICLES WHICH ARE MASKED OR UNMASKED WITH RESPECT TO A CELL RECEPTOR The invention relates to recombinant viral particles containing a peptide possessing properties of masking and of unmasking with respect to a biological mechanism, notably with respect to a mechanism of cellular interaction.
The invention also relates to the application of the aforesaid viral particles, notably for cell targeting in gene transfer.
Retroviruses and therefore retrovirus vectors initiate their infectious cycle by recognizing specific cell surface molecules, called retrovirus receptors, with envelope glycoproteins expressed on the surface of the retroviral particles. This recognition then leads to fusion between the viral and cellular membranes, a process that is complex and poorly understood, which is also mediated by a second function of the envelope glycoprotein.
The possibility of altering the specificity of the interaction with the surface of the target cell has been demonstrated previously, notably by means of genetic modifications introduced in the retroviral envelope glycoprotein.
A certain number of works have shown that such modifications do not involve disturbing effects in the complex processes that permit the retroviral envelope glycoprotein to become mature, to be expressed on the cell surface, and to be incorporated selectively in the virions. Moreover, it has now been proved that these modifications can lead to the specific recognition of cells by interaction with the surface molecules corresponding to these polypeptides. Finally, in certain cases, this recognition permits continuation of the infectious cycle and integration of the transgene in the targeted cell with, in the best possible case, an efficiency that is greatly reduced relative to the efficiency that is provided by an unmodified retroviral envelope via its normal retrovirus receptor. Hence it is concluded, on the one hand, that certain target surface molecules cannot be utilized as a receptor for initiating infection and, on the other hand, when they can be so utilized, the processes following the primary interaction take place with extremely low efficiency. These conclusions are true with regard to "one stage targeting" strategies, i.e. approaches in which the aim of the primary interaction is to lead directly to continuation of the infectious cycle, without the intervention of auxiliary processes.
Retrovirus vectors are now the most-used vectors for gene transfer and in particular for gene therapy, as we require stable integration and expression of the transgene. Other gene transfer vectors exist (adenovirus vectors, liposomes, vectors derived from herpesviruses, or vectors derived from AAVs), but do not permit stable and efficient integration of the transgene. Whereas most of the gene therapy protocols examined up to now and using retroviral vectors are based on the explantation of the patient's cells, their transgenesis and expansion in vitro, followed by their reimplantation, it would be highly desirable to be able to transfer a therapeutic gene in the in vivo context by means of retroviral vectors. For that, the retroviral particle carrying the therapeutic gene would have to be endowed with certain additional characteristics, and more particularly, the ability to recognize very specifically the target cells of the gene transfer. In fact, the surface molecules that are recognized naturally by the retroviruses for initiation of infection are expressed very widely on most of the cells. This does not permit precise discrimination of the cells in which one wishes to effect a gene transfer.
Several works have had the aim of altering the infection tropism of retroviral vectors. Some of these works are based on biochemical modifications of retroviral particles, others on genetic modifications of the retroviral envelope glycoproteins, which guarantees that all the retroviral particles will be altered. In this last context, the works have consisted of "single-stage targeting", for which the modified viral particle attaches to the targeted cell surface molecule leading to continuation of the infectious cycle.
However, the efficiency of the retroviral vectors altered in this way is very low relative to what is required for obtaining a tool that can be used for purposes of therapy.
It is possible that the development of targeting strategies could not succeed with the "single-stage" system, for reasons already mentioned above: inefficiency of the gene fusion process of the chimeric envelope glycoproteins after their binding on the targeted surface molecule, and the impossibility of utilizing certain surface molecules as retroviral receptors.
A certain number of human gene therapy protocols will require retroviral vectors that are capable of effecting gene transfer in vivo, by direct inoculation of the recombinant retroviral particles. Among the improvements that this presupposes relative to the retroviral vectors developed until now, we may cite: improvement of the infectious titres, improvement of the stability of the viral particles in the serum and more generally in the various body fluids, the possibility of infecting quiescent cells, the possibility of discrimination of the target cells of gene therapy.
The invention has the aim of proposing means for discriminating the target cells of gene therapy. It is essential, for certain applications in gene therapy, to guarantee that gene transfer will only have taken place in the cells to be treated, and not in other categories of cells. For example, when we wish to confer a selective advantage on normal cells with respect to a chemotherapy, it is imperative that the transferred gene conferring this advantage has not been introduced into cancer cells.
2a- In a first aspect, the present invention provides the use of a peptide for transferring genes into a eukaryotic target cell, this peptide containing from about 10 to about 200 amino acids, in which at least 30% of the amino acids consist of proline residues, these proline residues being arranged regularly so as to induce turnings of the polypeptide chain at about 1800 ("p-turn" or "reverse-turn"), these turnings being regularly spaced and forming a polyproline p-turn helix, in a polypeptide construction containing, on the N-terminal side (upstream) of the said peptide, and N-terminal (upstream) protein domain capable of recognizing a targeted surface molecule or an antigen expressed on a cell surface, and on the C-terminal side (downstream) of the said peptide, a C-terminal (downstream) protein domain capable of recognizing a suitable receptor (auxiliary S. receptor) located on the aforesaid eukaryotic cell, this peptide being capable of facilitating or inhibiting interaction between the C-terminal (downstream) protein domain and the auxiliary receptor, inhibition of this interaction occurring for as long as the N-terminal (upstream) protein domain has not interacted with the targeted receptor 15 and promotion of interaction between the C-terminal (downstream) protein domain and the auxiliary receptor occurring when the N-terminal (upstream) protein domain has interacted with the targeted receptor.
In a second aspect, the present invention provides peptide sequences chosen from those containing, or constituted of, one of the following sequences: 20 PRO (4070A), PRO(MoMLV), PRO3, PRO+, APRO, APROp, APRO+, MOAPRO, MOAAPRO, EMOPRO, EMOPROp, EMOPRO+, EAPRO, EAPROp, EAPRO+, EMOAPRO, EMOAPROp, EMOAPRO+, EAAPRO, EAAPROp, EAAPRO+, EL3, EL3-V, AMOEL3, AMOEL3-V, AMOEL5, APRO4-P, APRO4-int, APRO4-vrb, PROP, PROint, PRO-vrb.
In a third aspect, the present invention provides peptide sequence containing a peptide of about 10 to about 200 amino acids, in which at least 30% of the amino acids consist of proline residues, these proline residues being arranged regularly so as to induce turnings of the polypeptide chain at about 1800 ("p-turn" or "reverse-turn"), these turnings being regularly spaced and forming a polyproline p-turn helix, 2b an N-terminal protein domain (upstream) of the said peptide, capable of reacting with a suitable receptor (targeted receptor) located on a eukaryotic cell, this protein domain permitting specific binding of a recombinant (retro)viral particle containing the said Nterminal protein domain and a C-terminal protein domain (downstream) of the said peptide, capable of interacting with a suitable auxiliary (retro)viral receptor ((retro)viral receptor) located on the said eukaryotic cell, this interaction performing the role of auxiliary mechanism of entry of the (retro)viral particle into the said eukaryotic cell, the process of cell entry of the said recombinant (retro)viral particle into the said eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to take place when the N-terminal (upstream) protein domain has recognized and bound the targeted receptor of the Seukaryotic cell with the said recombinant (retro)viral particle, leading, through the agency of the aforesaid peptide, to a mechanism of unmasking or of accessibility of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain, 15 and, in the case when recognition does not occur between the recombinant viral particle and the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) protein domain, there is produced a mechanism of masking or of non-accessibility, through the agency of the aforesaid peptide, of the (retro)viral receptor with respect to S; the C-terminal (downstream) protein domain.
20 In a fourth aspect, the present invention provides recombinant (retro)viral particle capable of infecting a eukaryotic cell, this cell possessing a targeted receptor and an auxiliary receptor of the aforesaid (retro)viral particle, comprising a substantially intact envelope glycoprotein, containing a peptide of about 10 to about 200 amino acids, in which at least 30% of the amino acids are constituted of proline residues, these proline residues being arranged regularly so as to induce turnings of the polypeptide chain at about 1800 ("p-turn" or "reverse turn"), these turnings being regularly spaced and forming a polyproline p-turn helix, a protein domain on the N-terminal side (upstream) of the aforesaid peptide, capable of interacting with the aforesaid targeted receptor, this peptide domain permitting specific 30 binding of the (retro)viral particle and 2c a protein domain on the C-terminal side (downstream) of the aforesaid peptide, capable of interacting with the aforesaid (retro)viral receptor, this interaction performing the role of auxiliary mechanism of entry of the (retro)viral particle into the eukaryotic cell, the process of cell entry of the recombinant (retro)viral particle into the eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to take place when the N-terminal (upstream) protein domain has recognized and bound the targeted receptor of the eukaryotic cell with the recombinant (retro)viral particle, leading through the agency of the aforesaid peptide to a mechanism of unmasking or of accessibility of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain, and, in the case when recognition does not occur between the recombinant viral particle and the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) protein domain, there is produced a mechanism of masking or of non-accessibility, through the agency of the aforesaid peptide, of the retroviral receptor with respect to the ;C-terminal (downstream) protein domain.
15 In a fifth aspect, the present invention provides a nucleic acid coding for a peptide or for a recombinant particle according to the invention.
SIn a sixth aspect, the present invention provides a method of selective transfer in vitro or ex vivo of a nucleic acid into target eukaryotic cells present among other nontarget cells, comprising the administration, to the target and non-target cells, of a recombinant (retro)viral particle according to the invention, containing the nucleic acid to be transferred.
In a seventh aspect, the present invention provides a pharmaceutical composition containing as active substance a (retro)viral particle according to the invention, and also containing a gene to be transferred, in combination with a physiologically suitable pharmaceutical vehicle.
In an eighth aspect, the present invention provides use of a (retro)viral particle according to the invention containing a nucleic acid, for the manufacture of a medicament for selectively transferring the nucleic acid into a target eukaryotic cell present among other non-target cells.
The invention relates to a two-stage mechanism, in which the second stage is dependent on realization of the first stage.
The invention relates to an alternative that is beneficial with regard to performance in targeting, particularly because it combines specific recognition of the target cell and entry into the target cell connected with a natural retroviral mechanism, known for its efficiency.
The invention relates more particularly to a two-stage targeting mechanism: the first stage permitting recognition of a targeted surface molecule by means of the new N-terminal binding domain, inserted in an envelope glycoprotein, the second stage permitting conditional recognition of a normal retroviral receptor via a domain inherent in the initial envelope glycoprotein and thus permitting a relay in the process of entry of the viral particle into the cell, the term "conditional" signifying that the relay in the entry mechanism can only be effected if the viral particle has previously interacted with the initial surface molecule, which in turn guarantees that the infection is truly targeted.
The invention relates to new peptides for carrying out the first stage in a twostage mechanism and which perform the role of "masking" with respect to the second stage, for as long as the first stage has not taken place and permitting the second stage, i.e. performing the role of unmasking with respect to the second stage if, and only if, the first stage has taken place.
The present invention also relates to the construction of chimeric envelope glycoproteins using these novel peptides.
The invention relates to the use of a peptide for transfer of genes into a target eukaryotic cell, this peptide containing from about 10 to about 200, especially from about 15 to about 150 amino acids, and preferably about 20 amino acids, in which at least 30% of the amino acids are made up of proline residues, these proline residues being regularly arranged so as to induce turnings of the polypeptide chain to about 180° ("P-turn" or "reverse-turn"), these turnings being evenly spaced and forming a polyproline helix with 1 type turning ("polyproline P-turn helix"), in a polypeptide construction containing, on the N-terminal side (upstream) of the said peptide, an N-terminal (upstream) protein region capable of recognizing a targeted surface molecule or an antigen expressed on a cell surface, especially a suitable receptor (targeted receptor) located on the said eukaryotic cell, and on the C-terminal side (downstream) of the said peptide, a C-terminal (downstream) protein region capable of recognizing a suitable receptor (auxiliary receptor) located on the aforesaid eukaryotic cell, this peptide being capable of promoting or inhibiting interaction between the Cterminal (downstream) protein region and the auxiliary receptor, s inhibition of this interaction occurring for as long as the N-terminal (upstream) protein domain has not interacted with the targeted receptor and promotion of interaction between the C-terminal (downstream) protein domain and the auxiliary receptor occurring when the N-terminal (upstream) protein domain has interacted with the targeted receptor.
In the case of a peptide of 20 amino acids (APRO defined below), this b-turn polyproline helix contains four P turnings and therefore 4 turns, and moreover is incompatible with an oa-helix or P-sheet secondary structure. Advantageously, the polyproline helix with 0 type turning positioned between the two domains of the chimeric protein (N-terminal domain and auxiliary domain) possesses intrinsically: 1) an elastomeric force, 2) the property of self-assembly with other polyproline helices, probably in connection with the trimeric nature of the envelope, 3) the property of transmitting, to the auxiliary domain, a distortion that is induced by binding of the Nterminal domain with its receptor, causing activation of the auxiliary domain.
The invention also relates in general to any two-stage mechanism, in which the second stage can only be effected if the first stage has taken place, and relates for example to an enzymatic mechanism involving a chimeric protein which is only to occur if the chimeric protein is able to recognize its substrate.
The expression "N-terminal (upstream) protein domain capable of recognizing a targeted surface molecule, or an antigen expressed on a cell surface", means that: 1) the interaction between this N-terminal protein domain and the targeted surface molecule can be characterized by a dissociation constant (of nanomolar order with respect to interaction between wild-type retroviral envelope glycoprotein and retroviral receptor); 2) the soluble form of this N-terminal protein domain not associated in the construction of the chimeric envelope glycoprotein) possesses binding characteristics similar to this same protein domain when it is inserted at the N-terminal position in the chimeric envelope glycoprotein; 3) the chimeric envelope glycoprotein containing the N-terminal protein domain can be characterized according to classical techniques of virology (e.g binding test; cf "Examples").
The following may be mentioned as examples of targeted surface molecule or of antigen expressed on a cell surface: markers for differentiating the various haematopoietic lineages, in particular markers expressed on immature cells and/or haematopoietic stem cells (example: CD34), markers expressed on tumour cells (example: carcino-embryonic antigens), markers present specifically on various differentiated tissues (example: receptor of growth factors, of peptide hormones).
As an example of a targeted surface molecule, we may mention in particular a receptor which will be designated as targeted receptor hereinafter.
For convenience of terminology, the expression "targeted receptor" will be used in the following to encompass any targeted surface molecule or any antigen expressed on a cellular surface.
