Chimeric Viral Envelopes

π All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to chimeric polytropic viral envelope polypeptides and uses thereof, as well as to polynucleotides encoding said chimeric polypeptides and constructs comprising said polypeptides and/or polynucleotides.

The present invention also relates to chimeric retroviral envelope polypeptides, polynucleotides and vectors encoding said chimeric retroviral envelope polypeptides, virus particles and cells harbouring said chimeric envelope polypeptides. The present invention further relates to methods of targeting receptors, methods of treatment and methods for delivery of agents using said chimeric retroviral envelope polypeptides.

Background of invention

Retroviruses Retroviruses are RNA viruses. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The family Retroviridae are enveloped single- stranded RNA viruses that typically infect mammals, such as, for example, bovines, monkeys, sheep, and humans, as well as avian species.

Retroviral envelope proteins

Retroviruses carry their genomes as two copies of a single RNA molecule and the simplest retroviruses contain the gag, pro.pol and env genes.

The first step in the replication cycle of a retrovirus is its entry into a host cell (see figure 1). The envelope protein (env) is responsible for binding of the retrovirus to a specific cell surface receptor. A retroviral receptor is a membrane integral protein in the plasma membrane of the host cell and as such has a function unrelated to virus infection. However, retroviral envelopes that use non-protein receptors are known, e. g.

the vesicular stomatitis virus.

Retroviruses can be thought of as a protein-package comprising RNA wrapped in a lipid membrane that contains glycoproteins. The lipid bi-layer is derived from the cell membrane after budding and is thought to be associated with a viral gene product, a peripheral membrane protein called Matrix (MA). Traversing through the lipid bi-layer is another viral gene product, the envelope protein, which consists of two subunits: the transmembrane (TM) and the surface unit (SU). The function of the envelope protein is binding of the virus to its target cell and mediating fusion of the viral and cellular membranes.

The retroviral envelope protein can be seen as a nano-device that mediates receptor- dependent fusion of biological membranes. When the envelope protein is attached to the lipid-bilayer membrane surrounding the virus, the net result of fusion with a cellular membrane is entry of the nucleoprotein core of the virus into the cytoplasm. Such fusion is triggered by the envelope protein's recognition of a receptor on the plasma membrane or an endosomal membrane. Natural receptors for retroviral infection are integral membrane proteins with multiple membrane-spanning domains. For the gammaretroviruses such as murine leukemia viruses, several natural receptors are known to have transporter functions for e.g. amino acids. When expressed on the plasma membrane of a cell, the viral envelope protein may also mediate cell to cell fusion. The dynamics of the fusion process is generated by the viral envelope protein which is produced in an activated state and has "one shot" to trigger membrane fusion.

The ability of redirecting the retroviral fusion machinery to a desired receptor would have wide biotechnological and potentially also nanotechnological applications. However, the regulatory mechanisms that interconnect receptor binding with fusion are poorly understood, which has made intelligent engineering of the envelope protein difficult. Many attempts at redirecting the receptor-specificity have found that incorporation of a ligand into the envelope protein may cause receptor-dependent binding without activation of the fusion machinery.

SL3-2 Murine leukaemia virus envelope polypeptide

In an amino acid sequence alignment between SL3-2 and MCF-247, a region has been found to display differences in the 15 amino acids long stretch upstream of the proline

rich region. This region has been named VR3 by the present inventors. Further, a sequence alignment of MLVs from different sub-families show conserved amino acids at positions 203-208 WGLRLY and at positions 214-215 DP based on SL3-2 sequence, thus defining a 13 amino acid stretch (see figures 4-5).

In the present context, the term "VR3 region" comprises all of the amino acids found between the residue found at two positions after the conserved tryptophan 197 and the residue before the conserved aspartic acid 214 (according to the sequence shown in SEQ ID NO:2) including these two positions.

Tropism of murine leukaemia virus (MLV)

The MLVs are a group of gammaretroviruses that has been divided into families based on their host range and interference properties. The families are the ecotropic, amphotropic, xenotropic and polytropic subfamilies. Ecotropic viruses are defined by their usage of the mCAT-1 receptor (Wang et al. 1991 ). Ecotropic viruses are able to infect only murine cells. Examples of ecotrpic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human and other species through the Pit-2 receptor (Kavanaugh et al. 1994). One example of an amphotopic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (XpM) receptor. However, the xenotropic and polytropic viruses differ in their species tropism. Xenotropic viruses such as NZB-9-1 infect human and other species but not murine species, whereas polytropic viruses infect murine, human and other species as exemplified by the mink cell focus-forming viruses (MCF) for example the MCF 247 virus. However, the polytropic SL3-2 virus has a host range as the mouse ecotropic viruses in that it infects and replicates in mouse cells, but are impaired in its ability to infect and replicate in mink cells or human cells. The SL3-2 envelope protein virus utilizes the polytropic (Xpr1 ) receptor.

Retroviral vectors in therapy Retroviral vector particles are useful agents for introducing polynucleotides into cells, such as eukaryotic cells. The term "introducing" as used herein encompasses a variety of methods of transferring polynucleotides into a cell, such methods including transformation, transduction, transfection, and transinfection.

Retroviruses typically have three common open reading frames, gag, pol, and env,

which encode the structural proteins, encode enzymes including reverse transcriptase, and encode envelope proteins, respectively. Typically, retroviral vector particles are produced by packaging cell lines that provide the necessary gag, pol, and env gene products in trans. (Miller, et al., Human Gene Therapy, Vol. 1, pgs. 5-14 (1990)). This approach results in the production of retroviral vector particles which transduce mammalian cells, but are incapable of further replication after they have integrated into the genome of the cell.

Thus, retroviral vector particles have been used for introducing polynucleotides into cells for gene therapy purposes. In one approach, cells are obtained from a patient, and retroviral vector particles are used to introduce a desired polynucleotide into the cells, and such modified cells are returned to the patient with the engineered cells for a therapeutic purpose. In another approach, retroviral vector particles may be administered to the patient in viva, whereby the retroviral vector particles transduce cells of the patient in vivo. Chimeric retroviruses have also been suggested in order to induce immune reactions against viruses, however no positive data have been reported showing this effect in humans.

Viral interference Among viruses such as the murine γ-retroviruses a phenomenon termed receptor interference has been used to classify viruses based on their tropism (Sommerfelt et al. 1990). Upon infection the virus synthesize de novo envelope proteins for the production of new viral particles. Some of these envelope proteins will engage the receptor via an unknown mechanism and shield the receptor (Figure 2). This shielding prevents the recurrence of an infective event by an exogenous virus. In cell culture the interference is very effective in that complete block of infection can be observed.

HIV-1 is somewhat different with regard to receptor usage. For HIV-1 entry to occur a two-step binding mechanism is required. First the HIV-1 envelope protein binds the CD4 receptor (primary receptor) (Eckert et al 2001). This event initiates a conformational change that exposes a region termed V3 (Variable loop 3) which is responsible for a second interaction with a co-receptor (either CCR-5 or CXCR-4) (Huang et al 2005). This co-receptor interaction is absolutely required for infection to occur. In cell culture the same degree of receptor interference is not observed by HIV-1 infection, which may be due to the dual receptor requirement.

The retroviral phenomenon of superinfection resistance (SIR) defines an interference mechanism that is established after primary infection, preventing the infected cell from being superinfected by a similar type of virus.

In most cases, virus-encoded proteins are responsible for the phenomenon of SIR. A simple form of SIR is receptor occupancy by viral Env proteins, preventing the binding of a second virus, but many additional mechanisms have been described. SIR is furthermore not restricted to retroviruses.

Uses of chimeric retroviral envelopes

Ecotropic and amphotropic MLVs have been widely used as research tools. Ecotropic viruses are usually chosen because of safety concerns, while the amphotropic viruses have the ability to infect human cells. Different packaging cell lines that express the ecotropic or amphotropic envelopes have been designed to fulfil these different requirements.

Several functional chimeric envelopes have already been described but none of these can mediate transduction at efficiencies comparable to the efficiencies obtained with wild type envelope proteins. The described functional chimeric MLV-envelopes can be divided into two groups. The first group has the heterologous ligand inserted in the N- terminal of the SU-protein and can mediate transduction without co-expression of wild type envelope, whereas the other group has the ligand inserted internally in SU and is dependent of co- expressed wild type envelope. Peptide linkers and a single chain antibody specific for the human major histocompatibility complex class I(MHC-I) molecule have e. g. been inserted at four internal positions in Akv-env.

The first attempts to direct virus particles towards receptors not normally recognised by retroviruses were done by antibody-bridging and by usage of chemical modifications. By cross-linking monoclonal antibodies against SU and the transferring receptor with a sheep anti-mouse kappaiight chain antibody binding of the virus to human HEp2 cells, and subsequent internalisation was shown. However, internalisation of the virus by this infection route was not followed by establishment of the proviral state.

Others used a similar approach to target the attachment of ecotropic viruses by streptavidin bridging biotinylated antibodies against SU and against specific membrane

markers expressed on human cells. By this method human cells expressing MHC class I, MHC class II, epidermal growth factor and insulin were successfully infected, whereas this method did not prove feasible for promoting infection of cells expressing transferrin, high density lipoprotein and galactose receptors.

Also, chemically coupled galactose residues to ecotropic Env, making the virus particles capable of infecting human hepatoma cells through the asialoglycoprotein receptor, have been tried.

Infection of human cells by an ecotropic virus displaying chimeric-envelope proteins on the surface of the virion is also known to a person skilled in the art. This can be achieved by e. g. substituting a part OfMoMLV SU with a sequence encoding theerythropoietin hormone (EPO), insertion of a sequence encoding human heregulin for infection of human breast cancer cells overexpressing the human epidermal growth factor receptor, substitution of an internal fragment of SU with a single-chain variable fragment (ScFv) derived from a monoclonal antibody recognising the human low density lipoprotein receptor which gave a chimeric envelope capable of infecting human cells.

In these reports with chimeric envelopes, targeted infection was only obtained when wild type env was co-expressed with the chimeric construct from the packaging cell line.

This indicates that functional domains are contained within the ecotropic envelope, which is necessary for mediating infection beyond the point of receptor binding.

The obtained targeting efficiencies with chimeric envelopes reported until now are considerably lower than the efficiencies obtained with wild type envelopes. The reasons for these low transduction efficiencies of target cells are probably diverse, including the choice of insertion site, stability of the chimeric envelope protein, the tertiary protein structure and the choice of target cells. Furthermore, the choice of ligand is probably also very important for obtaining infection, as several chimeric envelopes have failed to promote infection. One more positive example relates to insertion of a short nondisruptive peptide (RDG) known to bind to several integrins displayed on the surface of cells (Golan TJ and Green-MR, 2002).

The above-described examples all utilised the ecotropic envelope. One advantage of using this envelope is that it is restricted in infecting human cells as the surface protein part of the envelope does not recognise a human receptor. The concept is that if the envelope can be engineered to bind to a human receptor by inserting a heterologous sequence in the envelope mediating this binding, the otherwise intact fusogenic properties of the envelope would mediate the fusion.