The expression "C-terminal (downstream) protein domain capable of recognizing a suitable receptor (auxiliary receptor)" means that the C-terminal protein domain can interact with the auxiliary receptor, this interaction being characterized by a dissociation constant which is of nanomolar order if the C-terminal protein domain is derived from a retroviral envelope glycoprotein and if the auxiliary receptor is the retroviral receptor used by this same glycoprotein, this interaction permitting the triggering of the gene fusion process in a mechanism that is strictly similar to the natural process, i.e. outside of the context of a chimeric envelope glycoprotein.
The peptide that is the subject of the invention is such that, positioned between two protein domains (an N-terminal protein domain relative to the said peptide and a Cterminal protein domain relative to the said peptide), it can induce the function of the Cterminal protein domain (for example binding if that is the function of this C-terminal domain) if, and only if, the N-terminal protein domain has been mobilized in its function (for example binding).
Non-induction of the function of the C-terminal protein domain by the peptide of the invention corresponds to the mechanism of "masking" of the peptide of the invention, whereas induction of the finction of the C-terminal protein domain by the peptide of the invention corresponds to the mechanism of "unmasking" of the peptide of the invention.
That is why the peptide of the invention will also be designated hereinafter as "masking/unmasking peptide".
The invention relates to the use of a peptide according to the invention, in the construction of a glycoprotein with targeting and gene-fusion activity, essentially intact, carried by a viral or non-viral recombinant gene-transfer vector capable of infecting a eukaryotic cell, the said eukaryotic cell possessing a targeted receptor and an auxiliary receptor permitting facilitation of entry of the said viral or non-viral vector into the eukaryotic cell, the aforesaid glycoprotein comprising: the aforesaid peptide, a protein domain on the N-terminal (upstream) side of the said peptide, capable of interacting with the above-mentioned targeted receptor, this protein domain permitting specific binding of the aforesaid gene-transfer vector and a protein domain on the C-terminal (downstream) side of the said peptide, capable of interacting with the aforesaid auxiliary receptor, this interaction performing the role of auxiliary mechanism of entry of the aforesaid gene-transfer vector into the eukaryotic cell, the process of cell entry of the viral or non-viral recombinant vector into the eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to occur when the N-terminal (upstream) protein domain has recognized and bound the viral or non-viral recombinant vector with the targeted receptor of the eukaryotic cell, leading, by means of the aforesaid peptide, to a mechanism of "unmasking" or of accessibility of the auxiliary receptor with respect to the C-terminal (downstream) protein domain, and, in the case when recognition does not occur between the aforesaid gene-transfer vector and the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) protein domain, a mechanism of "masking" or of non-accessibility is produced, by means of the aforesaid peptide, of the auxiliary receptor with respect to the C-terminal (downstream) protein domain.
The expression glycoprotein with targeting and gene-fusion activity denotes a glycoprotein which is: 1) capable of being incorporated efficiently on (retro)viral particles carrying a transgene, 2) capable of specifically recognizing the targeted cell-surface molecule and of specifically redirecting the binding of the (retro)viral particle which carries it to this molecule, 3) capable of causing fusion, after fixation on the molecular target, of the membrane of the (retro)viral particle and the cytoplasmic membrane of the cell, according to the mechanism used naturally by the (retro)virus from which the envelope glycoprotein was derived.
The expression "substantially intact" refers to a viral glycoprotein that retains all its necessary determinants for preserving the post-translation processes: oligomerization, the properties of viral incorporation and of fusion, as required. However, certain changes (such as mutations, deletions, additions) can be made to the glycoprotein without significantly affecting its functions and the glycoproteins containing these minor changes are regarded as substantially intact for the needs of the invention. In particular, the glycoprotein may lack some amino acids (for example about I to 10), especially at the N-terminal end, but will generally be of the same size as the wild-type protein and possesses essentially the same biological properties as the wild-type protein.
The expression "viral recombinant gene-transfer vector" means any virus capable of infecting cells of the eukaryotic type, and preferably a virus that is suitable for gene therapy, such as an adenovirus or a retrovirus (for example a type C retrovirus).
The expression "non-viral recombinant gene-transfer vector" means macromolecular complexes combining the DNA containing the transferred gene, its regulatory sequences, and molecules belonging to the class of lipids, carbohydrates, or proteins, which possess functional properties capable of: 1) targeting deposition of DNA on the surface of the target cell, 2) introducing this DNA into the targeted cell, and 3) introducing this DNA into the nucleus of the targeted cell.
The expression "process of cell entry of the viral recombinant gene-transfer vector" means all of the events leading to introduction of the transported gene into the cytoplasm of the targeted cell following initial contact between the surface of this cell and the gene-transfer vector.
As an example, for retroviral vectors, in relation to a defined cellular target for which a "targetable" surface molecule is known sufficiently specific relative to the other tissues) and a ligand for the surface molecule (ligand or single-chain antibody), a gene coding for the envelope glycoprotein targeting this surface molecule can be constructed genetically. This is accomplished by fusing (from N to C-terminal) a signal peptide, the ligand, the "masking/unmasking" peptide, and the rest of the retroviral envelope. An expression vector for this chimeric molecule is inserted into a "semitranscomplementing" cell line expressing the gag and pol proteins of the MLV virus (coding for the viral capsid and the enzymes of replication of the retrovirus). A "transcomplementing" line is obtained, which can then be used for producing retroviral vectors if a plasmid carrying this retroviral vector is additionally introduced, as occurs with the conventional transcomplementing lines expressing normal retroviral envelopes.
The invention also relates to the use of a peptide according to the invention, in the construction of an essentially intact (retro)viral envelope glycoprotein, carried by a recombinant (retro)viral particle capable of infecting a eukaryotic cell, the said envelope glycoprotein preferably being of polymeric form, and especially of trimeric form, each monomer of the polymeric form being in its turn of heterodimer form, the said eukaryotic cell possessing a targeted receptor and an auxiliary receptor permitting facilitation of entry of the aforesaid (retro)viral particle ((retro)viral receptor) into the eukaryotic cell, the envelope glycoprotein comprising: the aforesaid peptide, a protein domain on the N-terminal side (upstream) of the aforesaid peptide, capable of interacting with the aforesaid targeted receptor, this interaction permitting specific binding of the (retro)viral particle and a protein domain on the C-terminal side (downstream) of the aforesaid peptide, capable of interacting with the aforesaid (retro)viral receptor, this interaction performing the role of auxiliary mechanism of entry of the (retro)viral particle into the eukaryotic cell, the process of cell entry of the (retro)viral recombinant particle into the eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to occur when the N-terminal (upstream) protein domain has recognized and bound the targeted receptor of the eukaryotic cell with the (retro)viral recombinant particle, leading, via the aforesaid peptide, to a mechanism of "unmasking" or of accessibility of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain, and, in the case when recognition does not occur between the viral recombinant particle and the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) protein domain, a mechanism of "masking" or of non-accessibility is produced, by means of the aforesaid peptide, of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain.
The (retro)viral envelope glycoproteins are trimers of heterodimers with surface subunit (SU) and transmembrane subunit This concept of trimerization is fundamental for the functionality of the (retro)viral envelope. The envelope glycoproteins of the invention are preferably of trimeric form.
According to an advantageous embodiment of the invention, the N-terminal (upstream) protein domain is chosen from the following polypeptides: single-strand antibodies recognizing cell-surface molecules, any ligand for a cell-surface molecule, especially polypeptide hormones, cytokine, growth factors.
According to an advantageous embodiment of the invention, the C-terminal (downstream) protein domain corresponds to a (retro)viral envelope glycoprotein, essentially intact, including the natural binding domain, the functions of fusion and of attachment of the wild-type envelope glycoprotein from which is derived the envelope glycoprotein carried by the recombinant (retro)viral particle.
According to an advantageous embodiment of the invention, the peptide originates from the envelope glycoprotein of type C retroviruses, and in that the virus is preferably chosen from: the ecotropic MLV virus, the amphotropic MLV virus, the xenotropic MLV virus, the MCF MLV virus, the MLV 10Al virus, GALV (Gibbon Ape Leukemia Virus), SSAV (Simian Sarcoma Associated Virus), FeLV A, FeLV B, FeLV C (FeLV: Feline Leukemia Virus), and especially in that the peptide is chosen from those containing or consisting of one of the following sequences: PRO (4070A), PRO(MoMLV), APRO, PRO+, APRO+, PROP, APROp, APRO4-3, APRO4-int, APRO4-vrb, PROp, PRO-int, PRO-vrb.
The invention relates to the use of a peptide derived or adapted from bovine elastin and chosen from those containing or consisting of one of the following sequences: EL3, EL3-V, ELS.
The invention also relates to peptide sequences chosen from those containing or consisting of one of the following sequences: PRO (4070A), PRO(MoMLV), PROP, PRO+, APRO, APROP, DPRO+, MOAPRO, MOAAPRO, EMOPRO, EMOPROP, EMOPRO+, EAPRO, EAPROP, EAPRO+, EMOAPRO, EMOAPROB, EMOAPRO+, EAAPRO, EAAPROB, EAAPRO+, AMOEL3, AMOEL3- V, PRO (4070A), PRO(MoMLV), PROP, PRO+, APRO, APROI, APRO+, EL3, EL3-V, EL5 are masking/unmasking peptides of the invention.
AMOPRO, AMOAPRO, AMOEL3, AMOEL3-V, AMOEL5 correspond to Ram-1 targeting envelopes.
MOAPRO, MOAAPRO correspond to Rec-1 targeting envelopes.
EMOPRO, EMOPROP, EMOPRO+, EAPRO, EAPRO3, EAPRO+, EMOAPRO, EMOAPROP, EMOAPRO+, EAAPRO, EAAPRO, EAAPRO+ correspond to EGFR targeting envelopes.
The invention also relates to a polypeptide sequence containing a peptide of about 10 to about 200, especially from about 15 to about 150 amino acids, and preferably about 20, in which at least 30% of the amino acids consist of proline residues, and these proline residues are regularly arranged so as to induce turnings of the polypeptide chain at about 1800 ("p-turn" or "reverse-turn"), these turnings being regularly spaced and assembling themselves into a polyproline p-turn helix, an N-terminal protein domain (upstream) of the aforesaid peptide, capable of reacting with a suitable receptor (targeted receptor) located on a eukaryotic cell, and this protein domain permits specific binding of a recombinant (retro)viral particle containing the said N-terminal protein domain and a C-terminal protein domain (downstream) of the aforesaid peptide, capable of interacting with a suitable auxiliary (retro)viral receptor ((retro)viral receptor) located on the said eukaryotic cell, and this interaction performs the role of auxiliary mechanism of entry of the (retro)viral particle into the said eukaryotic cell, the process of cell entry of the said recombinant (retro)viral particle into the said eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to occur when the N-terminal (upstream) protein domain has recognized and bound the targeted receptor of the eukaryotic cell with the said recombinant (retro)viral particle, leading, by means of the aforesaid peptide, to a mechanism of unmasking or of accessibility of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain, and, in the case when recognition does not occur between the recombinant viral particle and the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) protein domain, there is produced a mechanism of masking or of non-accessibility, by means of the aforesaid peptide, of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain.
The invention also relates to a recombinant (retro)viral particle capable of infecting a eukaryotic cell, this cell containing a targeted receptor and an auxiliary receptor of the aforesaid (retro)viral particle, including a substantially intact envelope glycoprotein, especially of polymeric form and preferably of trimeric form, each monomer of the polymeric form preferably being itself of heterodimer form, containing a peptide of about 10 to about 200, especially of about 15 to about 150 amino acids, and preferably of about 20, in which at least 30% of the amino acids are made up of proline residues, these proline residues being regularly arranged so as to induce turnings of the polypeptide chain at about 1800 ("p-turn" or "reverse-turn"), these turnings being regularly spaced and assembling themselves into a polyproline 0-turn helix, a protein domain on the N-terminal side (upstream) of the aforesaid peptide, capable of interacting with the aforesaid targeted receptor, this peptide region permitting specific binding of the (retro)viral particle and a protein domain on the C-terminal side (downstream) of the aforesaid peptide, capable of interacting with the aforesaid (retro)viral receptor, this interaction performing the role of auxiliary mechanism of entry of the (retro)viral particle into the eukaryotic cell, the process of cell entry of the recombinant (retro)viral particle into the eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to occur when the N-terminal (upstream) protein domain has recognized and bound the targeted receptor of the eukaryotic cell with the recombinant (retro)viral particle, leading, via the aforesaid peptide, to a mechanism of unmasking or of accessibility of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain, and, in the case when recognition does not occur between the recombinant viral particle and the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) protein domain, there is produced a mechanism of masking or of non-accessibility, via the aforesaid peptide, of the retroviral receptor with respect to the C-terminal (downstream) protein domain.
The invention also relates to a recombinant (retro)viral particle characterized in that the N-terminal (upstream) protein domain is chosen from the following peptides: single-strand antibody recognizing cell surface molecules, any ligand for a cell surface molecule, especially polypeptide hormones, cytokine, growth factors.
The invention also relates to a recombinant (retro)viral particle characterized in that the C-terminal (downstream) protein domain corresponds to a polypeptide of (retro)viral origin possessing functions of binding, of fusion and of attachment of the wild-type envelope glycoprotein from which is derived the envelope glycoprotein carried by the recombinant (retro)viral particle, and can originate from natural regions possessing functions of binding, of fusion and of attachment of the envelope glycoproteins derived from retroviruses MLV-A, GALV, FeLVB, or viruses such as adenoviruses, herpesviruses, AAV (Adeno Associated Virus), or more generally from viral glycoproteins derived from viruses of eukaryotic origin, especially orthomyxoviruses (such as influenza viruses) or paramyxoviruses (such as The invention also relates to a recombinant (retro)viral particle characterized in that the peptide is derived from the envelope glycoprotein of type C retroviruses, and in that the peptide is preferably derived from a virus chosen from: ecotropic MLV virus, amphotropic MLV virus, xenotropic MLV virus, MLV MCF virus, MLV 10A1 virus, GALV (Gibbon Ape Leukemia Virus), SSAV (Simian Sarcoma Associated Virus), FeLV A, FeLV B, FeLV C (FeLV: Feline Leukemia Virus), and especially in that the peptide is chosen from those containing or consisting of one of the following sequences: PRO (4070A), PRO(MoMLV), APRO, PRO+, APRO+, PROP, APROP, APRO4-0, APR04int, APRO4-vrb, PROP, PRO-int, PRO-vrb.
The invention also relates to a recombinant (retro)viral particle characterized in that: the peptide originates from the envelope glycoprotein of type C retroviruses, and in that the virus is preferably chosen from: ecotropic MLV virus, amphotropic MLV virus, xenotropic MLV virus, MLV MCF virus, MLV 10A1 virus, GALV (Gibbon Ape Leukemia Virus), SSAV (Simian Sarcoma Associated Virus), FeLV A, FeLV B, FeLV C (FeLV: Feline Leukemia Virus), and especially in that the peptide is chosen from those containing or consisting of one of the following sequences: PRO (4070A), PRO(MoMLV), APRO, PRO+, APRO+, PROp, APROP, APRO4-, APRO4-int, APRO4-vrb, PROP, PRO-int, PRO-vrb, the N-terminal (upstream) protein domain is chosen from the following peptides: single-strand antibodies recognizing cell surface molecules, any ligand for a cell surface molecule, especially polypeptide hormones, cytokine, growth factors, the C-terminal protein domain corresponds to a polypeptide of (retro)viral origin possessing functions of binding, fusion and attachment of the wild-type envelope glycoprotein from which is derived the envelope glycoprotein carried by the recombinant (retro)viral particle, and can originate from natural regions possessing functions of binding, of fusion and of attachment of the envelope glycoproteins derived from retroviruses MLV-A, GALV, FeLVB, or from viruses such as adenoviruses, herpesviruses, AAV (Adeno Associated Virus), or more generally from viral glycoproteins derived from viruses of eukaryotic origin, especially orthomyxoviruses (such as influenza viruses) or paramyxoviruses (such as 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 (downstream) protein domain.
The invention also relates to a nucleic acid coding for a peptide or for a recombinant particle according to the invention.
The invention also relates to a method of selective in vitro or ex vivo transfer of a nucleic acid into eukaryotic target cells present among other non-target cells, comprising the administration to the target and non-target cells, of a recombinant (retro)viral particle according to the invention, containing the nucleic acid to be transferred.
The invention also relates to a pharmaceutical composition containing, as active substance, a (retro)viral particle according to the invention, and also containing a gene to be transferred, together with a physiologically suitable pharmaceutical vehicle.
With regard to genes to be transferred that are important for gene therapy, these are for example IFN, IL2, p53, VEGF, TNF, CFTR, HSV-TK, lacZ, GFP, gene of various cytokines, other types of suicide genes including conditional suicide genes, other genes with antiviral activity, other genes with antitumour activity, other marker genes and any gene for therapy of a mono- or multi-genic disease. As an example, the pathologies most specifically involved are: most mono- or multi-genic diseases (mucoviscidosis, myopathy, lysosomal diseases, various forms of cancer, viral diseases (AIDS), etc.).
For a proper understanding of the mechanism of the invention (see Fig. we must bear in mind that the envelope glycoproteins according to the invention (also denoted by "chimeric envelopes") possess, as well as an additional recognition region, the functions corresponding to their own particular regions; that is (see Fig. 2), 1) the natural binding domain located in the N-terminal part of the surface subunit (SU) of the wild-type envelope glycoprotein, and therefore just downstream of the supernumerary binding domain and 2) the fusion domain located in the C-terminal part of the subunit (SU) and in the transmembrane subunit (TM) of the envelope glycoprotein complex. For the chimeric envelopes constructed previously (EMO and AMO envelopes, for example), on the basis of the general structure shown diagrammatically in Fig. 2, the natural binding domain is functional. If the retroviral receptor that it recognizes is expressed at the surface of the target cell, then this domain will recognize it, and will permit infection to proceed. Then there will be no possibility of specific targeting, even if a surface molecule specifically recognizing the supernumerary binding domain is also expressed.
However, depending on the peptide inserted between the supernumerary binding 13 domain and the natural binding domain, it is possible for the functionality of the natural binding domain to be adjusted considerably and, for some of these peptides, there can be effective prevention of its accessibility for recognition of the retroviral receptor (first action). It will be possible for this site to be unmasked, and hence rendered accessible to interaction with the normal retroviral receptor, if and only if the supernumerary binding domain has previously interacted with the targeted surface molecule. This second action is also medicated by the peptide separating the two domains. Here the normal retroviral receptor plays the role of auxiliary molecule.
Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".
Symbols on the diagrams: Fig. 1 represents the two-stage entry process of the targeting viral particle. The 15 viral particles are generated with targeting envelope glycoproteins composed of an N-terminal domain (ligand, single-strand antibody etc.), of the masking/unmasking peptide, and a C-terminal domain The stages giving rise to introduction of the virion into the targeted cell involve a mechanism that is coordinated by the masking/unmasking peptide 20 Fig. 2 is a schematic representation of some of the targeting envelopes investigated. The position of some functional regions is shown. Vertical arrows: sites of proteolytic cleavage. SU: surface subunit, TM: transmembrane subunit, SP: signal peptide, PRO: polyproline 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 grey boxes: sequences defined from the env gene of MoMLV, Light grey boxes: sequences derived from the env gene of MLV- 4070A, White boxes: other sequences derived from MLVs. Black boxes: spacer peptides derived from the polyproline region. All the env genes are expressed starting from the same promoter (LTR) and polyadenylation signal (pA) starting from the subgenomic mRNA using the retroviral splicing sites, donor (SD) and acceptor with -13aan identical intron sequence of 190 not containing the end of the pol gene (APOL). The position of some restriction sites is shown.
Fig. 3 shows the sequence of the spacer peptides and of the binding domains investigated. Sequence of the spacer peptides in the series AMO, AS208 and fused with the various spacer peptides, and the whole is fused with codon 7 of the SU of the envelope of the MoMLV. Sequence of the spacer peptides in the series MOA. The binding domain at Rec-1 is fused with the various spacer peptides, and the whole is fused with codon 5 of the SU of the envelope of the amphotropic MLV. Sequence of the spacer peptides in the series EMO and EA. The binding domain EGF is fused with the various spacer peptides, and the whole is fused with codon 5 of the SU of the envelope of the amphotropic MLV or with codon 7 of the SU of the envelope of the MoMLV.
oooo o *o o oo Fig. 4 shows detection of membrane expression of the envelopes of the EMO series. Populations of transfected cells, selected using phleomycin, are marked with (black histograms) or without (white histograms) anti-hEGF antibodies, then with anti- IgG mouse antibodies combined with FITC.
Fig. 5 shows expression and viral incorporation of the chimeric envelopes of the AMO series. Immunoblots on lysates of TELCeB6 cells transfected by the plasmids expressing the chimeric envelopes (see Fig. 2 and Fig. 3A) and on deposits of viral particles purified by ultracentrifugation. The immunoblots are detected with an anti-SU antiserum (top part) or with an anti-p30-CA antiserum (bottom part, size less than 46 KD). The positions of the p30-CA (CA) and, for the MO wild-type envelopes, of the precursor (PR) and of the surface protein (SU) of the envelope complex are shown.
Fig. 6 shows binding tests on human cells of the envelopes of series EMO (A) and AMO The background noise of fluorescence is provided by incubation of human cells with the ecotropic envelope (white histograms), Fig. 7 shows the amino-acid and nucleotide sequence of PRO(4070A).
Fig. 8 shows the amino-acid and nucleotide sequence of PRO(MoMLV).
Fig. 9 shows the amino-acid and nucleotide sequence of PROp(MoMLV).
Fig. 10 shows the amino-acid and nucleotide sequence of PRO+(4070A).
Fig. 11 shows the amino-acid and nucleotide sequence of APRO.
Fig. 12 shows the amino-acid and nucleotide sequence of APRO3.
Fig. 13 shows the amino-acid and nucleotide sequence of APRO+.
Fig. 14 shows the amino-acid and nucleotide sequence of AMOPRO.
Fig. 15 shows the amino-acid and nucleotide sequence of AMOAPRO.
Fig. 16 shows the amino-acid and nucleotide sequence of MOAPRO.
Fig. 17 shows the amino-acid and nucleotide sequence of MOAAPRO.
Fig. 18 shows the amino-acid and nucleotide sequence of EMOPRO.
Fig. 19 shows the amino-acid and nucleotide sequence of EMOPROI.
Fig. 20 shows the amino-acid and nucleotide sequence of EMOPRO+.
Fig. 21 shows the amino-acid and nucleotide sequence of EAPRO.
Fig. 22 shows the amino-acid and nucleotide sequence of EAPRO3.
Fig. 23 shows the amino-acid and nucleotide sequence of EAPRO+.
Fig. 24 shows the amino-acid and nucleotide sequence of EMOAPRO.
Fig. 25 shows the amino-acid and nucleotide sequence of EMOAPRO3.
Fig. 26 shows the amino-acid and nucleotide sequence of EMOAPRO+.
Fig. 27 shows the amino-acid and nucleotide sequence of EAAPRO.
Fig. 28 shows the amino-acid and nucleotide sequence of EAAPROp.
Fig. 29 shows the amino-acid and nucleotide sequence of EAAPRO+.
Fig. 30 shows the amino-acid and nucleotide sequence of AMOEL3.
Fig. 31 shows the amino-acid and nucleotide sequence of EL3.
Fig. 32 shows the amino-acid and nucleotide sequence of AMOEL3-V.
Fig. 33 shows the amino-acid and nucleotide sequence of EL3-V.
Fig. 34 shows the amino-acid and nucleotide sequence of Fig. 35 shows the amino-acid and nucleotide sequence of ELS.
Fig. 36 shows the amino-acid and nucleotide sequence of APRO4-beta.
Fig. 37 shows the amino-acid and nucleotide sequence of APR04-int.
Fig. 38 shows the amino-acid and nucleotide sequence of APRO4-vrb.
Fig. 39 shows the amino-acid and nucleotide sequence of PRO-beta.
Fig. 40 shows the amino-acid and nucleotide sequence of PRO-int.
Fig. 41 shows the amino-acid and nucleotide sequence of PRO-vrb.
EXAMPLES:
EXAMPLE 1: The retroviruses utilize a certain number of cell surface molecules, called viral receptors, for initiating the infectious process Apart from some notable exceptions, especially in the case of human immunodeficiency viruses, most of the receptors utilized by the other retroviruses and in particular the type C mammalian retroviruses are distributed over most cell types of the host organism. For example, the amphotropic murine leukemia virus (MLV-A) is capable of infecting the majority of mammalian cells because its receptor, the phosphate transporter Ram-1, is expressed on almost all the cells.
The type C mammalian retroviruses are currently used for making retroviral vectors, in particular for purposes of gene transfer in humans, in gene therapy. Certain gene therapy procedures would be facilitated if the retroviral vectors were capable of very accurately recognizing the true target cells of gene transfer. For this, a certain number of research groups, including ours, have developed various strategies aiming to modify the recognition between the viral particle and the cell surface. This interaction essentially involves the retroviral envelope glycoprotein; it therefore seems logical to make genetic changes to this protein so as to enable it to recognize cell surface molecules specifically expressed on the target cells of gene transfer.
Two types of strategies permitting such changes have been developed recently.
In the first strategy, the natural binding domain of the retroviral envelope glycoprotein for its receptor was altered by insertion or substitution of peptides of reduced size that are able to bind a cell surface molecule. This work has demonstrated the feasibility of cell targeting for gene transfer In the second approach, polypeptides (ligands, single-strand antibodies) capable of binding various cell surface molecules were inserted at the N-terminal end of the SU subunit of the envelope glycoprotein (10) (13) (15) In general, investigation of the virions generated with these various types of targeting envelopes sliowed that it was possible for the binding of viral particles to be redirected specifically and efficiently towards new surface molecules. Some factors limiting the efficacy of targeting were also identified. The first seems to depend on physiological properties of the surface molecule targeted (dimerization, internalization, intracellular transport ("trafficking") process) the second is connected with the low intrinsic gene-fusion capacity of the chimeric envelopes generated by N-terminal insertion of ligands It was observed that this low gene-fusion capacity can be partially overcome by introducing a spacer peptide between the new binding domain and the envelope However, the best infectious titres obtained are 100 times lower than can be obtained with retroviral vectors bearing a wild-type envelope. Moreover, it is possible that these results obtained in a particular targeting model (targeting of Ram-I) cannot be extended to other types of targeting envelope glycoproteins. It therefore seemed essential to develop alternative strategies to solve these problems.
Furthermore, a general finding made with the targeting envelope glycoproteins generated by N-terminal insertions is that the natural binding domain of the supporting envelope is always functional. To the extent that the target cells are human cells in gene therapy, this functionality of the natural binding domain does not pose problems of "background noise" of infection because the supporting glycoprotein used is the ecotropic envelope of the MoMLV virus which does not recognize a receptor on the cells of higher mammals. However, it seemed interesting to characterize these chimeric envelope glycoproteins that are able to recognize two different surface molecules, to see what influence the spacer peptide could have in this recognition, and to assess the relative contributions of the two types of interaction in the infectious process.
These observations, which form the subject of the work described below, led to the development of a two-stage targeting strategy, firstly involving specific recognition between the ligand inserted at the N-terminal end of the targeting glycoprotein, and then an auxiliary mechanism making it possible to facilitate entry of the virus specifically bound to the good cellular target by means of the natural retroviral receptor. To avoid any problem of background noise of infection connected with direct interaction between the natural binding domain and the natural retroviral receptor, masking/unmasking spacer peptides were also developed, inserted between the targeting site and the supporting envelope glycoprotein, and which are able to mask the natural binding domain for as long as the viral particle has not interacted with the targeted surface molecule. Realization of this interaction induces unmasking of the natural binding domain and interaction between the natural binding domain and the natural retroviral receptor (auxiliary mechanism) which then takes over for introducing the virus into the cell.
Equipment and Methods: Cell lines.
The cell line TELCeB6 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). The TELacZ cells express the retroviral vector MFGnlslacZ which is able to transduce a nuclear P-galactosidase. The TELCeB6 cells permit production of retroviral capsids (non-infectious, as they are devoid of envelopes) transporting the nlsLacZ retroviral marker vector. Cells A431 (ATCC CRL1555) and TE671 (ATCC CRL8805) are cultivated in DMEM medium (Gibco-BRL) supplemented with 10% of foetal calf serum (Gibco-BRL). Cells CHO, CERD9 and CEAR13 (9) are cultivated in DMEM medium (Gibco-BRL) supplemented with 10% of foetal calf serum and proline (Gibco-BRL). The NIH-3T3 cell lines and NIH-3T3 derivatives are cultivated in DMEM medium (Gibco-BRL) supplemented with 10% of newborn calf serum (Gibco-BRL).