Retroviruses Retroviruses are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The family Retroviridae are enveloped single-stranded RNA viruses that typically infect mammals, such as, for example, bovines, monkeys, sheep, and humans, as well as avian and murine species. Retroviruses are unique among RNA viruses in that their multiplication involves the synthesis of a DNA copy of the RNA which is then integrated into the genome of the infected cell.

The Retroviridae family comprises a number of retroviruses such as the lentiviruses exemplified by HIV-1 , HIV-2 and SlV, and the gammaretroviruses such as the leukaemia viruses for example murine leukaemia viruses (MLVs), and feline leukaemia viruses.

Retroviruses are defined by the way in which they replicate their genetic material. During replication the RNA is converted into DNA. Following infection of the cell a double-stranded molecule of DNA is generated from the two molecules of RNA which are carried in the viral particle by the molecular process known as reverse transcription. The DNA form becomes covalently integrated in the host cell genome as a provirus, from which viral RNAs are expressed with the aid of cellular and/or viral factors. The expressed viral RNAs are packaged into particles and released as infectious virion.

The retrovirus particle is composed of two identical RNA molecules. Each wild-type genome has a positive sense, single-stranded RNA molecule, which is capped at the 5 ¹

end and polyadenylated at the 3' tail. The diploid virus particle contains the two RNA strands complexed with gag proteins, viral enzymes (pol gene products) and host tRNA molecules within a 'core ^' structure of gag proteins. Surrounding and protecting this capsid is a lipid bilayer, derived from host cell membranes and containing viral envelope (env) proteins. The env proteins bind to a cellular receptor for the virus and the particle typically enters the host cell via receptor-mediated endocytosis and/or membrane fusion.

After the outer envelope is shed, the viral RNA is copied into DNA by reverse transcription. This is catalyzed by the reverse transcriptase enzyme encoded by the pol region and uses the host cell tRNA packaged into the virion as a primer for DNA synthesis. In this way the RNA genome is converted into the more complex DNA genome.

The double-stranded linear DNA produced by reverse transcription may, or may not, have to be circularized in the nucleus. The provirus now has two identical repeats at either end, known as the long terminal repeats (LTR). The termini of the two LTR sequences produces the site recognized by a pol product-the integrase protein— which catalyzes integration, such that the provirus is always joined to host DNA two base pairs (bp) from the ends of the LTRs. A duplication of cellular sequences is seen at the ends of both LTRs, reminiscent of the integration pattern of transposable genetic elements. Integration is thought to occur essentially at random within the target cell genome. However, by modifying the long-terminal repeats it is possible to control the integration of a retroviral genome.

Transcription, RNA splicing and translation of the integrated viral DNA is mediated by host cell proteins. Variously spliced transcripts are generated. In the case of the human retroviruses HIV-1/2 and HTLV-I/II viral proteins are also used to regulate gene expression. The interplay between cellular and viral factors is important in the control of virus latency and the temporal sequence in which viral genes are expressed.

Murine Leukaemia viruses are a family of simple retroviruses isolated from laboratory mice. Retroviruses carry their genomes as two copies of a single RNA molecule and the simplest retroviruses contain the gag, pro, pol and env genes. These genes are

found in the same order in all known retroviruses, reflecting the phylogenetic relationship of retroviruses.

Retroviral integration can activate genes in the vicinity of the integration site. In this way, retroviruses have been used to identify oncogenes since activation of these genes result in tumour growth. In much the same way the integration of a provirus can disrupt the expression of genes, hence inactivation of a tumour suppressor gene may contribute to tumour formation. A high number of integrations are desirable in such studies since not all integrations result in tumour generation and multiple hits are required. Very few integration events are expected to be near oncogene or tumour suppressor genes. Tumour formation might also involve multiple gene regulations.

Retroviral infections usually result in a single integration event since the envelope protein blocks receptors on an infected cell. This is the basis of the superinfection resistance (also called interference) phenomenon in which a virus-infected cell shows resistance to superinfection by viruses, which utilise the same receptor for entry. Thus, use of viruses with different receptor usage increases the number of integration events. Entry by different receptors may even provide access to retroviral disease induction in different mouse tissues.

The integration mechanism of retroviruses can be used to introduce any DNA sequence into a host genome, if the appropriate cis elements of the retroviral genome are maintained in the transducing vector and the DNA sequence can be encompassed in the vector (less than 9000 bp). Therefore retroviral vectors are attractive tools for gene therapy. Most simple retroviral receptors are found on many different cell types of the same species. That is why vector systems utilising wild type envelopes from simple retroviruses cannot be used to introduce genes in a selective manner into specific cells/tissues.

The retroviral envelope protein is a nano-device that mediates receptor-dependent fusion of biological membranes. When the envelope protein is attached to the lipid- bilayer membrane surrounding the virus, the net result of fusion with a cellular membrane is entry of the nucleoprotein core of the virus into the cytoplasm. Such fusion is triggered by the envelope protein's recognition of a receptor on the plasma membrane or an endosomal membrane. Natural receptors for retroviral infection are integral membrane proteins with multiple membrane-spanning domains. For the gammaretroviruses such as murine leukemia viruses, several natural receptors are

known to have transporter functions for e.g. amino acids. When expressed on the plasma membrane of a cell, the viral envelope protein may also mediate cell to cell fusion. The dynamics of the fusion process is generated by the viral envelope protein which is produced in an activated state and has "one shot" to trigger membrane fusion.

The ability of redirecting the retroviral fusion machinery to a desired receptor would have wide biotechnological and potentially also nanotechnological applications. However, the regulatory mechanisms that interconnect receptor binding with fusion are poorly understood, which has made intelligent engineering of the envelope protein difficult. Many attempts at redirecting the receptor-specificity have found that incorporation of a ligand into the envelope protein may cause receptor-dependent binding without activation of the fusion machinery.

Several functional chimeric envelopes have already been described but none of these can mediate transduction at efficiencies comparable to the efficiencies obtained with wild type envelope proteins. The described functional chimeric MLV-envelopes can be divided into two groups. The first group has the heterologous ligand inserted in the N- terminal of the SU-protein and can mediate transduction without co-expression of wild type envelope, whereas the other group has the ligand inserted internally in SU and is dependent of co- expressed wild type envelope. Peptide linkers and a single chain antibody specific for the human major histocompatibility complex class I(MHC-I) molecule have e. g. been inserted at four internal positions in Akv-env.

The first attempts to direct virus particles towards receptors not normally recognised by retroviruses were done by antibody-bridging and by usage of chemical modifications. By cross-linking monoclonal antibodies against SU and the transferring receptor with a sheep anti-mouse kappa light chain antibody binding of the virus to human HEp2 cells, and subsequent intemalisation was shown. However, internalisation of the virus by this infection route was not followed by establishment of the proviral state.

Others used a similar approach to target the attachment of ecotropic viruses by streptavidin bridging biotinylated antibodies against SU and against specific membrane markers expressed on human cells. By this method human cells expressing MHC class I, MHC class II, epidermal growth factor and insulin were successfully infected, whereas this method did not prove feasible for promoting infection of cells expressing

transferrin, high density lipoprotein and galactose receptors.

Also, chemically coupled galactose residues to ecotropic Env, making the virus particles capable of infecting human hepatoma cells through the asialoglycoprotein receptor, have been tried.

Infection of human cells by an ecotropic virus displaying chimeric-envelope proteins on the surface of the virion is also known to a person skilled in the art. This can be achieved by e. g. substituting a part OfMoMLV SU with a sequence encoding theerythropoietin hormone (EPO), insertion of a sequence encoding human heregulin for infection of human breast cancer cells overexpressing the human epidermal growth factor receptor, substitution of an internal fragment of SU with a single-chain variable fragment (ScFv) derived from a monoclonal antibody recognising the human low density lipoprotein receptor which gave a chimeric envelope capable of infecting human cells.

In these reports with chimeric envelopes, targeted infection was only obtained when wild type env was co-expressed with the chimeric construct (from theti ) 2 packaging cell line). This indicates that functional domains are contained within the ecotropic envelope, which is necessary for mediating infection beyond the point of receptor binding.

The obtained targeting efficiencies with chimeric envelopes reported until now are considerably lower than the efficiencies obtained with wild type envelopes. The reasons for these low transduction efficiencies of target cells are probably diverse, including the choice of insertion site, stability of the chimeric envelope protein, the tertiary protein structure and the choice of target cells. Furthermore, the choice of ligand is probably also very important for obtaining infection, as several chimeric envelopes have failed to promote infection. One more positive example relates to insertion of a short nondisruptive peptide (RDG) known to bind to several integrins displayed on the surface of cells (Golan TJ and Green-MR, 2002).

The above-described examples all utilised the ecotropic envelope. One advantage of using this envelope is that it is restricted in infecting human cells as the surface protein part of the envelope does not recognise a human receptor. The concept is that if the

envelope can be engineered to bind to a human receptor by inserting a heterologous sequence in the envelope mediating this binding, the otherwise intact fusogenic properties of the envelope would mediate the fusion.

The present invention provides improved chimeric envelope proteins with novel ligands and ligand insertion sites within the envelope polypeptide that are advantageous over prior art chimeric envelopes, for example in relation to improving therapeutic efficacy of gene therapies.

Summary of invention

The present invention provides an isolated chimeric viral envelope polypeptide comprising:

(i) a first polypeptide sequence consisting of the polypeptide sequence of a gamma retrovirus envelope polypeptide, or a homologue or fragment thereof;

wherein into said first polypeptide sequence has been inserted or attached at one end:

(ii) a second polypeptide sequence comprising a receptor-binding domain of a second, different viral envelope polypeptide, or a fragment or homologue thereof, wherein said second polypeptide sequence further optionally comprises one or more flexible linker sequence(s).

Said first polypeptide sequence preferably has a sequence that is at least 70 % identical to the amino acid sequence shown in SEQ ID NO:2, or is a fragment of a sequence that is at least 70 % identical to the amino acid sequence shown in SEQ ID NO:2, and can for example be a polytropic murine leukaemia virus. In one preferred embodiment, the inserted receptor-binding domain is the V-3 loop domain of HIV-1 or a fragment or homologue thereof.

The present invention further relates to isolated polynucleotides encoding the chimeric viral envelope polypeptide, as well as vectors and replication-competent retroviruses comprising the chimeric viral envelope polypeptides. Stable cell lines are also provided,

which may be used a packaging cell lines producing the replication-competent retroviruses.

Pharmaceutical compositions comprising the polypeptides and/or retroviruses of the present invention are also provided, which can be used in various therapeutic methods, including gene therapy and methods for prevention of viral infection.

Without being bound by theory, it is believed that the chimeric polypeptides trigger a type of artificial "superinfection resistance" in an individual thus treated. Thus, in the case of HlV treatment, one can for example use an engineered SL3-2 envelope that contains the V3 region of HIV to block the HIV co-receptors CCR-5 and/or CXCR-4 and thereby prevent or reduce HIV infection. The inventors have furthermore found that the envelope proteins can interfere with cell-cell fusion caused by the HIV-envelope.