Chimeric envelopes.
The DNA fragments coding for the polypeptides recognizing either EGFR (EGF receptor) or Ram-1 (MLV-A receptor) were generated after PCR (polymerase chain reaction) by using oligonucleotides containing restriction sites. These polypeptides were introduced at the N-terminal of the SU protein of MLV (surface protein gp70) in which the Sfil and NotI restriction sites were created at codon +6 A schematic diagram of the various env genes used in this article is shown in Fig. 2. Briefly, a DNA fragment derived from PCR amplification, coding for the 53 amino acids of human EGF was generated using a cDNA matrix (ATCC 59957) and two oligonucleotides: OUEGF:
ATGCTCAGAGGGGTCAGTACGGCCCAGCCGGCCATGGCCAATAGTGAC
TCTGAATGTCC)
with an SfiI restriction site and OLEGF:
ACCTGAAGTGGTGGGAACTGCGCGCGGCCGCATGTGGGGGTCCAGACT
CC)
containing a NotI site. After digestion by Sfil and NotI, these fragments were cloned in a gene coding either for the SU protein of MoMLV in the case of the chimeric protein EMO, or SU of the 4070A virus for the chimeric protein EA For the AMO construct a site NotI was created at the end of the recognition domain of the receptor in the 4070A envelope (called AS208), and the nucleotide (nt) 750 (14) using a PCR fragment generated from the XhoI site (nt 594) up to nt 750 before the proline-rich region, owing to two oligonucleotides: 805FC TCCAATTCCTTCCAAGGGGC) upstream of XhoI and 806FC ACCCCCACATGCGGCCGCTCCCACATTAAGGACCTGCCG) containing a NotI restriction site. The chimeric envelope is constructed by cloning of the XhoI/NotI PCR fragment and of the NotI/ClaI fragment, isolated from the env EMO gene (coding for the SU and TM- P15E transmembrane proteins of MoMLV), between the XhoI/ClaI sites of the env gene 4070A MLV.
The resulting constructs are recovered in the form of a BglII/ClaI fragment (corresponding to positions 5408 and 7676 in MoMLV) and cloned at sites BamHI and Clal of an FBMOSALF expression plasmid in which a selection marker gene (8) fused to the polyadenylation sequences of the PGK (phosphoglycerate kinase) gene and was introduced downstream of the LTR 3' of the MLV-C57 virus.
For EMO, EA, or AMO, the new recognition site was separated from the rest of the MLV envelope by a spacer peptide consisting of three alanines, supplied by the NotI cloning site In three other series of targeting envelopes (derived from envelopes EMO, EA or AMO), spacer amino acids were introduced either after the recognition domain of EGFR (EGF), or after the recognition domain of Ram-I (AS208) as described below.
The series of envelopes targeting Ram-1 was generated by introducing different spacers between the recognition domain of Ram-1 and the MoMLV envelope (Fig. 3A).
For AMOPRO, a region of 59 amino acids rich in proline originating from SU 4070A (amphotropic) (nucleotides 751 to 927) (14) was used. A shorter proline-rich region, also isolated from the envelope MLV 4070A (nt 751-789) was used for AMOAPRO.
This region corresponds to the 13 amino acid spacer of product v-mpl (originating from the virus of myeloproliferating leukemia) (18) located between its region derived from env and the equivalent of the cellular gene mpl.
In the case of AMO1, the first 208 amino acids, derived from the envelope of MLV 4070A, were fused to amino acid 1 of the SU of MoMLV. For AMOIFx, a 4 amino acid site corresponding to the cleavage site of blood coagulation factor Xa (Ile- Glu-Gly-Arg) (12) was inserted after the Ram-1 recognition site and fused to the +1 codon of the SU of MoMLV. The strategy used for these constructs is described above.
Briefly, an oligonucleotide (5'-TCCAATTCCTTCCAAGGGGC-3'), located just upstream of the XhoI site of the env gene of 4070A (nt 594) was used in combination with one or other of the following two oligonucleotides bearing the Not I site: 5'-AGTATGCGGCCGCTGGGGGTGGCTGTGGGACAC-3' and 5'-TATCTGCGGCCGCGTCGGGTAATACTGGGTTGG-3' so as to generate by PCR, using an env 4070A matrix, 3' fragments for the AMOPRO and AMOAPRO envelopes respectively.
These PCR fragments were submitted to digestion by Xhol and NotI and cloned in the open FBAMOSALF plasmid in Xhol/NotI, a plasmid expressing an AMO type of envelope. The plasmids expressing the envelopes AMOFx, AMOI and AMOIFx were 19 generated by cloning the NdeI/NotI fragment of FBAMOSALF containing the Ram-1 recognition site) in a series of plasmids (13) expressing the modified MoMLV envelopes so as to create a NotI site at codon 1 or at codon 6 with (AMOIFx, AMOFx) or without (AMO1) the Xa sequence. Envelopes derived from AMO and containing other types of spacer peptides were constructed. All of these spacer peptides are shown in Fig. 3A.
The MOAPRO and MOAAPRO envelopes were generated according to a method similar to that of the AMOPRO and AMOAPRO envelopes. The FBEASALF plasmid, expressing the EA envelopes, was opened at NedI/NotI. This DNA was used for cloning two fragments: the 5' NdeI/BamHl fragment from digestion of the FBMOSALF plasmid (expressing the ecotropic MO envelopes) and containing, in addition to LTR5' and the retroviral leader sequence, the N-terminal end of the env gene of the MoMLV virus (position 6565), 3' fragments were generated by PCR using the env gene of MoMLV as matrix, as oligonucleotide ACTGGGCTTACGTTTGT-3') upstream of the BamH site, and as oligonucleotide 3' (5'-TATGTGCGGCCGCCGGTGGAAGTTGGGTAGGGG-3') or (5'-TATGTGCGGCCGCGTCTGGCAGAACGGGGTTTGG-3') for constructing the MOAPRO and MOAAPRO envelopes, respectively. These PCR fragments were digested with BamHI and NotI, and co-ligated with the 5' fragment. The sequence of the spacer peptides for these two constructs is shown in Fig. 3B.
FBEMOSALF, expressing the EMO chimeric envelopes was submitted to digestion by BaaHII, filling by Klenow enzyme and digestion by NdeI. The resulting 1.8 Kb fragment, containing the LTRS', the leader sequence, the end of the pol gene and human EGF, was isolated and inserted either in FBAMOAPROSALF or FBAMOPROSALF (plasmids expressing the AMOAPRO and AMOPRO chimeric envelopes respectively) in which the NdeI/EcoRI fragment was eliminated and the EcoRI site was filled so as to generate the plasmids expressing the envelopes EMOAPRO+ and EMOPRO+, respectively. Plasmids expressing the envelopes EMOI, EMO1FX were also generated. The sequence of the spacer peptides for these two constructs is shown in Fig. 3C.
The plasmids expressing the EAPRO+ and EAAPRO+ envelopes were generated by replacing the Sfil/Not fragment of the FBEASALF plasmid by the SfiI/NotI fragments obtained from plasmids expressing the EMOPRO+ and EMOAPRO+ envelopes.
Finally for these various envelopes EMOAPRO+, EMOPRO+, EAPRO+ and EAAPRO+, the spacer peptides were reduced in their N-terminal part. For this, a DNA fragment was generated by PCR using as matrix the EMO gene, oligonucleotide 5' ACCATCCTCTAGACGGACATG-3') upstream of the XbaI site preceding the initiator codon and as oligonucleotide 3' CGCAGTTCCCACCACTTCAGGTCTCGGTACTGAC-3') containing a BamHI site.
This DNA was digested with XbaI/BamHI and cloned in one or other of the plasmids expressing the EMOPRO+ or EMOAPRO+ envelopes, after removing the XbaI/NotI fragments beforehand, by co-ligation with the BamHI/NotI fragments obtained from the plasmids expressing the MOAPRO and MOAAPRO envelopes. This results in two plasmids that are able to express the EMOPROP and EMOAPRO3 envelopes, respectively (Fig. 3C), in which EGF is fused just upstream of the BamHI site of the envelope of the MoMLV virus (nt 6537), (17) before the proline-rich region and leaving intact the potential b sheet. One or other of the SfiI/NotI fragments resulting from these last two constructs was then introduced into the FBEASALF plasmid after prior removal of the Sfil/Not fragment; this results in two plasmids capable of expressing the EAPROP and EAAPROp envelopes, respectively (Fig. 3C).
In another construction series (EMOPRO, EMOAPRO, EAPRO, EAAPRO), the potential P sheet was removed, and EGF was fused directly at the level of the prolinerich region (Fig. 3C).
Production of viruses.
The plasmids expressing the envelopes were transfected by the calcium phosphate precipitate method (16) in the TeLCeB6 cell line. The cells were submitted to selection with phleomycin (50 mg/ml), then the resistant clones were trypsinized in the bulk.
These confluence cells were used for recovering the viral supernatants after incubation over night in DMEM medium in the presence of FCS These supernatants are submitted to ultracentrifugation with the aim of obtaining samples for analysis in Western blots, in binding tests and in infection tests. Immunoblots. The virus-producing cells are lysed for 10 min at 4°C in buffer of Tris-HCL 20mM (pH containing triton X100 SDS 0.05%, deoxycholate 5 mg/ml, NaCI 150 mM and PMSF 1 mM. After centrifugation for 10 min at 10 000 g, for deposition of the cell nuclei, the supernatants are frozen at -70 0 C until analysis. These viral samples are obtained by ultracentrifugation of the viral supernatants (10 ml) in a SW41 Beckman rotor (30 000 rpm, 1 h at 4°C).
The deposits are resuspended in 100 ml of PBS (phosphate buffered saline) and frozen at -70 0 C. The samples (30 mg of cellular lysates or 10 ml of purified viruses) are mixed in a ratio of 5:1 with buffer of 375 mM Tris-HCl (pH 6.8) containing SDS bmercaptoethanol 30%, glycerol 10% and bromophenol blue 0.06%, then boiled for 3 min and analysed on acrylamide 10%/SDS gels. After transferring the proteins onto nitrocellulose membrane, immunologic marking is effected in TBS (Tris base saline, pH 7.4) in the presence of skimmed milk 5% and Tween Antibodies (Quality Biotech Inc., USA) obtained from goat antiserum, directed against gp70-SU of RLV (Rauscher Leukemia Virus) or p30 of RLV were used at a dilution of 1:1000 or 1/10000 respectively. The blots were developed using a conjugated antibody of rabbit origin f, 1- 21 directed against goat immunoglobulins (DAKO, UK) using an electrochemoluminescence kit (Amersham Life Science).
Binding tests.
The target cells were washed with PBS and separated by incubation for 10 min at 37°C with Versene 0.02% in PBS. These cells are rinsed with PBA (PBS containing 2% of FCS and sodium azide 106 cells are then incubated in the presence of viruses for 30 min at 4°C for the EMO envelope series or 45 min at 37 0 C for the AMO envelope series. After rinsing with PBA, the cells are incubated in the presence of 83A25 monoclonal antibodies (Evans et al., 1990) for 30 min at 4 0 C. After rinsing twice with PBA, the cells are incubated for 30 min at 4 0 C in the presence of conjugated anti-rat antibodies combined with FITC (Dako; UK). 5 min before the two final rinsings in PBA, the cells are counterstained with propidium iodide (20 mg/ml). The fluorescence of the live cells is analysed in a FACS (FACScalibur, Beckton Dickinson).
Infection tests.
The target cells are inoculated in 24-well culture plates at a density of 3.104 cells per well. Various dilutions of the viral supernatants, containing Polybrene at 4 mg/ml, are added to the cells for 3 to 5 h at 37 0 C. The supernatants are then replaced with fresh medium and the cells are incubated for 24 to 48 h at 37 0 C. X-gal staining is then carried out as described previously The viral titres are estimated as reported previously in number of colonies per ml (lacZ i.u./ml).
In order to block the EGFRs, the target cells are incubated for 30 min at 37 0 C in a medium containing 10" M of human recombinant EGF (236-EG, R&D Systems,
UK).
The cells are then rinsed and infections are carried out as described previously. To block 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.
Construction of the mutant envelopes.
Two series of modified envelopes capable of recognizing either the retroviral receptor Ram-I or the EGF receptor were generated. A first envelope targeting Ram-1, AMO, was constructed by insertion, at the N-terminal of the envelope of MoMLV (by fusion with codon of a polypeptide recognizing Ram-I (AS208, Fig.
3A) and corresponding to the first 208 amino acids of the SU of MLV-A The sequence coding for EGF was inserted in the env gene of MLV in position +6 of the SU of MoMLV (Fig. It had previously been demonstrated that this insertion site permits expression of a single-chain antibody fragment on the surface of virions In the case of the chimeric envelope EMO (Fig. human EGF was inserted in the envelope of MoMLV at the same position, whereas for the envelope EA, insertion was effected in the amphotropic envelope of MLV in position For the AMO, EMO and EA envelopes, the new binding domains were separated from the recognition domain of the retroviral receptor by a spacer peptide corresponding to three alanines. For the two types of parental envelopes targeting Ram-I or targeting EGFR, various constructs were then generated by insertion of spacers of different sizes and structures. The protein sequences of these different spacers are shown in Fig. 3A in the case of the envelopes targeting Ram-1 and in Fig. 3C for the envelopes targeting
EGFR.
The plasmids expressing the various envelopes, including the ecotropic (MO) and amphotropic control envelopes, were transfected into the cell line TELCeB6 which expresses the proteins coded by the gag and pol genes, as well as a retroviral vector nlsLacZ Expression and incorporation of the envelopes in the virions.
The protein lysates of the corresponding cells were analysed for the expression of envelopes by means of antibodies directed against the SU of MLV (Fig. 5) for most of the envelopes of the AMO series (not shown for the other chimeric envelopes). For all the chimeric envelopes, the precursors and the mature form SU of the envelopes could be detected at the expected size and at a level similar to the wild-type envelopes, suggesting that these chimeric envelopes are normally produced and matured.
Expression on the cell surface was determined by analyses of the producing cells in the FACS, using antibodies directed against the SU or using an anti-EGF monoclonal antibody. The cells transfected by the various envelopes can be marked by the anti-SU antibody (not shown). Only the cells expressing the EGF envelopes fusion envelopes can be marked by means of anti-EGF monoclonal antibodies (Fig. This demonstrates expression of the chimeric envelopes on the cell surface and correct folding of the EGF on the chimeric glycoproteins.