The present invention in one aspect provides a chimeric viral envelope polypeptide comprising

(i) an envelope polypeptide, or fragment thereof,

(ii) a polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region, and optionally a linker sequence,

wherein the receptor binding domain of said envelope polypeptide has a sequence that is at least 36% identical to the amino acid sequence shown in SEQ ID NO: 60, or is a fragment of a sequence that is at least 36% identical to the amino acid sequence shown in SEQ ID NO: 60.

Aspects of the present invention also relate to a polynucleotide encoding the chimeric viral envelope polypeptide, a retroviral vector comprising the polynucleotide or fragment thereof, a virus particle expressing the chimeric viral envelope polypeptide.

Further aspects pertain to a virus particle comprising:

(i) the polynucleotide as described above,

(ii) an agent for delivery to the target cell, optionally selected from the group consisting of a therapeutic agent, a gene or gene product, a diagnostic label, a label for

bioimaging, or a toxic agents, which agent is operatively associated with a retroviral packaging sequence.

Also aspects relating to a cell transfected with a. the polynucleotide as described above or b. the retroviral vector as described above,

a stable cell line comprising cells as described above, and an animal model comprising such cells, a packaging cell line comprising the retroviral vector are disclosed in the present invention.

Aspects of the present invention relates to a method for targeting an agent to a G- protein coupled receptor, comprising the steps of:

(i) providing the chimeric envelope polypeptide (ii) causing said chimeric envelope polypeptide to contact a target cell wherein said target cell comprises a receptor for the ligand of said chimeric envelope polypeptide,

a method for specifically tethering a chimeric retroviral envelope polypeptide to a specific cell type, comprising the steps of: (i) providing an virus particle expressing a chimeric retroviral envelope polypeptide, said envelope polypeptide comprising a ligand capable of binding said specific cell-type,

(ii) allowing said chimeric envelope polypeptide to specifically contact a cell of said specific cell type,

(ii) allowing the outer membrane of the virus to undergo a hemi-fusion process with the outer membrane of said cell, and

a method for labelling one or more object of interest on a cell, comprising: (i) providing the labelled vector, polypeptide or recombinant retrovirus,

(ii) allowing said labelled vector, polypeptide or recombinant retrovirus to contact said object of interest on said cell.

Further aspects of the present invention relates to a method for quantifying the amount or number of an object of interest in a biological specimen, such as a cell, said method comprising the steps of:

(i) providing the labelled vector, chimeric envelope polypeptide or virus particle, (ii) allowing said labelled vector, chimeric envelope polypeptide or virus particle to contact said object of interest on said cell, and a method for screening for and analysis of drugs that target the envelope-receptor interaction, comprising the virus particle.

The present invention further discloses methods relating. to therapy such as a therapeutic method for treatment of an individual in need thereof, said method comprising administering the chimeric viral envelope polypeptide, the vector, or virus particle.

For example the invention relates to a therapeutic method for treatment of an individual in need thereof, said method comprising administering a si-RNA, a method for delivering an agent to a mammalian target cell in an individual in need thereof, comprising the steps of:

(i) providing the chimeric envelope polypeptide or virus particle (ii) causing said chimeric envelope polypeptide or virus particle to contact a target cell population of said individual's cells, wherein said target cell comprises a receptor for the ligand of said chimeric envelope polypeptide (iii) allowing the virus particle to bind said receptor.

Furthermore the present invention relates to aspects of an antibody capable of specifically binding a molecule, or a medicament comprising the chimeric envelope polypeptide, vector, or viral particle.

Description of Drawings

Figure 1A: Schematic depiction of a retrovirus, showing viral envelope polypeptide Figure 1 B: Schematic depiction of retroviral life cycle

Fig. 2 shows a schematic depiction of retroviral interference. Top: Normal infection mechanism via receptor binding. Bottom: Receptor shielding by endogenous expressed envelope.

Fig. 3:

- Depicted in panel A is a wild type replication competent virus.

- In panel B said replication competent virus has an insert ("ScFv") in the envelope for redirection of tropism, which can be any suitable insert sequence, such as those described herein

- Panel C and E are replication competent vectors where a heterologous translational cassette has been inserted into either the U3 region panel C or in the 3 un-translated region downstream of the envelope gene Panel E.

- Panel D and F are the same as panel C and E except the insert ("ScFv" - although in this case, can be any suitable sequence, as described herein) has been inserted in the envelope for redirection of tropism.

Panel G is a replication competent retroviral vector expressing an envelope gene from a rnono-cistronic mRNA by directing translation of the envelope gene by an internal ribosome entry site (IRES).

Panel H same as Panel G except that an insert (ScFv - but can be any suitable insert as described herein) has been inserted into the envelope gene for redirection of tropism.

In the present context ScFv can be any heterologous peptide sequence that mediates redirection of envelope tropism, such as the V3 peptide.

Fig. 4 shows an alignment of amino acid sequences of selected MLVs in the VR3 region. Residues corresponding to arginine 212 are shown in the rectangular box.

Fig. 5a and 5b show sequence alignments showing homology between various viral envelope polypeptides

Fig. 6 shows flow cytometric analysis of cell surface expression of CXCR4 cells transduced with a vector expressing SL3-2 envelope with an inserted V3 loop.

Fig. 7 shows a sequence alignment of V3 loop from different HIV-1 subtypes. The arrows indicate important highly conserved Cysteine residues. Genebank accession numbers are indicated.

Fig. 8-9 show syncytia formation of 293T cells expressing the HIV-1 envelope protein (indicated by fluorescent signal in the right hand panel) and D17 CD4 CXCR4 cells. The first panel ("panel 1") indicates D17 CD4 CXCR4 cells (without SL3-2 envelope), the following four panels ("panel 2"-"panel 5") are D17 CD4 CXCR4 cells transduced by different variants of the SL3-2 envelope with V3 inserted. Final panel ("panel 6) is a transfection control of the vector pLXSN (expressing egfp) without the HIV-1 envelope.

Fig 10 shows further results of Syncitia assays. The arrows indicate multi-nuclei cell syncytia. NIH cells stably expressing the HIV-1 envelope protein were co-cultured with different HeLa target cells (HeLa expresses CXCR-4 endogenously). Top left HeLa cells lacking the CD4 receptor. Bottom left HeLa cells with CD4. Middle, HeLa CD4 cells transfected with Moloney MLV envelope. Right, HeLa CD4 cells transfected with two variants of chimeric Moloney/V3 envelopes. We note a reduction in syncytia formation in the presence of V3 envelope, in particular in the upper right panel.

Fig. 11 shows an alignment of part of the envelope amino acid sequence of a number of gammaretroviruses. The border of the receptor binding domain (RBD) and the praline rich repeat (PRR) is shown. The position of the variable region A and B (VRA and VRB is indicated as is the variable region 3 (VR3).

Fig. 12 shows the titers of the constructs in which apelin has been inserted. The GPCR APJ mediates specific entry into D17 dog cells of an SL3-2 envelope virus engineered to harbor its cognate ligand at a critical position (see Fig. 4). The insertion site in AP@155 is suboptimal relative to AP@165. NIH cells are mouse cells that can be infected by the wt SL3-2 as well as the three mutants. SL3-2GI is a mutant that infects human cells, but not dog cells.

Fig. 13 shows a G-protein coupled receptor with the typical 7-transmembrane helix- organization.

Fig. 14 discloses A) the functional domains of a retroviral envelope protein. RBD, receptor-binding domain; CTD. C-terminal domain; TM, transmembrane protein, harboring a folded-back helix that is extended into the receptor membrane upon

activation, B) the crystal structure of the receptor binding domain and the apelin insertion site.

Fig. 15 shows a schematic illustration of the steps in the membrane fusion process directed by a retroviral envelope protein; note the intermediate hemi-fusion stage that allows the mixing of lipids in the outer, but not the inner leaflets of the two membranes.

Fig. 16 shows a schematic depiction of retroviral interference. Top: Normal infection mechanism via receptor binding. Bottom: Receptor shielding by endogenous expressed envelope.

Fig. 17 shows the results of Syncitia assays. The arrows indicate multi-nuclei cell syncytia. NIH cells stably expressing the HIV-1 envelope protein were co-cultured with different HeLa target cells (HeLa expresses CXCR-4 endogenously). Top left HeLa cells lacking the CD4 receptor. Bottom left HeLa cells with CD4. Middle, HeLa CD4 cells transfected with Moloney MLV envelope. Right, HeLa CD4 cells transfected with two variants of chimeric Moloneyλ/3 envelopes. We note a reduction in syncytia formation in the presence of V3 envelope, in particular in the upper right panel.

Fig. 18-20. NIH3T3 cells were incubated with SL3-2 containing a hexa histidine motif in position 165 (or control virus) on ice for 1h. Liposomes containing the FRET pair: NBD- PE and Rh-PE were subsequently incubated with the virus/cell mix on ice for another hour followed by incubation in 37oC for 1h. Fusion is expected to result in increase and dispersal of the green fluorescence. Fig. 18 and fig. 19 show hexahistidine tagged virus incubated with NTA containing liposomes. Fig 20 show control virus incubated with liposomes without NTA. All figures show overlays of green, red fluorescences with whitefield.

Fig. 21 shows that NTA holding liposomes have higher binding affinity for hexa histidine containing viral envelope (Green) than liposomes without NTA (Blue).

Fig. 22 shows that binding of NTA containing liposomes (green) is not higher than liposomes without NTA (blue) to control virus

Fig. 23: titer of the chimeric envelopes on mono-amine transporters

Fig. 24: Liposome fusion to mouse fibroblasts in the presence of hexa histidine-tagged virus. Note the confinement of green fluorescence in the plasma membrane of cells incubated with NTA-containing liposomes.

Fig. 25: Syncytia formation by HIV-envelope expressed in 293T cells and Jurkat cells (labeled with the red fluorescence dye R18). Notice the reduced size and frequency of syncytia in Jurkat cells expressing the SL3-2-V3 chimeric envelope (right panel).

Fig. 26. The SL3-2 chimeric envelopes can use two different receptors. The figure shows the titer of SL3-2AP@165 envelope on different cells. NIH3T3: Murine cells, NIH3T3 + MCF247: murine cells infected with MCF247, thus having a blocked polytropic receptor, D17: Dog cells, D17-APJ: Dog cells expressing APJ, D17-APJ-6uM Apelin 13: Dog cells expressing APJ incubated with 6uM apelin-13 peptide for 1h. The data show that infection can occur through both the polytropic Xpr1 and the heterologous APJ receptors.