To demonstrate incorporation of the chimeric envelopes in the retroviral particles, the supernatants of the TELCeB6 cell lines transfected with the various envelopes were submitted to ultracentrifugation and the deposits of viral particles were recovered. These deposits were analysed by immunoblots for their expression of products of the gag gene (CAp30) and of the envelope proteins (Fig. 5 for most of the envelopes of the AMO series, not shown for the other chimeric envelopes). With the aim of comparing the efficiency of viral incorporation between the various chimeric envelopes, identical quantities of viral particles (determined by marking the gag proteins by means of anti-CAp30 antibodies) were deposited on the gels.
The SU proteins could be detected for all the mutants, at the expected size but at a rate slightly less than was observed for the wild-type envelopes. In the case of the AMOG2X and AMOG3X envelopes only, the efficiency of incorporation is appreciably lower relative to the wild-type envelopes. As expected, no envelope expression was observed in the deposits from TELacZ supernatants (not expressing gag and pol proteins) transfected by the various envelopes. These results show that the chimeric SU proteins are associated with retroviral particles.
Binding of the envelopes to the receptors.
Human cells expressing the receptors Ram-I and/or of EGF were used for this investigation. These cells are incubated in the presence of viral preparations and the binding of the viral envelopes on the target receptor is determined by analysis with the FACS with the aid of antibodies directed against the SU (Fig. 6B). As expected, no binding is observed in the case of viruses expressing MO ecotropic envelopes on the various human cells (not shown), whereas the viruses that have chimeric envelopes targeting Ram-I are able to bind to the TE671 cells with an efficiency similar to that observed for the viruses expressing unmodified amphotropic envelopes. All the envelopes targeting Ram-1, derived from AMO, are able to bind to the TE671 cells with a similar efficiency. This binding can be inhibited after competition by AS208 fragment (the purified recognition domain of Ram-1) which suggests that this recognition is specific (results not presented).
The envelopes targeting EGFR (EMO series) are moreover able to bind to the A431 cells, on EGFR expressor (Fig. 6A). This binding seems specific since preincubation of the A431 cells in the presence of EGF (inducing endocytosis of the EGFRs) inhibits this binding (not shown).
Ram-I and Rec-1 cooperation in infection.
Transduction of the retroviral vectors pseudotyped by the various targeting envelopes was measured on cells expressing different types of receptors: human cells TE671 expressing the EGF and Ram-I receptors; 3T3 cells expressing murine EGF, Rec-1 and Ram-1 receptors; CEARI3 cells expressing Rec-1 and Ram-1; CERD9 cells expressing only Rec-1. The titrations were carried out as described previously As expected, it was shown that the viruses pseudotyped by MO ecotropic envelopes were not capable of infecting the TE671 cells, but did permit infection of murine cells 3T3, CEARI3 and Cerd9 (with titres of the order of 10' lacZ Conversely, the viruses bearing the amphotropic A envelopes are able to infect the murine cells 3T3, CEAR13 and TE671 (with titres of the order of 10' lacZ i.u./ml).
The viruses that have chimeric AMO envelopes are able to infect the TE671 cells at a titre of 4.103 lacZ i.u./ml (Table In comparison, despite a similar efficiency of binding to the receptor (Fig. 6B), the titres obtained with the wild-type envelopes are 000 times higher. Surprisingly, the viruses expressing AMOPRO envelopes, despite good efficiency of binding, proved incapable of infecting the human cells TE671.
Compared with the titres obtained for the AMO envelopes (Table the other types of spacers inserted in the envelopes of the AMO series permit an increase in titres from fold (for AMODPRO) to more than 100-fold (for AMOlFx) making it possible to reach titres of 4.105 lacZ i.u./ml. It has been shown that these infections take place via the targeted receptor Ram-1. This was demonstrated by an interference test on target cells chronically infected with MLV-A virus. These cells become specifically refractory to infection by viruses bearing envelopes targeting Ram-1 (results not shown). The viruses bearing the chimeric envelopes in which the site for binding to Ram-1 was separated from the SU of MoMLV by various spacers proved very infectious on 3T3 cells.
Compared with the titres obtained for the AMO envelopes, an increase from 200-fold (for AMOPRO) to more than 1000-fold (for AMOlFx) in the viral titres was measured (Table 1).
Infection of the 3T3's is effected via Rec-1 or via Ram-1 (Table This can be demonstrated by interference tests carried out on 3T3 cells chronically infected either by MLV-A (blocking Ram-1) or by MoMLV (blocking Rec-1). The viruses expressing the AMO envelopes seem to be capable of infecting the 3T3's indiscriminately depending on whether one or the other, or both, Rec-1 and Ram-I receptors are available on the target cell. Compared with these AMO viruses, the viral particles containing the other envelopes capable of targeting Ram-1 are far less capable of infecting the 3T3's when only one of the two receptors is available. For example, when 100 particles (according to the titre determined on intact 3T3's) containing the AMOFx envelopes are used for infecting interfering 3T3's, 4 viruses are capable of infecting the cells if only Rec-1 is available and 2 viruses are capable of infecting the cells if only Ram-1 is available. This indicates a considerable loss of infectivity (more than 94% of the viruses are not infectious) when only one receptor is available compared with when both receptors are available. This also suggests that the two receptors Ram-1 and Rec-1 cooperate in infecting the 3T3's. It appears that this phenomenon of cooperation is even more marked in the case of viruses bearing the AMOPRO envelopes. These last-mentioned viruses can infect the 3T3's with difficulty when only Rec-l is available and cannot infect them at all when only Ram-1 is available. However, when Rec-I and Ram-1 are both available, infection is possible and titres of the order of 6xl0 4 lacZ i.u./ml can be obtained (Table 1).
For better characterization of this cooperation effect, infection tests were carried out using CHO cells as targets (naturally devoid of Ram-1 and Rec-1 receptors) altered so as to express either Rec-1 only (Cerd9 cells), or Rec-1 and Ram-1 (Cearl3 cells) or TE671 cells expressing Ram-1 only. Furthermore, other envelopes derived from the AMO envelope were generated. These envelopes possess other types of spacer peptides (see Fig. 3A) after the site targeting Ram-1, in particular flexible spacers. The results of a typical experiment are shown in Table 2. For each envelope, cooperativity indices were calculated as the ratio of the titre obtained on the cell type expressing just one receptor to the titre obtained on the cell type expressing both types of receptors. An index of 1 therefore indicates that the titre is the same, whether there is just one or both receptors. This is obviously the case with ecotropic or amphotropic wild-type envelopes.
An index less than 1 indicates that the titre is less good when a single receptor is expressed relative to when both are, and that both receptors are needed to promote infection. The lower this index is, the greater is the requirement for two receptors. As suggested in Table 1, the infectivity of the virions with the original AMO envelopes is not affected, whether there is a single type of receptor or both types (Table In fact, the indices are even greater than 1 suggesting that the simultaneous presence of the two receptors hampers the infectious process, perhaps because the two binding domains hinder each other. The situation is different for viruses with the AMO1Fx envelopes even though, compared with the AMO virions, their infectivity is at least 100 times better in the TE671 cells that express Ram-I only. This increase in infectivity via Ram-I can be explained by the increased size of the spacer peptide separating the two binding domains: it is possible that the AS208 site induces less steric hindrance with respect to the rest of the glycoprotein and that these envelopes can more easily induce the gene-fusion process. Moreover, the Cerd9 cells expressing Rec-I only are infected relatively easily by the AMO I Fx virions. However, in accordance with the results in Table 1, infection is facilitated by a factor of 10 when both molecules Ram-I and Rec-I are co-expressed (index of about 0.1) compared with when only one or the other of the two receptors is present. The envelopes with the "flexible" spacers (AMOGIFx, AMOG2, AMOG2Fx and AMOG3) seem to behave like the AMO1Fx envelopes with regard to infection via Rec-I expressed alone. However, infectivity by Ram-I expressed alone (RamID) tends to decrease as a function of the length of the spacer. This probably reflects a decrease in transmission of the gene-fusion signal following binding on Ram-I owing to the increase in distance between the AS208 domain and the fusion domain. With these envelopes as well, infection is favoured when the two receptors are co-expressed on the surface of the same cell.
As for the AMOIFx envelopes, but non-symmetrically (RamID similar, but Rec[D very different), the virions containing the AMOAPRO envelope can infect cells efficiently when Ram-I is expressed alone. For this envelope as well, infectivity is increased about 10-fold when Rec-I is also present on the cell surface. This difference is not due to the mere fact that the AMOAPRO virions utilize Rec-1 preferentially for infection. In fact, infection of cells on which Rec-I alone is available is extremely slight (Table I) or even undetectable (Table 2) compared with when Ram-I and Rec-I are coexpressed. The ReclD index is less than 105 (Table This also demonstrates that the two receptors can synergize infection. These results also suggest that the domain of binding to the ecotropic receptor Rec-I is not accessible when the AMOAPRO envelope is expressed on viral particles, and only becomes accessible if these virions interact with Ram-1 beforehand. It can also be suggested that following binding with Ram-1, the domain for binding to Rec-I is unmasked and recruited for facilitating the infectious process. It is possible that this masking/unmasking takes place according to an allosteric type of mechanism causing a change in conformation of the chimeric glycoprotein that is induced by the Ram-I/AS208 interaction and which involves the spacer peptide. It is likely that this mechanism is strongly dependent on the amino acid composition of the spacer peptide. With comparable size, there is a difference of at least 1000 times in the RecID's when the AMOAPRO virions are compared with the virions containing the envelopes with the flexible spacers AMO1Fx, AMOGIFx and AMOG2. The APRO peptide contains 5 prolines probably arranged in a type II polyproline helix, whereas the AMOGIFx and AMOG2 envelopes contain essentially glycines.
Similarly to the AMOAPRO virions, the virions containing the AMOPRO envelopes require the simultaneous presence of the two types of receptors for infecting the cells. The infectious titres in cell types co-expressing the two receptors are, however, lower than that observed with the AMOAPRO virions, though it is not possible to exclude the hypothesis that the lesser extent of incorporation of these envelopes is responsible for this result. Even more markedly than with AMOAPRO, the AMOPRO viruses cannot infect the cells when either one of the two receptors is expressed alone (Table The two indices RamID and RecID are in fact less than 10 These results suggest that: 1) interaction of the AMOPRO virions with Ram-i when it is expressed alone is not sufficient to trigger the changes in conformation of the glycoprotein permitting its gene-fusion. Furthermore, it is possible that the PRO spacer peptide is either too rigid, or too long to favour such a transition, 2) the domain for binding with Rec-1 is not accessible for interaction with Rec-l and to take over in the entry process as long as the AMOPRO virion has not interacted with Ram-1.
For the purpose of better discrimination of whether the masking of the binding domain located downstream of the targeting site is a unique property of the AS208 peptide conjugated to the PRO spacer peptide, the inverse construction was effected.
The MOAPRO envelopes contain the binding domain of the ecotropic envelope followed by the proline-rich region of this same envelope, the whole being fused at the N-terminal end of the amphotropic envelope (Fig. The results shown in Table 2, show that in a similar manner to the virions containing the AMOPRO envelopes, the MOAPRO virions can infect the cells expressing only either one of the receptors Rec-l or Ram-I with difficulty, or not at all. It even seems that the Ram-I domain in the MOAPRO envelope is even less accessible (RamID less than 7x10' 5 than the Rec-1 domain is in the AMOPRO envelope (RecID less than 5.6x10 4 The MOAPRO envelopes can efficiently infect the cells expressing the two types of receptors, with titres of the order of 105 lacZ i.u./ml, suggesting that, for this envelope as well, the presence of the two receptors synergizes the infectious process.
These results, taken together, suggest that the spacer peptide inserted between the targeting domain and the rest of the retroviral envelope exercises control over the accessibility of the domain located downstream of the said peptide and over the activation of 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 PRO spacer peptide would finally perform the same role as the proline-rich region in question and which is located, in the unmodified glycoprotein, between the binding domain to the receptor and the fusion domain. This role would be masking of the domain downstream (fusion domain for the wild-type envelope or binding domain for the chimeric envelope) and subsequent unmasking for interaction of the domain upstream with its receptor. In the case of the wild-type envelope, this unmasking would lead to activation of fusion, whereas in the case of chimeric envelopes, unmasking would lead to accessibility of the binding domain to the viral receptor. If the receptor is expressed at the cell surface, there can then be interaction, and this then triggers activation of the fusion domain, explaining why the simultaneous presence of the two receptors synergizes infection.
These results make it possible to propose a two-stage targeting strategy for which a targeting envelope is constructed with various domains, whose functions are activated and coordinated by means of specific spacer peptides containing proline-rich sequences. These chimeric envelope glycoproteins can be conceived as follows, with, from N-terminal to C-terminal, a "targeting" domain capable of recognizing a cell surface molecule specifically expressed on the targeted tissue or targeted cell (for example a single-chain antibody or a ligand for a surface receptor); a spacer peptide capable of masking an auxiliary region which is in turn capable of facilitating penetration of the virus when it is activated. Such an auxiliary domain can be an entire retroviral envelope, i.e. a structure capable of mediating and taking over from viral infection by means of interaction with a ubiquitous retroviral receptor, which therefore has a very strong likelihood of being co-expressed with the targeted surface molecule. Ideally, the auxiliary domain should be masked until the viral particle has specifically interacted with the targeted surface molecule. For example, in the case of the AMOPRO and AMOAPRO envelopes, the targeted surface molecule is Ram-I whereas the auxiliary domain is the ecotropic envelope.
I EGFR and Rec- I cooperation in infection.
To verify whether the PRO and APRO spacer peptides could mediate the masking/unmasking mechanism in the case of another type of targeting envelope, another two-stage targeting model was explored by means of the EGF receptor. The results obtained with the targeting of Ram-I made it possible to propose C-terminal ends of the masking/unmasking spacer peptides. However, it was not possible to define their Nterminal ends exactly. That is why, in the first place, the EMOPRO+ and EMOAPRO+ envelopes were constructed (Fig. 3B), in which the PRO and APRO spacer peptides contain in addition, at the N-terminus, 41 amino acids derived from the amphotropic envelope and located immediately upstream of the proline-rich region. For the EMOPRO+ and EMOAPRO+ envelopes, the targeting domain is EGF, whereas the auxiliary domain is the ecotropic envelope. These two envelopes were compared with the EMO envelope (Fig. 2 and 2B) which does not contain a spacer peptide.