Fig. 27-30 shows that the chimeric SL3-2AP@165 envelope can induce syncytia in XC cells using APJ but not Xpr-1. XC or APJ expressing XC cells were co-cultured with 293T cells transfected with R-peptide less SL3-2AP@165 envelope expression vector for 24h. Syncyita is best visible in the green fluorescent as large star-shaped cells. No syncytia are visible without APJ expression in XC cells. Fig. 27: Fluorescence picture of XC-APJ cells and fluorescent SL3-2AP@165 expressing 293T cells. Fig. 28: XC cells and fluorescent SL3-2AP@165 expressing 293T cells. Fig. 29: fluorescence picture of XC cells and fluorescent SL3-2AP@165 expressing 293T cells. Fig. 30: XC-APJ cells and fluorescent SL3-2AP@165 expressing 293T cells.

Detailed description of the invention

Definitions:

The term "polypeptide" as used herein means a polymer of amino acids and does not refer to any particular length of polymer. Such term also includes post-translationally modified polypeptides or proteins (e.g., glycosylated, acetylated, phosphorylated, etc.).

"Polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides at least 2 bases in length. By "isolated nucleic acid sequence" is meant a polynucleotide that is not immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA or RNA which is incorporated into a viral vector. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double stranded forms of DNA.

The term polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, polynucleotide as used herein can also refer to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide includes DNAS or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.

It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.

The polypeptides, vectors, retroviruses, antibodies, and polynucleotides according to the present invention are preferably isolated and/or purified, and can for example be produced using recombinant methods known to one skilled in the art.

Sequence homology:

The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology) ; the higher the percentage, the more similar the two sequences will be.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described and present a detailed consideration of sequence alignment methods and homology calculations, such as VECTOR NTI.

The NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NBCI, Bethesda, Md. ) and on the Internet, for use in connection with the sequence analysis programsblastp,biastn,blastx,tblastn andtblastx. It can be accessed at http://www. ncbi. nlm. nih. gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www. ncbi.nim. nih.gov/BLAST/blast~help. html.

The term "Homologue" as described herein refers to a molecule characterised by possession of at least 40 % sequence identity (such as at least at least 45 % sequence identity, for example at least 50 % sequence identity, such as at least at least 55 % sequence identity, such as at least at least 50 % sequence identity, for example at least 55 % sequence identity, such as at least at least 60 % sequence identity, for example at least 65 % sequence identity, for example at least 70 % sequence identity, such as at least at least 75 % sequence identity, for example at least 80 % sequence identity, such as at least at least 85 % sequence identity, for example at least 87 % sequence identity, such as at least at least 90 % sequence identity, for example at least 91 % sequence identity, such as at least at least 92 % sequence identity, for example at least 93 % sequence identity, such as at least at least 94 % sequence identity, for example at least 95 % sequence identity, such as at least at least 96 %

sequence identity, for example at least 97 % sequence identity, such as at least at least 98 % sequence identity, for example at least 98.5 % sequence identity, such as at least at least 99 % sequence identity, for example at least 99.5 % sequence identity) counted over the full length alignment with the disclosed polypeptide or polynucleotide sequence using e.g. the NCBI Basic Blast 2.0, gappedblast with databases such as the nr or swissprot database. Alternatively, one may manually align the sequences and count the number of identical amino acids or nucleotides. This number divided by the total number of amino acids or nucleotide in your sequence multiplied by 100 results in the percent identity.

Chimeric viral envelope polypeptide

In a first aspect of the present invention is provided a chimeric viral envelope polypeptide comprising:

(i) a first polypeptide sequence consisting of the polypeptide sequence of a gamma retrovirus envelope polypeptide, or a homologue or fragment thereof;

wherein into said first polypeptide sequence has been inserted or attached at one end:

(ii) a second polypeptide sequence comprising a receptor-binding domain of a second, different viral envelope polypeptide, or a fragment or homologue thereof, wherein said second polypeptide sequence further optionally comprises one or more flexible linker sequence(s).

Said chimeric viral envelope polypeptide is preferably isolated and/or purified.

First polypeptide sequence

The first polypeptide sequence of the chimeric viral envelope polypeptide according to the present invention comprises or consists of the polypeptide sequence of a gamma retrovirus envelope polypeptide, or a homologue or fragment thereof.

Said first polypeptide can for example be a gamma retrovirus envelope polypeptide, wherein said gamma retrovirus is selected from the group consisting of ecotropic viruses, polytropic viruses, amphotropic viruses and xenotropic viruses. Thus, said gamma retrovirus can be selected from the group consisting of a polytropic,

amphotropic, or xenσtropic gamma retroviruses. In another embodiment, said gamma retrovirus is selected from the group consisting of a polytropic or amphotropic gamma retroviruses. In another embodiment, said gamma retrovirus is polytropic, such as selected from the group consisting of: SL3-2, MCF-247, MCF CI-3, ERV-1 , Friend MCF, Friend SFV, Invitro MCF, MCF 1223, MLV DBA/2, Mo-MCF, Ns-6(186)MCF, Rauscher sfv, "Endogenous from 129 GIx+ mice", Ampho-MCF, MCF(Ter-Grigorov), MCF (Brosclus), Friend MCF#2, R-XC, Gibbon ape leukemia virus (GaLV), Feline leukemia virus (FeLV) subtypes A, B, and C, Koala Retrovirus (KoRV) and Xeno R- MCI-1. These viral types are described in more detail in Example 5.

In another embodiment, said gamma retrovirus is amphotropic, such as selected from the group consisting of: 10A1 and AKV. These viral types are described in more detail in Example 5.

In another embodiment, said gamma retrovirus is xenotropic, such as selected from the group consisting of: DG-75 Xeno, Xeno NZ8-9-1 , Xeno CWM-S-5X, Xeno Bxv-1- related, and 40701. These viral types are described in more detail in Example 5.

In another embodiment, said gamma retrovirus is ecotropic, such as selected from the group consisting of: SL3-3, Friend, Maloney, Friend fass and Consensus virus. These viral types are described in more detail in Example 5.

In another embodiment, said gamma retrovirus is mouse leukaemia virus (MLV), such as a polytropic MLV or SL3-2. Thus, in one embodiment of the present invention is provided a chimeric viral envelope polypeptide wherein the first polypeptide sequence comprises or consists of of the polypeptide sequence of the SL3-2 murine leukaemia virus envelope polypeptide, or fragment or homologue thereof.

In one embodiment, the first polypeptide is an envelope protein from the Murine

Leukaemia Virus (MLV) strain SL3-2, which is capable of infecting murine cells through usage of the polytropic receptor encoded by theRmcl locus, but lacks the ability of infecting human cells expressing the corresponding xenotropic receptor encoded by the RMC 1 locus.

It is preferred that the first polypeptide sequence has a sequence that is at least 80 % identical to the amino acid sequence shown in SEQ ID N0:2 (the SL3-3 envelope polypeptide), or is a fragment of a sequence that is at least 80 % identical to the amino acid sequence shown in SEQ ID NO:2. Thus, the first polypeptide sequence can have a sequence that is at least 85 % identical to the amino acid sequence shown in SEQ ID NO:2, or is a fragment of a sequence that is at least 85 % identical to the amino acid sequence shown in SEQ ID NO:2, For example, said first polypeptide sequence can have a sequence that is at least 90 % identical to the amino acid sequence shown in SEQ ID NO:2, or is a fragment of a sequence that is at least 90 % identical to the amino acid sequence shown in SEQ ID NO:2. For example, said first polypeptide sequence can have a sequence that is at least 95 % identical to the amino acid sequence shown in SEQ ID NO:2, or is a fragment of a sequence that is at least 95 % identical to the amino acid sequence shown in SEQ ID NO:2. For example, said first polypeptide sequence can have a sequence that is at least 95 % identical to the amino acid sequence shown in SEQ ID NO:2, or is a fragment of a sequence that is at least 95 % identical to the amino acid sequence shown in SEQ ID NO:2. For example, said first polypeptide sequence has a sequence that is at least 98 % identical to the amino acid sequence shown in SEQ ID NO:2, or is a fragment of a sequence that is at least 98 % identical to the amino acid sequence shown in SEQ ID NO:2. For example, said first polypeptide sequence is the envelope polypeptide of a polytropic murine leukaemia virus. For example, said first polypeptide sequence can comprise or consist of SEQ ID NO: 2, or a fragment thereof.

It has also been found that changing specific amino acids within the VR3 region of this MLV SL3-2 envelope polypeptide, or a polytropic homologue thereof, enables alteration of the host tropism of said envelope polypeptide. The present inventors have pinpointed exactly which amino acid that is essential for this host tropism shift. Thus, in the case that the first polypeptide is homologous to SEQ ID NO:2, one embodiment is that said first polypeptide includes at least one substitution in the VR3 region, or a region homologous thereto. In the present context, the term "VR3 region"comprises all of the amino acids found between the residue found at two positions after the conserved tryptophan 197 and the residue before the conserved aspartic acid 214 (according to the sequence shown in SEQ ID NO : 2) including these two positions. In one embodiment of the present invention, said first polypeptide includes at least one substitution in the region homologous to the VR3 region, such as 1 ,2, 3,4, 5 or 6

substitutions in the VR3 region. Examples of substitutions which are likely to provide the same effect are alanine, asparagine, aspartic acid, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamin, serine, threonine, valine, tryptophan or tyrosine.

In one preferred embodiment, the substitution changes the arginine to glycine. In another preferred embodiment the substitution results in a methionine. For example, said substitution can be at position 212 in SEQ ID NO: 2, or a region homologous thereto. It is preferred that said at least one substitution alters the host tropism of a virus or an infectious particle comprising said polypeptide, in a manner described in more detail in WO 03/097674 (Pipeline Biotech A/S).

Examples of suitable first polypeptide sequences (with the insert site marked for insertion of the second polypeptide sequence) have SEQ ID NO: 33-41 , 49, 51 , 53, 55, 57 or 59, or are homologues and/or fragments thereof. These are e.g. encoded by polynucleotides with SEQ ID NO: 48, 50, 52, 54, 56 or 58, or suitable homologues and/or fragments thereof.

Second polypeptide sequence Into the first polypeptide sequence of the chimeric viral envelope polypeptide according to the present invention is inserted or attached at one end: a second polypeptide sequence comprising a receptor-binding domain of a second, different viral envelope polypeptide, or a fragment or homologue thereof. Said second polypeptide sequence is preferably inserted within a site homologous to amino acids 80-106 in SEQ ID NO:2, such as within a site homologous to amino acids 80-106 in SEQ ID NO:2, such as within a site homologous to amino acids 80-100 in SEQ ID NO:2, or such as within a site homologous to amino acids 80-90 in SEQ ID NO:2, or such as within a site homologous to amino acids 90-106 in SEQ ID NO:2, or such as within a site homologous to amino acids 90-95 in SEQ ID NO:2, or such as within a site homologous to amino acids 80-82 in SEQ ID NO:2, or such as within a site homologous to amino acids 80-84 in SEQ ID NO:2.

In another preferred embodiment of the present invention, said second polypeptide sequence is inserted within a site homologous to within amino acids 152-181 in SEQ ID NO:2, such as within a site homologous to amino acids 152-164 in SEQ ID NO:2, or

such as within a site homologous to amino acids 152-160 in SEQ ID NO:2, or such as within a site homologous to amino acids 160-170 in SEQ ID NO:2, or such as within a site homologous to amino acids 165-175 in SEQ ID NO:2, or such as within a site homologous to amino acids 175-181 in SEQ ID NO:2, or such as within a site homologous to amino acids 160-165 in SEQ ID NO:2, or such as within a site homologous to amino acids 152-158 in SEQ ID NO:2.