The infection tests were carried out with cells expressing Rec-1 alone (Cerd9 cells) or with cells co-expressing Rec-1 and EGFR (3T3 cells). The results of a typical experiment are presented in Table 3. As expected from the results obtained with the AMO envelopes, the viruses containing the EMO envelopes can efficiently infect the Cerd9 and 3T3 cells, indicating that the binding domain to Rec-1 in these envelopes is not masked. In comparison with the EMO viruses, the viral particles containing the EMOPRO+ and EMOAPRO+ envelopes can only infect the Cerd9 cells with difficulty (between 1000 and 10 000 times less well than the EMO viruses). However, when Rec- 1 and EGFR are co-expressed, even though this does not affect the titre of the EMO virions, the viral particles containing the EMOPRO+ and EMOAPRO+ envelopes are and 60 times more infectious, respectively, compared with when Rec-1 is expressed alone.
In relation to the results obtained with the AMOPRO and AMOAPRO envelopes, masking is apparently effected less well, leading to non-negligible infectivity on Cerd9 cells. This is perhaps due to the fact that the PRO+ and APRO+ spacer peptides are not optimized for their function, but perhaps also to the fact that the Cerd9 cells express a few EGF receptors which would contribute to activation of the EMOPRO+ and EMOAPRO+ envelopes.
Table I Titres (lacZ iuiml) obtained for the viruses containing the envelopes targeting Ram- I in interference tests env TE671 MTa 3T3-MLV-Aa~b 3T3-MoMLVa~b Ram-1C Ram-i Rec-1c Rec-1C Ram-1C 14O <1 92. 000, 000 (100) 46,000.000 (100) A 10, 000,000 12,000, 000 (100) 240 8,000,000 (100) AMO 4,000 24 (100) 32 (266.7) 8 (S0) AMOFx 230,000 44 0. 000 (100) 8,000 6,000 (2) AMOI 330,C00 1, 920,000 (100) 78,000 62,000 (4.8) AMO1Fx 400,000 1, 620, 000 (100) 60,000 74,000 (6.8) AMOtiPRO 150,000 2 60. 000 (100) 400 (0.29) 64,000 (34.3) AMOPRO 10 6 0, 000 100) 4 (0.013) <1 (0.002S) a: percentages calculated assigning a value of 100 to the titres obtained on 3T3 b: infection on 3T3 chronically infected by MILV-A (3)T3-MIL V-A) or by MoMILV (T3 -MoMLV) c: receptor available at the surface of the cell in question Table 2 Titres (lacZ i./ml) obtained for the viruses containing the envelopes targeting Ramn-I Spaceren peptide, TE671 CERD9 Ram! D RecID 3 13 16 18 19 23 26 62
MO
A
AMO
AXIOlFx
AMO&PRO
AMOGi Fx AMOG2 AMOG2Fx AIMOG 3 Fx
AMOPRO
M.OAPRO
2. Bx3.0 6 Sx1O* 5 10a2 6X10+ 4 4 -x1O+4 8x10 6x10*2 1. 8 I.
3.3x1o~s 2 .2xl10 2 1 .6x1O+ 4 SX1O* 3 4 Sx10, 3 2.7xlC+ 3 1 .2X1O+ 3 IXlO+2 .7x1C+ 0 1x1O+ 0 2. BXIO* 6 .2xI10 0 6 .2x10 0
O
2. 2xl10 5 6 .2x10 0
O
8.7x,0+ 3 3. 1x3.O, 3 1.2xlO*3 7. 2x!0+ 3 cjxj1C 2 1 .7x10* 3 1x10, 2. 2X.0* 0 2 7xl10 I 2. 6xl 1 1 I11V
I
3 .4xI10 1 2x10- I. 10 4 <7xlO*s 1. 2x1O0-5 6 .2x10- 2 3. 7x1O* 0 3. 3x10- 4 2. 2x10 1 3. 9x10- 1 2x3 0- 1 1. 3x10< 0 <S .6x10- 4 1. 3x10 2 Table '3 Titres (lacZ i.u./mI) obtained for the viruses containing the envelopes targeting EGFR env 3T3 CeRD9 RecID
MO
EMOtAPRO EMO PRO E7O01 9.2x10 6 3. 5x,0 4 9. 6xI0 2 2xI06 1 .3X10 7 8 5x10 2
I
31. 7X10- 2 S. 2x10- 2 1 7X101 3X10 6 EXAMPLE 2: With the aim of characterizing the cooperation between the Rec-I and Ram-1 receptors, as well as the peptides that are capable of regulating this cooperation of receptors, a new series of type AMO chimeric envelope glycoproteins (see preceding example) was constructed: in order to verify whether the infection obtained with the AMOPRO and AMODPRO envelopes passes, in a second stage, through an interaction with Rec-1, the binding domain with Rec-I was inactivated by point mutagenesis (D84K mutation) (MacKrell et al., J. Virology, 70:1768-1774 (1996)) in the AMOPRO and AMODPRO envelopes as well as in the AMOG1X control envelope which does not require the cooperation of receptors to permit infection (Valsesia-Wittmann et al., The EMBO Journal 16:1214-1223. (1997)).
in order to demonstrate the role of the type II polyproline helix structure for the cooperating peptides, the envelopes AMOEL3 and AMOEL5 were constructed. These envelopes have respectively 3 and 5 turns of a type II polyproline helix as 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.
Material and Methods.
The 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 CCC CGG AGC CCC TAC CCC AGG TGC GGC CGC GGT GAC CAT AAA) were hybridized together. .The resulting bicatenary DNA fragment was digested with the Eael restriction enzyme and cloned in the FBAMOSALF expression plasmid previously opened at NotI.
The result was the plasmid FBAMOEL3SALF (see sequence of the gene env AMOEL3 in Fig. 30) containing the peptide EL3 the peptide sequence of which is shown in Table 4 (see nucleotide sequence in Fig. 31).
The oligonucleotides UpE15: (5'-GAT GTA CCT GGG GTA GGC GCC CCT GGA GTC GGG GCT CCT GGG GTA GGA TTC AT) and LowEl5: (5'-ATG AAT CCT ACC CCA GGA GCC CCG ACT CCA GGG GCG CCT ACC CCA GGT ACA TC) were hybridized together. The resulting bicatenary DNA fragment was digested with EcoNI restriction enzyme and cloned in the FBAMOEL3SALF expression plasmid, previously opened at EcoNI. The result is the plasmid FBAMOEL5SALF (see sequence of the gene env AMOEL5 in Fig. 32) containing the peptide EL5, the peptide sequence of which is shown in Table 4 (see nucleotide sequence in Fig. 33).
The oligonucleotides DELASTIN3-V Upper: (5'-GTC ACC GCG GCC GTC CCT GGG GTA GGG GTG CCG GGG GTA GGG GTG CCT GGG GTG GCC ATA TAA) 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) were hybridized together. The resulting bicatenary DNA fragment was digested with the EaeI restriction enzyme and cloned in the FBAMOSALF expression plasmid, previously opened at NotI.
The result is the plasmid FBAMOEL3-VSALF (see sequence of the gene AMOEL3-V in Fig. 34) containing the EL3-V peptide, the peptide sequence of which is shown in Table 4 (see nucleotide sequence in Fig. The oligonucleotides DELASTIN3-I Upper: (5'-GTC ACC GCG GCC GTC ATA GGG GTA GGG GTG ATT GGG GTA GGG GTG ATC GGG GTG GCC ATA TAA) and DELASTIN3-I Lower (5'-TTA TAT GGC CAC CCC GAT CAC CCC TAC CCC AAT CAC CCC TAC CCC TAT GAC GGC CGC GGT GAC) were hybridized together. The resulting bicatenary DNA fragment was digested with the EaeI restriction enzyme and cloned in the FBAMOSALF expression plasmid, previously opened at NotI.
This resulted in the plasmid FBAMOEL3-ISALF containing the peptide EL3-I, the peptide sequence of which is shown in Table 4.
The oligonucleotides UpXhoD84K: (5'-AGG CTG CTC GAG AAA ATG CGA AGA ACC TTT AAC CTC CC) and LoXhoD84K: (5'-ATT TTC TCG AGC AGC CTG GGC TGC TGC CCC C) were synthesized. Starting from the oligonucleotides 805FC and LMOADeltaPRO3 (see sequence above), the pairs 805FC/LoXhoD84K or UpXhoD84K/LMOADeltaPRO3 were used independently for PCR amplification of two DNA fragments starting from the FBAMOSALF matrix. These two DNAs were digested by the enzymes NotI/XhoI and XhoI/BamHI respectively and co-ligated in one or other of the three plasmids FBAMOSALF, FBAMODeltaPROSALF, and FBAMOProSALF previously opened at NotI and BamHI. The resulting plasmids express respectively the envelopes AMOD84K, AMODeltaProD84K, and AMOProD84K.
Two DNA fragments of 2005 bp and 241 bp were isolated from the plasmid FBAMOGIX (Valsesia-Wittmann et al., The EMBO Journal 16:1214-1223. (1997)) by digestion with the restriction enzymes NdeI/XhoI and XhoI/BstEII respectively. These two inserts were cloned in the plasmid FBAMOD84KSALF previously digested by the enzymes Ndel and BstEII, resulting in a plasmid capable of expressing the AMOGI XD84K envelope.
Results and Discussion.
Expression and viral incorporation of the chimeric envelopes. The expression plasmids for the envelopes AMO, AMODeltaPRO, AMOPRO, AMOEL3, AMOEL3-V, AMOEL3-I, AMOIFX, AMOG1X, AMOD84K, AMODeltaPROD84K, AMOPROD84K, AMOGIXD84K, AMODeltaPRO2 (Valsesia-Wittmann et al., The EMBO Journal 16:1214-1223. (1997)), and AMODeltaPRO4 (Valsesia-Wittmann et al., The EMBO Journal 16:1214-1223. (1997)) were introduced by transfection into the cells of the TELCeB6 line (Cosset et al., Journal of Virology 69:7430-7436. (1995b)). After selection by phleomycin, the phleomycin-resistant colonies were combined for each DNA and virions were generated and analysed following the procedures originally described (Cosset et al., Journal of Virology 69:6314-6322. (1995a)).
These various chimeric envelopes are normally expressed and matured in the cells, and, moreover, efficiently incorporated on the viral particles (results not shown).
The binding tests that were carried out show that these retroviruses can bind specifically on human cells by means of the targeted surface molecule Ram-1 (results not shown).
These various viruses were used for infecting cells expressing either Rec-1 only (Cerd9), or Ram-1 only (CHO-Ram-1), or the two molecules Ram-1 and Rec-1 (Cearl3). The results of titration of these viruses are presented in Table 4.
These results can be summarized as follows: in an AMO envelope, substitution of the spacer peptide by three beta-turns of a synthetic (AMOEL3) or natural polyproline helix, described in the literature (AMOEL3- V, from bovine elastin) confers, with regard to capacity for masking the function of the ecotropic envelope and for regulating the cooperation of the Ram-1 and Rec-1 receptors, a phenotype similar to the viruses bearing the "AMO" envelopes containing the cooperating spacer peptides DeltaPRO2, DeltaPRO, DeltaPRO4, or PRO. Since the peptides derived from elastin (AMOEL3-V and AMOEL3) are arranged as a type II polyproline helix, it can be suggested on the basis of the results obtained that regulation of the cooperation of the Ram-1 and Rec-I receptors by the DeltaPro and Pro peptides is probably due to their presumed secondary structure, as a type II polyproline helix.
Moreover, mutations introduced into the spacer peptide derived from elastin (AMOEL3- V) and having the purpose of destroying the folding of the peptide into a type II polyproline helix (AMOEL3-I, mutations obtained by replacing the proline of each betaturn with an isoleucine) lead to cancellation of receptor cooperation.
destruction of the capacity for binding to the ecotropic receptor (D84K mutations) stops receptor cooperation for the envelopes containing cooperating spacer peptides, especially PRO (see results AMOPRO vs AMOPROD84K), but does not affect the functionality of the control envelopes bearing the flexible spacer peptide GIX (see results AMOGIX vs AMOGIXD84K). We deduce from this that binding to the ecotropic receptor is necessary for infection, in a second stage, following fixation on the Ram-I receptor.
Note that the present results show that in the case of the retroviruses generated with the chimeric envelope AINO Pro, the binding domain to the ecotropic receptor is masked (Valsesta-Wittmann et al., The EMBO Journal 1&:1214-1223. (1997)). The results, taken together, are therefore compatible with a model of two-stage interaction in which: in its "~naive"' configuration, i.e. when it has not been permitted to interact with a cell, the "AMIOPRO" retrovirus can potentially interact with the targeted "primary" receptor (the Ram- I molecule), but cannot directly interact with the auxiliary receptor (the Rec-l molecule). This masking seems to be due to a first property of the Pro spacer peptide.
-when this virus is permitted to interact with Ram-I, a local change in conforma~tion occurs at the level of the Pro spacer peptidle which will make the binding domain to Rec-I accessible. This change in conformation is due to a second property of the Pro spacer peptide.
if the Rec-I receptor is present at the surface of the same cell that has Ram-I and on which the virus is bound. then in a second stage, this receptor will serve as an entry molecule for the virus.
Table 4. Results of titration.
sequence of the spacer peptid&e Cear13 b C HO- R-aniTEICcrdgb A NI AMOD84K AMODchtaPro2 AMO I FX AMODeltaPro AMODcItarroD84K N\'G AAA llQV N~VG PRVPIGPNPAA
IPHQV
NVG PRVPIGPNPAA
PHQ\'
NVG AAAIEGRZASPGSS
PHQV
NVG PR\'PIGPNPVLPDAAA
PHQV
NVG PRVPIGPNPVLPDAAA
PHQV
+4-I- +1- AMOEL3 N\'G AAAPGVGAPGVGAPGVAA PH-QV AMOEL3-\' NVG AAVPGVG\'PGVGVPGVAA
PHQV
AMOEL3-l NVG AAVIGVGVIGVGVIGVAA
PHQV
AMOG IX NVG AAAGGGGSIEGRASPGSS
PHQV
AMOG I XD84K NVG AAAGGGGSIEGRASPGSS
PHQV
AMODcltaPro4 NVG PRVPIGPNPVLPDQR.LPSSAA
PHQV
AMOELS NVG AAAPGVGAPGVGAPGVGAPGVGAPGVAA PIiQV AMOPRO N\'G PRVPIGPNPVLPDQRLPSSPIEIVPAPQPPSP..