In another preferred embodiment of the present invention, said second polypeptide sequence is inserted into a site homologous to a.a. 192-213 in SEQ ID NO:2, such as within a site homologous to amino acids 192-202 in SEQ ID NO:2, or such as within a site homologous to amino acids 198-203 in SEQ ID NO:2, or such as within a site homologous to amino acids 205-213 in SEQ ID NO:2, or such as within a site homologous to amino acids 200-213 in SEQ ID NO:2.

In another preferred embodiment of the present invention, said second polypeptide sequence is inserted into a site homologous to a.a. 229-281 in SEQ ID NO:2, such as within a site homologous to amino acids 229-259 in SEQ ID NO:2, or such as within a site homologous to amino acids 239-269 in SEQ ID NO:2, or such as within a site homologous to amino acids 249-281 in SEQ ID NO:2, or such as within a site homologous to amino acids 259-281 in SEQ ID NO:2, or such as within a site homologous to amino acids 271-281 in SEQ ID NO:2, or such as within a site homologous to amino acids 235-245 in SEQ ID NO:2, or such as within a site homologous to amino acids 245-255 in SEQ ID NO:2.

The inserted sequence can be inserted between two contiguous amino acids of the insert site, or can replace one or more amino acids at said insert site, such as replacing one, two, three or more amino acids at the insert site, such as replacing 1-10 amino acids at the insert site.

The second polypeptide sequence comprises a receptor-binding domain of a second, different viral envelope polypeptide.

Said receptor-binding domain of said second, different viral envelope polypeptide is in one embodiment a co-receptor-binding domain, or a fragment or homologue thereof.

In one preferred embodiment of the present invention, said receptor binding region is a receptor binding region of a human virus, such as e.g. Vesicular stomatitis virus (VSV) (Protein G), cytomegalovirus envelope (CMV), HIV, or influenza virus hemagglutinin (HA).

For example, said receptor-binding domain of said second, different viral envelope polypeptide can be a fragment or homologue of the influenza hemmaglutinin or the V3 domain of HIV.

Thus, in one embodiment of the present invention, the second, different viral envelope polypeptide is the V3-loop domain of HIV or a fragment or homologue thereof. Said HIV may for example be a CXCR-4 tropic HIV and/or, a strain of HIV-1 or a strain of HIV-2.

Thus, in one preferred embodiment of the present invention the receptor-binding domain of the second, different viral envelope polypeptide has a sequence selected from the group consisting of: any of SEQ ID NO: 9-32, or a fragment or homologue thereof. For example, said sequence can be selected from the group consisting of a fragment or homologue of any of SEQ ID NO: 9-16. In another embodiment, said sequence can be selected from the group consisting of a fragment or homologue of any of SEQ ID NO: 9-12. In another embodiment, said sequence can be selected from the group consisting of a fragment or homologue of any of SEQ ID NO: 16-24. In another embodiment, said sequence can be selected from the group consisting of a fragment or homologue of any of SEQ ID NO: 16-20. In another embodiment, said sequence can be selected from the group consisting of a fragment or homologue of any of SEQ ID NO: 21-25. In another embodiment, said sequence can be selected from the group consisting of a fragment or homologue of any of SEQ ID NO: 25-32. In another embodiment, said sequence can be selected from the group consisting of a fragment or homologue of SEQ ID NO: 32.

In another embodiment of the present invention, the receptor binding region is a hepatitis B virus surface protein binding region, preferably binding to a liver cell.

In another embodiment of the present invention, the receptor binding region is the receptor binding region of gp46 of HTLV-I virus, preferably binding to a T cell.

Optionally, a portion of the first retroviral envelope protein is deleted and the second polypeptide is inserted into said deleted portion. Preferably, the only portion of the retroviral envelope protein that is deleted is (i) a portion or all of the receptor binding region, (ii) a portion of the receptor binding region and a portion or all of the hinge region, or (iii) all of the receptor binding region and a portion or all of the hinge region. Thus, in one embodiment of the present invention, a portion of the receptor binding region of the first polypeptide sequence is deleted, for example all of the receptor binding region of the retroviral envelope protein is deleted, for example all of the receptor binding region and a portion of the hinge region of the first polypeptide are deleted.

Flexible linker sequence

The second polypeptide sequence of the chimeric viral envelope polypeptide further optionally comprises one or more flexible linker sequence(s) of one or more amino acid residues as known by one skilled in the art - for example 2-30 amino acid residues, such as 2-20 amino acid residues, such as 2-10 amino acid residues. The linker sequences are preferably placed at the N-terminal and/or C-terminal of the insert region, preferably whereby such linkers increase rotational flexibility and/or minimize steric hindrance of the modified envelope polypeptide. Thus, in one embodiment of the present invention, a linker sequence is positioned at each end of the second polypeptide sequence, that is to say at either end of the second polypeptide sequence. Any suitable linker sequence known to one skilled in the art can be used: examples of suitable linker sequences include, but are not restricted to, linkers described by Argos et al., 1990 (Argos, 1990). One preferred linker sequence has the polypeptide sequence SGGSG. Other preferred linkers can for example be QGIYQC or CG or

QGIYQC or CG, or homologues thereof with one, two or more amino acid substitutions.

Preferred sequences of the chimeric viral envelope polypeptide according to the present invention In one embodiment of the present invention, the chimeric viral envelope polypeptide has a sequence comprising or consisting of any of SEQ ID NO: 6-8 or 45-47, or a fragment or homologue thereof. Thus, the chimeric viral envelope polypeptide can comprise or consist of SEQ ID NO: 6, or a homologue thereof. In another embodiment, said chimeric viral envelope polypeptide can comprise or consist of SEQ ID NO: 7, or a homologue thereof. In another embodiment, said chimeric viral envelope polypeptide

can comprise or consist of SEQ ID NO: 8, or a homologue thereof. In another embodiment, said chimeric viral envelope polypeptide can comprise or consist of SEQ ID NO: 45, or a homologue thereof. In another embodiment, said chimeric viral envelope polypeptide can comprise or consist of SEQ ID NO: 46, or a homologue thereof. In another embodiment, said chimeric viral envelope polypeptide can comprise or consist of SEQ ID NO: 47, or a homologue thereof.

Polynucleotide

The present invention further discloses isolated nucleic acid sequences capable of encoding the envelope polypeptide sequences of the present invention.

As known to a person skilled in the art, a codon of an amino acid can be generated by various nucleic acid sequences, thus the present invention relates to all isolated nucleic acid sequences capable of encoding an envelope polypeptide having an amino acid sequence as described in the present application. Thus, the present invention relates to an isolated polynucleotide comprising or consisting of a polynucleotide encoding the chimeric viral envelope polypeptide according to the present invention.

Thus, in one embodiment, said polynucleotide has a sequence comprising or consisting of SEQ ID NO: 3, or a homologue thereof. In another embodiment, said polynucleotide has a sequence comprising or consisting of SEQ ID NO: 4, or a homologue thereof. In another embodiment, said polynucleotide has a sequence comprising or consisting of SEQ ID NO: 5, or a homologue thereof. In another embodiment, said polynucleotide has a sequence comprising or consisting of SEQ ID NO: 42, or a homologue thereof. In another embodiment, said polynucleotide has a sequence comprising or consisting of SEQ ID NO: 43, or a homologue thereof. In another embodiment, said polynucleotide has a sequence comprising or consisting of SEQ ID NO: 44, or a homologue thereof.

The polynucleotides may be constructed by genetic engineering techniques known to those skilled in the art. For example, a first expression plasmid may be constructed which includes a polynucleotide encoding the unmodified envelope. The plasmid then is engineered such that a polynucleotide encoding the second polypeptide is inserted between two codons encoding consecutively numbered amino acid residues of the first envelope polypeptide, or is engineered such that a polynucleotide encoding a portion of

the unmodified envelope is removed, whereby such portion may be replaced with a polynucleotide encoding the second polypeptide. The polynucleotide encoding the second polypeptide may be contained in a second expression plasmid or may exist as a naked polynucleotide sequence. The polynucleotide encoding the second polypeptide or the plasmid containing such polynucleotide is cut at appropriate restriction enzyme sites and cloned into the first expression plasmid which also has been cut at appropriate restriction enzyme sites. The resulting expression plasmid thus includes a polynucleotide encoding the chimeric envelope polypeptide. Such polynucleotide then may be cloned out of the expression plasmid, and into a vector, such as a retroviral plasmid vector. The resulting vector, which includes the polynucleotide encoding the modified envelope protein, and which also may include a polynucleotide encoding a heterologous protein or peptide, is transfected into an appropriate packaging cell line to form a producer cell line for generating the modified envelope protein, such as for generating the retroviral vector particles of the present invention. Alternatively, a naked polynucleotide sequence encoding the modified envelope protein can be transfected into a "pre-packaging" cell line including nucleic acid sequences encoding the gag and pol proteins, thereby forming a packaging cell line, or is transfected into a packaging cell line including nucleic acid sequences encoding the gag, pol, and wild-type (i.e., unmodified) env proteins, thereby forming a packaging cell line including nucleic acid sequences encoding wild-type env protein and the modified envelope protein. Such packaging cells then may be transfected with a retroviral plasmid vector, which may include a nucleic acid sequence encoding a heterologous protein or peptide, thereby forming a producer cell line for generating retroviral vector particles including the modified envelope protein. Such a polynucleotide thus may be contained in the above-mentioned retroviral vector particle, or in a producer cell for generating the above-mentioned retroviral vector particle.

The polynucleotide according to the present invention can be comprised in a suitable vector known to one skilled in the art. Thus, one aspect of the present invention relates to a vector comprising a polynucleotide encoding the chimeric viral envelope polypeptide according to the present invention. A vector in the present context preferably comprises all vectors capable of directing expression of any given envelope by directing expression of vector DNA into RNA, poly-adenylation of said RNA, splicing of said RNA, if necessary, export out of the nucleus of said RNA, and finally translation

of said RNA outside of the nucleus. The vector can for example be a plasmid, or a recombinant virus particle.

Thus, the vector can also in one embodiment comprise the chimeric viral envelope polypeptide encoded by said polynucleotide, preferably as part of the viral envelope. The virus particle is preferably replication competent. For example, the virus can comprise a heterologous translation cassette, such as a heterologous translation cassette comprising or consisting of an IRES-gene element.

A replication competent retrovirus can further comprise all genes necessary for replication of a retrovirus, and for RNA being exported out of the cell and packaged in proteins expressed by said proteins. Said RNA further comprises all RNA and DNA elements necessary for said RNA to be reverse transcribed into double stranded DNA and integrated into the host genome, as exemplified in figure 3 panel A. In panel B this replication competent retrovirus further comprises a heterologous peptide inserted into the envelope gene for redirection of host cell tropism. Only the ScFv is depicted in figure 3, but other insertions could be similarly useful.

The exemplified replication competent retroviral vector further comprises a replication competent virus where a heterologous gene is being expressed from a position in the U3 region of the virus, panel C and D, or from a position in the 3 prime untranslated region downstream of the envelope and upstream of the downstream LTR, panel E and F. Said replication competent vectors can further be redirected in host cell tropism by insertion of an ScFv or any heterologous peptide in the envelopes, panel D and F. Only the ScFv is depicted in figure 3. Based upon this example, the text of the present application and common knowledge of a person of ordinary skill in the art will be able to make other useful embodiments.

The virus particle is preferably a retroviral vector being capable of transcribed into RNA and capable of being packaged into a retroviral particle, reverse transcribed into double stranded DNA and inserted into the host genome by the retroviral enzymatic machinery. For translation of said envelope an internal ribosome entry site (IRES) has been inserted upstream of the envelope in the exemplified retroviral expression vector, panel G and H. The host cell tropism of said retrovirus can further be redirected by inserting an ScVf or any heterologous peptide in the envelope, panel H. Only the ScFv

is depicted in figure 3, but other inserts could be similar useful.

A particular embodiment of the present invention relates to any of the replication competent vectors described in the present application and further comprising a heterologous translation cassette.

Thus, a presently preferred particular embodiment relates to a replication competent vector comprising a heterologous translation cassette, wherein said heterologous translation cassette comprises an IRES-gene element.

Another embodiment of the present invention relates to a vector according to the present invention further comprising at least one heterologous gene to be expressed.

In another embodiment, the present application relates to a vector according to the present invention further comprising at least one heterologous gene to be expressed, wherein said expression is directed by a IRES-element.

Embodiments relating to gene therapy

The recombinant virus particle can further comprise an agent for delivery to the target cell, optionally selected from the group consisting of a therapeutic agent or a gene or gene product, which agent is optionally operatively associated with a retroviral packaging sequence. Thus, the virus particle can in one embodiment comprise at least one heterologous gene to be expressed in the host after a gene therapy procedure. Said heterologous gene to be expressed can for example comprise the polynucleotide encoding the chimeric envelope polypeptide according to the present invention. The agent is preferably a therapeutic agent, such as a polynucleotide sequence, or a polynucleotide sequence encoding a therapeutic agent.

The polynucleotide encoding the therapeutic agent is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; the histone promoter; the pollll promoter, the .beta.- actin promoter; inducible promoters, such as the MMTV promoter, the metallothionein promoter; heat shock promoters; adenovirus promoters; the albumin promoter; the

ApoAI promoter; B19 parvovirus promoters; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex Virus thymidine kinase promoter; retroviral LTRS; human growth hormone promoters, and the MxIFN inducible promoter. The promoter also may be the native promoter which controls the polynucleotide encoding the therapeutic agent. It is to be understood, however, that the scope of the present invention is not to be limited to specific foreign genes or promoters.

The polynucleotides encoding the modified envelope polypeptide and the therapeutic agent may be placed into an appropriate vector by genetic engineering techniques known to those skilled in the art. When the modified vector is a retroviral vector particle, the polynucleotides encoding the modified envelope polypeptide and the therapeutic agent can e.g. be placed into an appropriate retroviral plasmid vector.

In one embodiment, the retroviral plasmid vector may be derived from Moloney Murine Leukemia Virus and is of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al., J. Virol., Vol. 61 , pgs. 1639-1649 (1987) and Miller, et al., Biotechniques, Vol. 7, pgs 980-990 (1989). Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon. The term "mutated" as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.

In another embodiment, the retroviral plasmid vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs. Preferred cloning sites are selected from the group consisting of Notl, SnaBI, Sail, and Xhol. In a preferred embodiment, the retroviral plasmid vector includes each of these cloning sites. Such vectors are further described in U.S. patent application Ser. No. 08/340,805, filed Nov. 17, 1994, and in PCT Application No. W091 /10728, published JuI. 25, 1991 , and incorporated herein by reference in their entireties.

When a retroviral plasmid vector including such cloning sites is employed, there may also be provided a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of

Notl, SnaBI, Sail, and Xhol located on the retroviral plasmid vector. The shuttle cloning vector also includes at least one desired polynucleotide encoding a therapeutic agent which is capable of being transferred from the shuttle cloning vector to the retroviral plasmid vector.

The shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.

The shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.

The retroviral plasmid vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and .beta.-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

Cell lines and animal models

In another embodiment, there is provided a packaging cell which includes a nucleic acid sequence encoding a modified chimeric envelope in accordance with the invention, and which may further include nucleic acid sequences encoding the gag and pol proteins. A producer cell for generating viral particles which includes a modified envelope in accordance with the invention can for example be produced by introducing into such packaging cell either a retroviral vector particle or a retroviral plasmid vector, in each case including a polynucleotide encoding a therapeutic agent. The producer

cell line thus generates infectious retroviral particles including the modified chimeric envelope and the polynucleotide encoding the therapeutic agent.

Thus, in another aspect of the present invention is provided a cell transfected with the vector according to the present invention. In one preferred embodiment, said cell is comprised in a stable cell line comprising such cells. The present invention further relates to "packaging cell lines" for producing the vectors of the present invention.

MLV based packaging cells are widespread tools for research. Packaging cells based on ecotropic viruses have the advantage of being harmless to humans and are used in bio- safety level 1 laboratories.

One embodiment of the present invention relates to a packaging cell construct comprising the vector comprising a nucleic acid coding for a polypeptide envelope as described in the present application, and optionally a non-viral or viral promoter and poly-adenylation signals.

Another embodiment of the present invention relates to use of any of the vectors according to the present invention for the generation of a packaging cell.

The packaging cell line can for example be engineered to produce the viral Gag, Pol and Env proteins from constructs that lack the packaging signal (to prevent them from being taken up by budding virions). Thus, when a vector is inserted into a packaging cell line, it will be packaged into budding virions and can be transferred into target cells. Representative examples of packaging cell lines include, but are not limited to, the

PE501 and PA317 cell lines disclosed in Miller, et al., Biotechniques, Vol. 7 pgs. 980- 990 (1989).

In one embodiment, the packaging cell line is a "pre-packaging" cell line which includes polynucleotides encoding the gag and pol retroviral proteins, but not the envelope, or env, protein. Examples of such "pre-packaging" cell lines include, but are not limited to, GP8 cells, GPL cells, and GPNZ cells as described in Morgan, et al., J. Virol., Vol. 67, No. 8, pgs. 4712-4721 (August 1993). Such cell lines, upon transduction with the retroviral plasmid vector, generates infectious retroviral particles including the modified, or chimeric, envelope and a polynucleotide encoding the therapeutic agent.

In another embodiment, a retroviral plasmid vector which includes a polynucleotide encoding a modified polynucleotide encoding a modified envelope polypeptide in accordance with the invention and a polynucleotide encoding a therapeutic agent is used to transduce a packaging cell line including nucleic acid sequences encoding the gag, pol, and wild-type (i.e., unmodified) env retroviral proteins. Examples of such packaging cell lines include, but are not limited to, the PE501 , PA317 (ATCC No. CRL 9078), .psi.-2, .psi.-AM, PA12, T19-14X, VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, and use of liposomes, such as hereinabove described, and CaPO ₄ precipitation. Such producer cells generate infectious retroviral vector particles which include the modified envelope, the wild-type retroviral envelope, a polynucleotide encoding the modified, or chimeric, envelope, and a polynucleotide encoding a therapeutic agent.

In another preferred embodiment, said cell is comprised in an animal model using methods known to one skilled in the art. Said model is preferably a mouse,

Antibodies

The present invention further relates to an antibody capable of specifically binding one of the molecules provided in the present invention, such as a chimeric envelope polypeptide according to the present invention, and/or a retroviral particle expressing said chimeric envelope polypeptide.

The term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and

keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.

Therapeutic methods using any of the aspects of the present invention

Any of the polynucleotide, vectors and/or envelope polypeptides provided herein can be used in therapeutic methods and/or prophylaxis of disease, such as viral disease. These polynucleotides, vectors and/or envelope polypeptides are herein below described as "constructs", by which it is meant that any of the polynucleotides, vectors and/or envelope polypeptides can advantageously be used. Preferably, said construct is a retroviral particle as described herein.

Thus, the present invention relates in one embodiment to a therapeutic method for treatment of an individual in need thereof, said method comprising administering a construct according to the present invention to an individual in need thereof.

The present invention further relates to a method for prevention or reduction of a viral infection in an individual in need thereof, comprising the steps of:

(i) providing constructs (preferably virus particles) according to the present invention as disclosed herein;

(ii) causing said constructs to contact a target population of said individual's cells, wherein cells within said target population comprise a receptor capable of being specifically bound by said construct, (Ni) allowing the construct to bind said receptor.

It is preferred that said binding of a construct to said receptor blocks binding of other viral molecules to the cell bound by said construct.

In one embodiment, the bound construct (preferably a recombinant virus particle) is taken up into the cell which it has specifically bound.

The binding can also lead to prevention and/or reduction of syncitial formation between another, pathogenic virus and the cell bound by the construct of the present invention.

Another effect of the binding can be reduction in the expression level of the bound viral receptor (such as a viral co-receptor, such as the CXCR4 co-receptor) on the surface of the cell bound by said construct.

In one embodiment of the above method, an agent is delivered to said cell by said construct (preferably a virus particle). Said agent can for example be an anti-viral drug or a polynucleotide.

Where said agent is a polynucleotide, said method is advantageous to use for gene therapy. The polynucleotide introduced into the cell by said gene therapy method can for example be the polynucleotide according to the present invention encoding the chimeric envelope polypeptides described herein, however said polynucleotide can in equally be another anti-viral polynucleotide, such as encoding a polypeptide with antiviral activity. The term "introducing" as used herein encompasses a variety of methods of transferring polynucleotides into a cell, such methods including transformation, transduction, transfection, and transinfection.

Thus, retroviral vector particles of the present invention can be used for introducing polynucleotides into cells for gene therapy purposes. In one approach, cells are obtained from a patient, and retroviral vector particles are used to introduce a desired polynucleotide into the cells, and such modified cells are returned to the patient with the engineered cells for a therapeutic purpose. In another approach, retroviral vector particles may be administered to the patient in viva, whereby the retroviral vector particles transduce cells of the patient in vivo.

Methods for in vivo and ex vivo gene therapy are well known in the art, such as for example described in e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764; Friedmann, 1989, Science, 244:1275-1281; Mulligan, 1993, Science, 260:926-932, R. Crystal, 1995, Science 270:404-410, each of which are incorporated herein by reference in their entirety). An increasing number of these methods are currently being applied in human clinical trials (Morgan, R., 1993, BioPharm, 6(1):32-35; see also The Development of Human Gene Therapy, Theodore Friedmann, Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. ISBN 0-87969-528-5, which is incorporated herein by reference in its entirety).