LNTSYPPSTTSTPSTSPTSPSVPQPPPAAA
PHQV
A MOPROD84 K N VG PR\'PIG PNP\'LPDQRLPSSPIEIVPAPQQPPSP...
LNTSYPPSTTSTPSTSPTSPSVPQPPPAAA
PHQV
4-.
4-4- 4envelope. "PH-QV" represents the amino acids 7 to 10 of the envelope of MoMILV and "NVG" represents the last 3 amino acids of the binding domain to Ram-I1.
b: relative titres obtained on the cells indicated: Cearl3, expressing the receptors Ramn-I and Rec-l-; CHO-Ram- I, expressing Ram-I only;, Cerd9, expressing REPLACEME NT SHEET (RULE'26) Rec-I only.
EXAMPLE 3.
The development of strategies of targeting gene transfer by means of the construction of chimeric envelope glycoproteins, generated by N-terminal insertions of ligands, comes up against the difficulty, in particular, of low capacity, or even incapacity of interaction between virus and targeted surface molecule for activating fusion of these targeting envelopes (Cosset and Russell, Gene Therapy 3:946-956 (1996)). The possibility of causing two surface molecules to cooperate (Valsesia-Wittman et al., The EMBO Journal 16:1214-1223. (1997)), one being the targeted receptor or cell surface molecule of attachment, the other being a (retro)viral receptor specialized for fusion or auxiliary surface molecule, makes it possible to envisage a means of overcoming this problem of low gene-fusion capacity of chimeric envelopes and more generally of low efficiency of the targeting retroviruses. The cooperation of receptors was tested in three models of targeting, in which the following three cell surface molecules serve as points of attachment for the targeting retroviruses: receptor of EGF (epidermal growth factor), and (ii) class I molecule of human CMH. The binding domains for these two surface molecules are either growth factors (EGFR), or a single-strand antibody (CMH-I).
These ligands were inserted by fusion at the N-terminal end of the amphotropic MLV envelope (4070A) and various peptides from the proline-rich region carried by the SU subunit of the amphotropic MLV virus were inserted between the ligands and the 4070A envelope (see Table Materials and Methods DNA fragments coding for the spacer peptides DeltaPro2, DeltaPro3, DeltaPro4, and Pro (see Table 5) were generated by PCR using as DNA matrix the gene env 4070A, at 5' the oligonucleotide PRO-5-NE (5'-ATC GAG GTC ACC GCG GCC GCG GGA CCC CGA GTC CCC ATA GGG CCC) which is the same for the four PCR fragments and as oligonucleotides 3' the sequences AMODPRO(-H (5'-TAT GAG CGG CCG GGT TGG GCC CTA TGG GGA DPro3: (5'-TTA TAC GGC CGT GTC GGG TAA TAC TGG), AMODPRO(+H+S-A): (5'-TAT GTG CGG CCG AGG AAG GGA GTC TTT GGT C) and PRO-3-NE: (5'-ATA ATC GGC CGG GGG TGG CTG TGG GAC).
The corresponding DNA fragments were digested by the enzyme EagI and inserted separately in the plasmid FBEASALF (expressing the chimeric envelope glycoproteins EA) (Cosset et al., Journal of Virology 69:6314-6322. (1995a)) previously opened at the Notl restriction site. The resulting plasmids express the envelopes EADeltaPro2, EADeltaPro3, EADeltaPro4, and EAPro.
The Ndel/Notl fragment containing the promoter FB29 as well as the scFv anti- MHC-I provided with the signal peptide of the envelope glycoprotein of the MoMLV virus (Marin et al., Journal of Virology 70:2957-2962. (1996)) was cloned in the FBEASALF plasmid from which the Ndel/Notl fragment was removed beforehand. This results in the plasmid FB34ASALF capable of expressing a 4070 chimeric envelope with the scFv fused at its N-terminal end. This plasmid was then opened at NotI for inserting the spacer peptides DeltaPro2, DeltaPro3, DeltaPro4, and Pro (see Table 5) previously digested with the EagI enzyme. This results in a series of expression vectors for the envelopes 34DeltaPro2, 34DeltaPro3, 34DeltaPro4, and 34Pro.
Results and Discussion.
These various DNAs were introduced by transfection into the cells of the TELCeB6 line and retroviruses were generated following the usual procedure (see examples 1 and It was shown that these retroviruses correctly express the chimeric envelope glycoproteins and that the latter permit efficient redirection of binding of the viral particles on the specific cellular targets (results not shown).
The viruses produced with the chimeric envelopes of the various groups were used for infecting cells that only express the amphotropic receptor and not the targeted surface molecule. The results of titration of these viruses are shown in Table These results show that it is possible to mask the functions of the amphotropic envelope by means of fragments from the proline-rich region. In the case of chimeras effected with EGF, it is necessary to insert at least five beta-turns to obtain a significant masking effect, and insertion of the whole of the proline-rich region leads to complete inhibition. For the chimeras effected with scFv anti-MHC-I, three beta-turns are required to obtain a complete masking effect.
Table 5. Results of titration.
peptidea ligand forh: name sequence EGFR MHC-1 without' AAA PHQV 6e3 39e2 DeltaPro2 AAA GPRVPIGPNPAA PHQV 7e3 18el DeltaPro3 AAA GPRVPIGPNEVLPDTAA PHQV 1.2e3 el DeltaPro4 AAA GPRVPIGPPEVLPDQRLESSAA PHQV 7el <lcl Pro AAA GPRVPlGPNEVLEDQRLPSSPIEIVPAPQPI'.
SPLNTSYPPSTTSTPSTSPTSPSVPQPPPAA PIQV <lel lel a: peptide inserted between the targeting binding domain and the 4070A envelope. "AAA" codes for the Notl site used for effecting fusion in the chimeric envelope, "PHQV" represents the amino acids 4 to 7 of the amphotropic envelope. The REPLACEMENT SHEET (RULE 26) beta-turns are underlined.
b: titration on Cearl3 cells for the EGFR targeting envelopes (ligand: EGF) and for the targeting envelopes targeting MHC-I (ligand: scFv anti-MHC-I).
c: the ligand is directly fused at the end of the amphotropic SU (with the 4th amino acid), and does not have a spacer peptide.
EXAMPLE 4.
The previous investigations made it possible to delimit the C-terminal ends of the cooperating peptides and to determine the number of turns of type II polyproline helix necessary for obtaining a masking effect and a minimal cooperative effect. In the case of the model of the AMO chimeric envelopes (see above), a minimum of two turns of the helix is sufficient (Valsesia-Wittmann et al., The EMBO Journal 16:1214-1223. (1997)).
However, for chimeric envelopes generated with other binding domains than that for Ram-1 (in the case of AMO chimeras) and using the amphotropic envelope as support envelope, the cooperative effect is less marked, on the one hand because masking of the functions of the amphotropic envelope requires four turns of polyproline helix (see Table and on the other hand because activation of the functions of the amphotropic envelope is less strong following binding of the viruses on the targeted surface molecules. One possible explanation is that, in the model of the AMO chimeras, apart from the PRO spacer peptide, the binding domain to Ram-1 itself carries important determinants for inducing, in a concerted manner with this PRO peptide, activation of the functions of the ecotropic envelope. The binding domain for Ram-1 is in fact a fragment of retroviral envelope (derived from the amphotropic envelope) which is naturally located immediately upstream of the proline-rich region. In order to determine the presence and the importance of such regions in receptor cooperation, chimeric envelopes were constructed combining a targeting domain with the amphotropic envelope and, inserted between these two polypeptides, various peptides tested for their cooperative effect containing notably the proline-rich region (or a fragment of this region) combined with peptide fragments derived from the N-terminal domain of the amphotropic envelope.
Materials and Methods DNA fragments coding for the spacer peptides DeltaPro4-beta, DeltaPro4-int, DeltaPro4-vrb and.. were generated by PCR using as DNA matrix the gene env 4070A, at 3' the oligonucleotide AMODPRO(+H+S-A): (5'-TAT GTG CGG CCG AGG AAG GGA GTC TTT GGT C) and at 5' the oligonucleotides UPro-beta: (5'-ATG CTG GCG GCC GCG GAT CCT ATT ACC ATG TTC TCC CTG ACC CGG UPro-int: ATG CTG GCG GCC GCG AAC CCT CTA GTC CTA GAA TTC ACT GAT GC), and UPRO-vrb: (5'-ATG CTG GCG GCC GCG GAA ACC ACC GGA CAG GCT TAC TGG AAG CCC), respectively (see Figs. 36 to 38).
DNA fragments coding for the spacer peptides Pro-beta, Pro-int and Pro-vrb were generated by PCR using as DNA matrix the gene env 4070A, at 3' the oligonucleotide PRO-3-NE: (ATA ATC GGC CGG GGG TGG CTG TGG GAC) and at 5' the oligonucleotides UPro-beta: (5'-ATG CTG GCG GCC GCG GAT CCT ATT ACC ATG TTC TCC CTG ACC CGG UPro-int: (5'-ATG CTG GCG GCC GCG AAC CCT CTA GTC CTA GAA TTC ACT GAT GC), and UPRO-vrb: (5'-ATG CTG GCG GCC GCG GAA ACC ACC GGA CAG GCT TAC TGG AAG CCC), respectively (see Figs. 39 to 41).
These DNA fragments were digested with EagI enzyme and inserted either in the FBEASALF plasmid (see above) resulting in production of the expression vectors for the chimeric envelopes EADeltaPro4-beta, EADeltaPro4-int, EADeltaPro4-vrb, EAProbeta, EAPro-int and EAPro-vrb, or in the FB34ASALF plasmid (see above) resulting in production of the expression vectors for the chimeric envelopes 34ADeltaPro4-beta, 34ADeltaPro4-int, 34ADeltaPro4-vrb, 34APro-beta, 34APro-int and 34APro-vrb.
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K. L. Collins. 1994. Type C retrovirus inactivation by human complement is determined by both the viral genome and producer cell. J. Virol. 68:8001-8007.
20. Valsesia-Wittmann, A. Drynda, G. Deleage, M. Aumailley, Heard, O. Danos, G. Verdier, and Cosset. 1994. Modifications in the binding domain of avian retrovirus envelope protein to redirect the host range of retroviral vectors. J. Virol.
68:4609-4619.
21. Valsesia-Wittmann, F. J. Morling, B. H. K. Nilson, Y. Takeuchi, S. J.
Russell, and Cosset. 1996. Improvement of retroviral retargeting by using amino acid spacers between an additional binding domain and the N terminus of Moloney murine leukemia virus SU. J. Virol. 70:2059-2064.
22. VanZeijl, S. V. Johann, E. Cross, J. Cunningham, R. Eddy, T. B. Shows, and B. O'Hara. 1994. An amphotropic virus receptor is a second member of the gibbon ape leukemia virus receptor family. Proc. Natl. Acad. Sci. USA. 91:1168-1172.
23. Weiss, R. A. 1993. Cellular receptors and viral glycoproteins involved in retroviral entry, p. 1-108. In J. levy The Retroviridae, vol. 2. Plenum Press.

Claims (37)

1. Use of a peptide for transferring genes into a eukaryotic target cell, this peptide containing from about 10 to about 200 amino acids, in which at least 30% of the amino acids consist of proline residues, these proline residues being arranged regularly so as to induce turnings of the polypeptide chain at about 1800 ("13-turn" or "reverse-turn"), these turnings being regularly spaced and forming a polyproline 3-turn helix, in a polypeptide construction containing, on the N-terminal side (upstream) of the said peptide, and N- terminal (upstream) protein domain capable of recognizing a targeted surface molecule or an antigen expressed on a cell surface, and on the C-terminal side (downstream) of the said peptide, a C-terminal (downstream) protein domain capable of recognizing a suitable receptor (auxiliary receptor) located on the aforesaid eukaryotic cell, this peptide being capable of facilitating or inhibiting interaction between the C-terminal S. (downstream) protein domain and the auxiliary receptor, inhibition of this interaction :occurring for as long as the N-terminal (upstream) protein domain has not interacted with the targeted receptor and promotion of interaction between the C-terminal (downstream) protein domain and the auxiliary receptor occurring when the N-terminal (upstream) protein domain has interacted with the targeted receptor.
2. Use of a peptide according to claim 1, wherein the peptide for transferring genes into a eukaryotic target cell contains from about 15 to about 150 amino acids. a.o. 20 3. Use of a peptide according to claim 2, wherein the peptide for transferring genes into a eukaryotic target cell contains about 20 amino acids.
4. Use of a peptide according to any one of claims 1 to 3, wherein the polypeptide construction containing, on the N-terminal side (upstream) of the said peptide, an N- terminal (upstream) protein domain capable of recognizing a targeted surface molecule or an antigen expressed on a cell surface, is a suitable receptor (targeted receptor) located on the said eukaryotic cell. Use of a peptide according to any one of claims 1 to 4, in the construction of a Sglycoprotein with targeting and gene-fusion activity, essentially intact, carried by a viral 0 r non-viral recombinant gene-transfer vector capable of infecting a eukaryotic cell, and -43- this eukaryotic cell has a targeted receptor and an auxiliary receptor permitting facilitation of entry of the aforesaid viral or non-viral vector into the eukaryotic cell, the aforesaid glycoprotein comprising: the aforesaid peptide, a protein domain on the N-terminal side (upstream) of the said peptide, capable of interacting with the said targeted receptor, this protein domain permitting specific binding of the said gene-transfer vector and a protein domain on the C-terminal side (downstream) of the said peptide, capable of interacting with the said auxiliary receptor, this interaction performing the role of auxiliary mechanism of entry of the said gene-transfer vector into the eukaryotic cell, the process of cell entry of the viral or non-viral recombinant vector into the eukaryotic cell Sby means of the C-terminal (downstream) protein domain only being able to take place when the N-terminal (upstream) protein domain has recognized and bound the viral or Snon-viral recombinant vector with the targeted receptor of the eukaryotic cell, leading, through the agency of the aforesaid peptide, to a mechanism of "unmasking" or of accessibility of the auxiliary receptor with respect to the C-terminal (downstream) protein domain, and, in the case when recognition does not occur between the aforesaid gene-transfer vector S; and the targeted receptor of the eukaryotic cell by means of the N-terminal (up-stream) 20 protein domain, there is produced a mechanism of "masking" or of non-accessibility, through the agency of the aforesaid peptide, of the auxiliary receptor with respect to the C-terminal (downstream) protein domain.