It may be preferable to remove the majority of a target cell population prior to therapy, for example surgically.

In fact, any of the therapies described herein can be in vivo or ex vivo, that is to say that said contacting occurs outside the individual, and then the target population is returned to the individual's body.

The present invention further provides a therapeutic method for specifically tethering a chimeric retroviral envelope polypeptide to a specific cell type and preventing or reducing retroviral infection, comprising the steps of:

(i) providing an enveloped virus expressing a chimeric retroviral envelope polypeptide as disclosed herein,

(ii) allowing said chimeric envelope polypeptide to specifically contact a cell of said specific cell type, (iii) allowing the outer membrane of the virus to undergo a hemi-fusion process with the outer membrane of said cell.

Said specific cell type can for example be T cells, a cell expressing CXCR5 or CXCR4, or macrophage cells.

In another embodiment of the present invention is provided a method for treatment or prevention of a viral disease, comprising administering a construct as disclosed herein to an individual in need thereof. Said construct is preferably the viral envelope polypeptide as disclosed herein, or the recombinant virus particle as disclosed herein. Said viral disease is preferably selected from HIV (for example, HIV-1 or HIV-1)

Target cell populations for any of the therapeutic methods of the present invention In one preferred embodiment of the therapeutic method of the present invention, the target cell population comprises or consists of T cells. In another preferred embodiment of the therapeutic method of the present invention, said target population comprises or consists of cells expressing CXCR5 or CXCR4. In another preferred embodiment of the therapeutic method of the present invention, said target population comprises or consists of macrophage cells.

Further example of cells which may be targeted for binding, infection or transduction with the chimeric envelope polypeptides or vector particles of the present invention include, but are not limited to, T cell, endothelial cells, tumor cells, chondrocytes, fibroblasts and fibroelastic cells of connective tissues; osteocytes and osteoblasts in bone; endothelial and smooth muscle cells of the vasculature; epithelial and subepithelial cells of the gastrointestinal and respiratory tracts; vascular cells, connective tissue cells, and hepatocytes of a fibrotic liver, the reparative mononuclear and granulocytic infiltrates of inflamed tissues, liver cells, T-cells, lymphocytes, endothelial cells, T4 helper cells, or macrophages.

In another embodiment, the receptor binding region is a hepatitis B virus surface protein binding region, and the target cell is e.g. a liver cell.

Pharmaceutical formulations, administration and dosages In another aspect of the present invention is further disclosed a pharmaceutical formulation comprising any of the constructs described herein, such as a chimeric envelope polypeptide or viral particle as disclosed herein.

The constructs of the present invention may be directly administered to a desired target cell ex vivo, and such cells may then be administered to a patient as part of a gene therapy procedure.

Although the chimeric polypeptides and/or vector particles may be administered directly to a target cell, they may also be engineered such that they are resistant to inactivation by human serum, and thus may be administered to a patient by (e.g. intravenous) injection, and travel directly to a desired target cell or tissue without being inactivated by human serum.

The vector particles, may be concentrated from dilute vector stocks in vitro by contacting a dilute vector stock with an extracellular matrix component to which the modified viral surface protein will bind. Such binding enables one to obtain a concentrated stock of the vector particles.

In addition, the modified viral surface proteins of the present invention may be employed to form proteoliposomes; i.e., the modified viral surface protein forms a

portion of the liposome wall. Such proteoliposomes may be employed for gene transfer or for drug delivery to cells located at a site of an exposed extracellular matrix component.

Any of the constructs disclosed herein may be administered to a host in an amount effective to produce a therapeutic effect in the host. The host may be a mammalian host, which may be a human or non-human primate host. The exact dosage which may be administered is dependent upon a variety of factors, including the age, sex, and weight of the patient, the cells which are to be transduced, the therapeutic agent which is to be administered, and the severity of the disorder to be treated.

The constructs, such as viral particles, may be administered systemically, such as, for example, by intravenous, intracolonic, intratracheal, intraperitoneal, intranasal, intravascular, intrathecal, intraarterial, intracranial, intramarrow, intrapleural, intradermal, subcutaneous, intramuscular, intraocular, intraosseous and/or intrasynovial administration. The constructs also may be administered topically.

Rational drug design

Another aspect of the present invention relates to use of the polypeptides disclosed herein for rational drug design.

To facilitate understanding of the invention, a number of terms are defined below.

The term "polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides at least 2 bases in length. By "isolated nucleic acid sequence" is meant a polynucleotide that is not immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA or RNA which is incorporated into a viral vector. The nucleotides of the invention can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double stranded forms of DNA.

The term polynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others,

single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.

In addition, polynucleotide as used herein can also refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.

It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.

The term "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

A "detectable label" refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.

A "fragment" is a unique portion of the polynucleotide encoding the chimeric retroviral

envelope polypeptide of the present invention which is identical in sequence to but shorter in length than the parent sequence. Similarly the term 'fragment' refers to the the chimeric retroviral envelope polypeptide of the present invention A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide or amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.

The term "Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.

The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide

sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.

Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

The term "insertion" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.

The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double- stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.

The term "operably linked" refers to the situation in which a first nucleic acid sequence, amino acid sequence or ligand is placed in a functional relationship with a second nucleic acid sequence, amino acid sequence or ligand. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences or protein or ligands may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

Homologies Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described and present a detailed consideration of sequence alignment methods and homology calculations, such as VECTOR NTI. The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences will be.

The NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NBCI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at http://www.ncbi.nlm.nih.gov/BLAST/. A description of how to determine sequence identity using this program is available at http://www.ncbi. nlm.nih.gov/BLAST/blast_help.html.

Homologs of the disclosed polypeptides are typically characterised by possession of at least 94% sequence identity counted over the full length alignment with the disclosed amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Alternatively, one may manually align the sequences and count the number of identical amino acids. This number divided by the total number of amino acids in your sequence multiplied by 100 results in the percent identity.

Chimeric retroviral envelope polypeptide

In a first aspect of the present invention is provided a chimeric viral envelope polypeptide comprising (i) an envelope polypeptide, or fragment thereof, (ii) a polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region, and optionally a linker sequence, wherein the receptor binding domain of said envelope polypeptide has a sequence that is at least 36% identical to the amino acid sequence shown in SEQ ID NO: 60, or is a fragment of a sequence that is at least 36% identical to the amino acid sequence shown in SEQ ID NO: 60.

However, in other embodiments of the present invention the said receptor binding domain has a sequence that is for example at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 67%, such as at least 70%, for example at least 72%, such as at least 75%, for example at least 77%, such as at least 80%, for example at least 81 %, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% identical to the amino acid sequence shown in SEQ ID NO: 60. In another embodiment of the present invention said receptor binding domain of said envelope polypeptide is a fragment of a sequence that is for example at least at least 40%, such as at least 45%, for example at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 67%, such as at least 70%, for example at least 72%, such as at least 75%, for example at least 77%, such as at least 80%, for example at least 81 %, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% identical to the amino acid sequence shown in SEQ ID NO: 60.

The envelope polypeptide of the chimeric retroviral envelope according to claim 66 may derive from gamma retroviruses. In one embodiment the gammaretroviruses are murine leukaemia viruses, such as SL3-2 (SEQ ID NO: 60), for example FeLV-B, such as MCF 247, for example MCF CI-3, such as ERV-1 , for example Friend MCF, such as

.Friend SFV, for example Invitro MCF, such as MCF 1223, for example MLV DBA/2, such as Mo-MCF, for example Ns-6(186) MCF, such as Rauscher sfv, for example Endogenous from 129 GIX+ mice, such as Ampho-MCF, for example MCF (Ter- Grigorov), such as MCF (Broscius), for example Friend MCF #2, such as R-XC-, for example Xeno R-MCF-1 , such as DG-75 Xeno, for example Xeno NZB-9-1 , such as Xeno CWM-S-5-X, for example Xeno Bxv-1 related, such as 4070A, for example 10A1 , such as Akv, for example SL3-3, such as Friend. R is appreciated that each of these viruses may be used individually in the present invention.

Amino acid sequences and polynucleotides that represent particular embodiments of the present invention are listed in the sequence listing herein.

A chimeric viral envelope polypeptide comprising (i)an envelope polypeptide, or fragment thereof, (ii) a polypeptide sequence of a receptor binding region, ligand or a polypeptide sequence of a ligand binding region, and optionally a linker sequence according to the present invention has an altered host range mediated by a non-viral receptor of the target cell. The polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region of the chimeric envelope polypeptide enables the viral particle to enter a cell expressing the protein on its cell surface, for example a receptor or transporter, ion channel, symporter, antisporter which is recognised by the receptor binding region, ligand or polypeptide sequence of a ligand binding region.

Envelope polypeptide Retroviruses can be thought of as a protein-package comprising RNA wrapped in a lipid membrane that contains glycoproteins. The lipid bi-layer is derived from the cell membrane after budding and is thought to be associated with a viral gene product, a peripheral membrane protein called Matrix (MA). Traversing through the lipid bi-layer is another viral gene product, the envelope protein, which upon cleavage in the endoplasmatic reticulum by cellular proteases consists of two subunits: the n-terminal transmembrane (TM) subunit and the C-terminal surface subunit (SU). The function of the envelope protein is binding of the virus to its target cell and mediating fusion of the viral and cellular membranes. The SU is responsible for receptor recognition and binding. The TM is engaged in fusion of the viral and cellular membranes.