6. Use of a peptide according to any one of the claims 1 to 5, in the construction of an essentially intact (retro)viral envelope glycoprotein, carried by a recombinant (retro)viral particle capable of infecting a eukaryotic cell, the said eukaryotic cell containing a targeted receptor and an auxiliary receptor permitting facilitation of entry of the said (retro)viral particle ((retro)viral receptor) into the eukaryotic cell, the envelope glycoprotein comprising: the aforesaid peptide, -44- a protein domain on the N-terminal side (upstream) of the said peptide, capable of interacting with the said targeted receptor, this interaction permitting a specific binding of the (retro)viral particle and a protein domain on the C-terminal side (downstream) of the said peptide, capable of interacting with the said (retro)viral receptor, this interaction performing the role of auxiliary mechanism of entry of the (retro)viral particle into the eukaryotic cell, the process of cell entry of the recombinant (retro)viral particle into the eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to take place when the N-terminal (upstream) protein domain has recognized and bound the targeted receptor of the eukaryotic cell with the recombinant (retro)viral particle, leading, through the agency of the aforesaid peptide, to a mechanism of "unmasking" or of accessibility of a* S" the (retro)viral receptor with respect to the C-terminal (downstream) protein domain, and, in the case when recognition does not occur between the recombinant viral particle and the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) protein domain, there is produced a mechanism of "masking" or of non-accessibility, through the agency of the aforesaid peptide, of the (retro)viral receptor with respect to 9 the C-terminal (downstream) protein domain. 0.
7. Use of a peptide according to claim 6 wherein the said envelope glycoprotein is of polymeric form, each monomer of the polymeric form being in itself of heterodimer 20 form.
8. Use of a peptide according to claim 7 wherein the said envelope glycoprotein is of trimeric form.
9. Use of a peptide according to any one of the claims 1 to 8, wherein the N-terminal (upstream) protein domain is chosen from the following polypeptides: single-strand antibodies recognizing cell surface molecules, any ligand for a cell surface molecule. Use of a peptide according to claim 9 wherein the ligand for the cell surface molecule is a polypeptide hormone, cytokine or growth factor.
11. Use of a peptide according to any one of the claims 1 to 10 wherein the C-terminal (downstream) protein domain corresponds to a (retro)viral envelope glycoprotein, essentially intact, containing the natural binding domain, the functions of fusion and of attachment of the wild-type envelope glycoprotein from which the envelope glycoprotein carried by the recombinant (retro)viral particle is derived.
12. Use of a peptide according to any one of the claims 1 to 11, wherein the peptide comes from the envelope glycoprotein of type C retrovirues.
13. Use of a peptide according to claim 12, wherein the virus is chosen from: the ecotropic MLV virus, the amphotropic MLV virus, the xenotropic MLV virus, the MLV 10 MCF virus, the MLV 10A1 virus, GALV (Gibbon Ape Leukemia Virus), SSAV (Simian Sarcoma Associated Virus), FeLV A, FeLV B, FeLV C (FeLV: Feline Leukemia Virus). S14. Use of a peptide according to claim 12 or claim 13, wherein the peptide is chosen from those containing, or that are constituted of, one of the following sequences: PRO 15 (4070A), PRO(MoMLV), APRO, PRO+, APRO+, PROP, APROP, APRO4-P, APR04- Sint, APRO4-vrb, PROP,PRO-int, PRO-vrb.
15. Use of a peptide according to any one of the claims 1 to 14, wherein the peptide is derived or adapted from bovine elastin and is chosen from those containing, or that are constituted of, one of the following sequences: EL3, EL3-V,
16. Peptide sequences chosen from those containing, or constituted of, one of the following sequences: PRO (4070A), PRO(MoMLV), PROp, PRO+, APRO, APROp, APRO+, MOAPRO, MOAAPRO, EMOPRO, EMOPROP, EMOPRO+, EAPRO, EAPROp, EAPRO+, EMOAPRO, EMOAPROp, EMOAPRO+, EAAPRO, EAAPROp, EAAPRO+, EL3, EL3-V, AMOEL3, AMOEL3-V, AMOEL5, APRO4-P, APRO4-int, APRO4-vrb, PROP, PRO- int, PRO-vrb. -46-
17. Peptide sequence containing a peptide of about 10 to about 200 amino acids, in which at least 30% of the amino acids consist of proline residues, these proline residues being arranged regularly so as to induce turnings of the polypeptide chain at about 1800 ("p-turn" or "reverse-turn"), these turnings being regularly spaced and forming a polyproline p-turn helix, an N-terminal protein domain (upstream) of the said peptide, capable of reacting with a suitable receptor (targeted receptor) located on a eukaryotic cell, this protein domain permitting specific binding of a recombinant (retro)viral particle containing the said N- terminal protein domain and a C-terminal protein domain (downstream) of the said peptide, capable of interacting :with a suitable auxiliary (retro)viral receptor ((retro)viral receptor) located on the said eukaryotic cell, this interaction performing the role of auxiliary mechanism of entry of "the (retro)viral particle into the said eukaryotic cell, the process of cell entry of the said recombinant (retro)viral particle into the said eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to take place when the N-terminal (upstream) protein domain has recognized and bound the targeted receptor of the eukaryotic cell with the said recombinant (retro)viral particle, leading, through the agency of the aforesaid peptide, to a mechanism of unmasking or of accessibility of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain, 20 and, in the case when recognition does not occur between the recombinant viral particle and the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) protein domain, there is produced a mechanism of masking or of non-accessibility, through the agency of the aforesaid peptide, of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain.
18. Peptide sequence according to claim 17, wherein the peptide is about 15 to about 150 amino acids.
19. Peptide sequence according to claim 18, wherein the peptide is about 20 amino acids. Recombinant (retro)viral particle capable of infecting a eukaryotic cell, this cell S possessing a targeted receptor and an auxiliary receptor of the aforesaid (retro)viral -47- particle, comprising a substantially intact envelope glycoprotein, containing a peptide of about 10 to about 200 amino acids, in which at least 30% of the amino acids are constituted of proline residues, these proline residues being arranged regularly so as to induce turnings of the polypeptide chain at about 1800 p-turn" or "reverse turn"), these turnings being regularly spaced and forming a polyproline p-turn helix, a protein domain on the N-terminal side (upstream) of the aforesaid peptide, capable of interacting with the aforesaid targeted receptor, this peptide domain permitting specific binding of the (retro)viral particle and a protein domain on the C-terminal side (downstream) of the aforesaid peptide, capable of interacting with the aforesaid (retro)viral receptor, this interaction performing the role of auxiliary mechanism of entry of the (retro)viral particle into the eukaryotic cell, the process of cell entry of the recombinant (retro)viral particle into the eukaryotic cell by means of the C-terminal (downstream) protein domain only being able to take place S when the N-terminal (upstream) protein domain has recognized and bound the targeted 15 receptor of the eukaryotic cell with the recombinant (retro)viral particle, leading through the agency of the aforesaid peptide to a mechanism of unmasking or of accessibility of the (retro)viral receptor with respect to the C-terminal (downstream) protein domain, and, in the case when recognition does not occur between the recombinant viral particle Sand the targeted receptor of the eukaryotic cell by means of the N-terminal (upstream) 20 protein domain, there is produced a mechanism of masking or of non-accessibility, through the agency of the aforesaid peptide, of the retroviral receptor with respect to the C-terminal (downstream) protein domain.
21. Recombinant (retro) viral particle according to claim 20 wherein the envelope glycoprotein is of polymeric form.
22. Recombinant (retro) viral particle according to claim 20 wherein the envelope glycoprotein is of trimeric form.
23. Recombinant (retro) viral particle according to claim 21 or claim 22 wherein each monomer of the polymeric or trimeric form is of heterodimer form. -48-
24. Recombinant (retro) viral particle according to any one of claims 20 to 23, wherein the peptide is of about 15 to about 150 amino acids. Recombinant (retro) viral particle according to any one of claims 20 to 24 wherein the peptide is of about 20 amino acids.
26. Recombinant (retro)viral particle according to any one of claims 20 to 25, wherein the N-terminal (upstream) protein domain is chosen from the following peptides: single-strand antibodies recognizing cell surface molecules, any ligand for a cell surface molecule.
27. Recombinant (retro) viral particle according to claim 26 wherein the ligand for the cell surface molecule is a polypeptide hormone, cytokine or growth factor.
28. Recombinant (retro)viral particle according to one of the claims 20 to 27, wherein the C-terminal (downstream) protein domain corresponds to a polypeptide of (retro)viral origin possessing functions of binding, of fusion and of attachment of the wild-type envelope glycoprotein from which the envelope glycoprotein carried by the recombinant (retro)viral particle is derived, and can originate from the natural domains possessing the functions of binding, of fusion and of attachment of the envelope glycoproteins from retroviruses MLV-A, GALV, FeLV B, or from virues such as adenoviruses, herpesviruses, AAV (Adeno Associated Virus), or more generally from viral glycoproteins from viruses of eukaryotic origin.
29. Recombinant (retro)viral particle according to claim 28 wherein the viral glycoproteins from viruses of eukaryotic origin are from orthomyxoviruses (such as influenza viruses) or paramyxoviruses (such as Recombinant (retro)viral particle according to any one of the claims 20 to 29, wherein the peptide originates from the envelope glycoprotein of type C retroviruses.
31. Recombinant (retro)viral particle according to claim 30, wherein the peptide originates from a virus chosen from: the ecotropic MLV virus, the amphotropic MLV virus, the xenotropic MLV virus, the MLV MCF virus, the MLV 10A1 virus, GALV -49- (Gibbon Ape Leukemia Virus), SSAV (Simian Sarcoma Associated Virus), FeLV A, FeLV B, FeLV C (FeLV: Feline Leukemia Virus).
32. Recombinant (retro)viral particle according to claim 30 or claim 31, wherein the peptide is chosen from those containing, or constituted of, one of the following sequences: PRO (4070A), PRO(MoMLV), APRO, PRO+, APRO+, PROP, APROp, APRO4-P, APRO4-int, APRO4-vrb, PROp, PRO-int, PRO-vrb.
33. Recombinant (retro)viral particle according to any of the claims 20 to 32, wherein the peptide originates from the envelope glycoprotein of type C retroviruses.
34. Recombinant (retro)viral particle according to claim 33, wherein the virus is ;10 preferably chosen from: the ecotropic MLV virus, the amphotropic MLV virus, the xenotropic MLV virus, the MLV MCF virus, the MLV 10A1 virus, GALV (Gibbon Ape 9 Leukemia Virus), SSAV (Simian Sarcoma Associated Virus), FeLV A, FeLV B, FeLV 009* C (FeLV: Feline Leukemia Virus). o Recombinant (retro)viral particle according to claim 33 or claim 34, wherein the 15 peptide is chosen from those containing, or constituted of, one of the following 0OO* S: sequences: PRO (4070A), PRO(MoMLV), APRO, PRO+, APRO+, PROP, APROp, APRO4-P, APRO4-int, APRO4-vrb, PROP, PRO-int, PRO-vrb, the N-terminal (upstream) protein domain is chosen from the following peptides: single-strand antibodies recognizing cell surface molecules, any ligand for a cell surface molecule. the C-terminal protein domain corresponds to a polypeptide of (retro)viral origin possessing the functions of binding, of fusion and of attachment of the wild-type envelope glycoprotein from which the envelope glycoprotein carried by the recombinant (retro)viral particle is derived, and can originate from the natural domains possessing the functions of binding, of fusion and of attachment of the envelope glycoproteins from the retroviruses MLV-A, GALV, FeLV B, or from viruses such as adenoviruses, herpesviruses, AAV (Adeno Associated Virus), or more generally viral glycoproteins from viruses of eukaryotic origin.
36. Recombinant (retro)viral particle according to any one of claims 33 to 35 wherein the ligand for the cell surface molecule is a polypeptide hormone, cytokine or growth factor.
37. Recombinant (retro)viral particle according to any one of claims 33 to 36 wherein the viral glycoprotein from viruses of eukaryotic origin is from orthomyxoviruses (such as influenza viruses) or paramyxoviruses (such as
38. Recombinant (retro)viral particle according to one of the claims 20 to 37, wherein 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 10 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 S 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 (downstream) protein domain. 15 39. Nucleic acid coding for a peptide or for a recombinant particle according to any Sone of the claims 20 to 38.
40. Method of selective transfer in vitro or ex vivo of a nucleic acid into target eukaryotic cells present among other non-target cells, comprising the administration, to the target and non-target cells, of a recombinant (retro)viral particle according to one of the claims 20 to 38, containing the nucleic acid to be transferred.
41. Pharmaceutical composition containing as active substance a (retro)viral particle according to any one of the claims 20 to 38, and also containing a gene to be transferred, in combination with a physiologically suitable pharmaceutical vehicle.
42. Use of a (retro)viral particle according to any one of claims 20 to 38 containing a nucleic acid, for the manufacture of a medicament for selectively transferring the nucleic acid into a target eukaryotic cell present among other non-target cells. -51
43. Use of a peptide for transferring genes into a eukaryotic target cell, substantially as herein described with reference to any one of the examples but excluding comparative examples.
44. A peptide sequence containing a peptide of about 10 to 200 amino acids in which at least 130% of the amino acids consist of proline residues, substantially as herein described with reference to any one of the examples but excluding comparative examples. A recombinant (retro)viral particle capable of infecting a eukaryotic cell, substantially as herein described with reference to any one of the examples but excluding 10 comparative examples. V"o4 46. A nucleic acid coding for a peptide or for a recombinant particle capable of infecting a eukaryotic cell, this cell possessing a targeted receptor and an auxiliary *g receptor of the aforesaid (retro)viral particle, substantially as herein described with reference to any one of the examples but excluding comparative examples. *fe* 15 47. A method of selective transfer in vitro or ex vivo of a nucleic acid into target eukaryotic cells present among other non-target cells, substantially as herein described with reference to any one of the examples but excluding comparative examples.
48. A pharmaceutical composition containing as active substance a (retro)viral particle, substantially as herein described with reference to any one of the examples but excluding comparative examples.
49. Use of a (retro)viral particle capable of infecting a eukaryotic cell, this cell possessing a targeted receptor and an auxiliary receptor of the aforesaid (retro)viral particle, substantially as herein described with reference to any one of the examples but excluding comparative examples. DATED this 16th Day of August 2000 CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE Attorney: IVAN A. RAJKOVIC Fellow Institute of Patent Attorneys of Australia of BALDWIN SHELSTON WATERS
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