A number of regions of the envelope polypeptide have been identified in gammaretroviruses. In the present invention the receptor binding domain (RBD) of the envelope polypeptide is defined as the region delineated by the first amino acid of SEQ ID NO: 60 and the amino acid preceeding the proline rich region (PPR) corresponding to the amino acid number 214 of SEQ ID NO: 60, see Fig. 11). Within the RBD are found two variable regions A and B, see Fig. 11. It is appreciated that the position of RBD varies in the various retroviruses. The exact position of the regions varies from species of the gammaretroviruses as can be seen from Fig.11τ

Embodiments for insertion of the polypeptide sequence of a receptor binding domain, ligand, or polypeptide sequence of a ligand binding region. The polypeptide sequence of a receptor binding domain, ligand, or polypeptide sequence of a ligand binding region is inserted into an insert site within the envelope polypeptide. In one embodiment the polypeptide sequence of a receptor binding domain, ligand, or polypeptide sequence of a ligand binding region is inserted into the receptor binding domain of said envelope polypeptide or fragment thereof. As described above the recptor binding domain in the present invention is defined as the first amino acid of SEQ ID NO: 60 and the amino acid preceeding the proline rich region (PPR) corresponding to the amino acid number 214 of SEQ ID NO: 60. Thus, in one embodiment the insert site is in the region of SEQ ID NO: 60 delineated by amino acid number 1 and amino acid number 214. In another embodiment the insert site is in the region of SEQ ID NO: 60 delineated by amino acid number 1 and amino acid number 101. In another embodiment the insert site is in the region of SEQ ID NO: 60 delineated by amino acid number 102 and amino acid number 117, corresponding to the variable region A (VRA). In yet another embodiment the insert site is in the region of SEQ ID NO: 60 delineated by amino acid number 118 and amino acid number 156. In yet another embodiment the insert site is in the region of SEQ ID NO: 60 delineated by amino acid number 157 and amino acid number 173, corresponding to the variable region B (VRB). In a further preferred embodiment the insert site is in the region of SEQ ID NO: 60 delineated by amino acid number 174 and amino acid number 214. In one embodiment the insert site is in the region of SEQ ID NO: 60 delineated by amino acid number 155 and amino acid number 165. In a preferred embodiment of the present invention the insert site is at position 155 of SEQ ID NO: 60. Another preferred embodiment of the present invention the insert site is at position 155 of SEQ ID NO:

60. Yet another preferred embodiment of the present invention the insert site is at position 165 of SEQ ID NO: 60.

Tropism of murine leukaemia virus (MLV) The MLVs are a group of gammaretroviruses that has been divided into families based on their host range and interference properties. The families are the ecotropic, amphotropic, xenotropic and polytropic subfamilies. Ecotropic viruses are defined by their usage of the mCAT-1 receptor (Wang et al. 1991). Ecotropic viruses are able to infect only murine cells. Examples of ecotopic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human and other species through the Pit-2 receptor (Kavanaugh et al. 1994). One example of an amphotopic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xpr1) receptor. However, the xenotropic and polytropic viruses differ in their species tropism. Xenotropic viruses such as NZB-9-1 infect human and other species but not murine species, whereas polytropic viruses infect murine, human and other species as exemplified by the mink cell focus-forming viruses (MCF) for example the MCF 247 virus. However, the polytropic SL3-2 virus has a host range as the mouse ecotropic viruses in that it infects and replicates in mouse cells, but are impaired in its ability to infect and replicate in mink cells or human cells. The SL3-2 envelope protein virus utilizes the polytropic (Xpr1) receptor.

One embodiment of the present invention relates to a chimeric retroviral envelope polypeptide comprising an envelope polypeptide, or fragment thereof, and a polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region, wherein the envelope polypeptide is from a gamma retrovirus, for example the murine leukaemia viruses as listed herein but also the Feline-B virus is one example of a virus, in one embodiment according to the present invention the envelope polypeptide is from MLV. Another embodiment is a chimeric retroviral envelope polypeptide, wherein the envelope polypeptide is from for example ecotropic viruses, such as xenotropic viruses, for example amphotropic viruses, or such as polytropic viruses. In one particular embodiment of the present invention the envelope polypeptide is from the SL3-2 virus.

In one embodiment of the present invention the envelope polypeptide is derived from all viruses except ecotropic viruses

One aspect of the invention relates to a chimeric viral envelope polypeptide comprising (i)an envelope polypeptide, or fragment thereof, (ii) a polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region, and optionally a linker sequence, wherein the envelope polypeptide, or fragment thereof is defined according to the tropism of virus from where the originates. Thus, one embodiment of the present invention pertains to a chimeric viral envelope polypeptide comprising (i) an envelope polypeptide, or fragment thereof, (ii) a polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region, and optionally a linker sequence, wherein the envelope polypeptide, or fragment or homologue thereof is selected from the group consisting of envelope polypeptides from polytropic viruses. Examples of polytropic viruses are SL3-2, MCF- 247, MCF CI-3, ERV-1 , Friend MCF, Friend SFV, Invitro MCF, MCF1223, MLV DBA/2, Mo-MCF, Ns-6(186) MCF, Rauscher sfv, endogenous from 129 GIX+ mice, ampho- MCF, MCF(Ter-Grigorov), MCF (Broscius), Friend MCF#2 or R-XC. In one embodiment of the present invention the envelope polypeptide, or fragment or homologue thereof is selected from the group consisting of envelope polypeptides from SL3-2, MCF-247, MCF CI-3, ERV-1, Friend MCF, Friend SFV, Invitro MCF, MCF1223. In another embodiment of the present invention the envelope polypeptide, or fragment or homologue thereof is selected from the group consisting of envelope polypeptides from SL3-2, MLV DBA/2, Mo-MCF, Ns-6(186) MCF ₁ Rauscher sfv, endogenous from 129 GIX+ mice, ampho-MCF, MCF(Ter-Grigorov), MCF (Broscius), Friend MCF#2 or R-XC. In yet another embodiment of the present invention the envelope polypeptide, or fragment or homologue thereof is selected from the group consisting of envelope polypeptides from SL3-2, Friend MCF, Friend SFV, Invitro MCF, MCF1223, MLV DBA/2, Mo-MCF, Ns-6(186) MCF, Rauscher sfv, endogenous from 129 GIX+ mice, ampho-MCF, MCF(Ter-Grigorov). In a further embodiment of the present invention the envelope polypeptide, or fragment or homologue thereof is selected from the group consisting of envelope polypeptides from SL3-2, MCF-247, MCF CI-3, ERV-1. It is understood that the envelope polypeptide, or fragment or homologue thereof are individual embodiments of the present invention. In a particular embodiment of the present invention the envelope polypeptide, or fragment or homologue thereof is SL3- 2.

It has also been found that changing specific amino acids within the VR3 region of this MLV SL3-2 envelope polypeptide, or a polytropic homologue thereof, enables alteration

the host tropism of said envelope polypeptide. The present inventors have pin-pointed exactly which amino acid that is essential for this host tropism shift.

In the case that the first polypeptide is homologous to SEQ ID NO: 60, one embodiment is that said first polypeptide includes at least one substitution in the VR3 region, or a region homologous thereto. In the present context, the term "VR3 region" comprises all of the amino acids found between the residue found at two positions after the conserved tryptophan 197 and the residue before the conserved aspartic acid 214 (according to the sequence shown in SEQ ID NO: 60) including these two positions. In one embodiment of the present invention, said first polypeptide includes at least one substitution in the region homologous to the VR3 region, such as 1 , 2, 3, 4, 5 or 6 substitutions in the VR3 region. Examples of substitutions which are likely to provide the same effect are alanine, asparagine, aspartic acid, cysteine, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, proline, glutamin, serine, threonine, valine, tryptophan or tyrosine.

In one preferred embodiment the substitution changes the arginine to glycine. In another preferred embodiment the substitution results in a methionine. For example, said substitution can be at position 212 in SEQ ID NO: 60, or a region homologous thereto. It is preferred that said at least one substitution alters the host tropism of a virus or an infectious particle comprising said polypeptide, in a manner described in more detail in WO 03/097674 (Pipeline Biotech A/S).

Polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding regions

As used herein, the term "ligand" is used broadly herein to refer to a molecule that can bind to a protein, for example a receptor, a transporter, ion channel, or symporter, expressed on the surface of a target cell or, conversely, to a receptor that can bind a molecule expressed on the surface of a target cell. As used herein in the following, the term 'polypeptide sequence of a receptor binding region' is used broadly to refer to a polypeptide or fragment thereof that can bind to a receptor, transporter, ion channel, or symporter expressed on the surface of a target cell or, conversely, to a receptor, transporter, ion channel, or symporter that can bind a polypeptide sequence of a receptor binding region expressed on the surface of a target cell.

The term ligand and 'polypeptide sequence of a receptor binding region thus can be any molecule binding to a protein, for example a receptor, transporter, ion channels, or symporter expressed on the surface of a target cell. One embodiment of the present invention relates to the chimeric viral envelope polypeptide according to claim 66, wherein said receptor binding region, ligand or polypeptide sequence of a ligand binding region is selected from the group consisting of receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof for receptors or co-receptors. The said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue is for receptors, however, in another embodiment the said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue bind to co- receptors. For clarity the term ligand for is identical to the term ligand binds to a receptor or coreceptor.

According to the present invention said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof of the chimeric viral envelope polypeptide bind to any protein expressed on the surface of a target cell. In one embodiment the chimeric viral envelope binds to a G-protein-coupled receptor. However, the chimeric viral envelope may also bind to transporter molecules, for example monoamine transporters.

In one embodiment of the present invention the said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof is selected from the group consisting of apelin, substance P, neurokinin A, neurokinin B, neurotensin receptor 1 and neurotensin receptor 2, or a fragment or homologue thereof. In another embodiment of the present invention the said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof is selected from the group consisting of,substance P, neurokinin A, neurokinin B, neurotensin receptor 1 and neurotensin receptor 2 or a fragment or homologue thereof. Another embodiment of the present invention comprises the said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof selected from the group consisting of apelin, neurokinin A and neurokinin B or a fragment or homologue thereof. In yet another embodiment of the present invention the said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof is selected from the group consisting of neurokinin A and neurokinin B or a fragment or homologue thereof.

In yet another embodiment of the present invention the said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof is selected from the group consisting of neurotensin receptor 1 and neurotensin receptor 2 or a fragment or homologue thereof.

A further embodiment of the present invention the said receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof is the each of these ligands individually for example apelin, such as substance P, for example neurokinin A, or such as neurokinin B, for example neurotensin receptor 1 , such as neurotensin receptor 2 or a fragment or homologue thereof.

Further embodiments are found as list of G-protein-coupled receptors bound by the receptor binding region or said ligand or a fragment or homologue thereof elsewhere herein.

In one embodiment of the present invention the polypeptide sequence of a receptor binding region which binds to a coreceptor is a viral envelope polypeptide, or a fragment or homologue thereof.

Said polypeptide sequence of a receptor binding region may in one embodiment be a co-receptor-binding domain, or a fragment or homologue thereof.

In one preferred embodiment of the present invention, said receptor binding region is a receptor binding region of a human virus, such as e.g. Vesicular stomatitis virus (VSV) (Protein G), cytomegalovirus envelope (CMV), HIV, or influenza virus hemagglutinin (HA).

For example, said polypeptide sequence of said receptor binding region can be a fragment or homologue binding to the coreceptor CCR-5 or CXCR-4. However, said polypeptide sequence of said receptor binding region can be a fragment or homologue binding to each of the co-receptors as individual co-receptors CCR-5, or CXCR-4.

Thus, in one embodiment of the present invention, the second, different viral envelope polypeptide is the V3-loop domain of HIV or a fragment or homologue thereof. Said HIV may for example be a strain of HIV-1 or a strain of HIV-2.

In another embodiment of the present invention, the receptor binding region is a hepatitis B virus surface protein binding region, preferably binding to a liver cell.

In another embodiment of the present invention, the receptor binding region is the receptor binding region of gp46 of HTLV-I virus, preferably binding to a T cell.

Another embodiment of the present invention is the use of non-peptide ligands for G- protein-coupled receptors (GPCR) or other receptors. One example is the use of nitrilotriacetic acid (NTA) as an adaptor molecule to associate a non-peptide ligand with the SL3-2 envelope. NTA is a chelating agent and binds strongly to a Ni ²⁺ ion leaving two coordination sites for interaction with the nitrogen atoms on two neighbouring His residues in proteins.

Non-peptide ligands of a GPCR can be fused to the tail of one or more NTA molecules, such as 1 NTA molecule and/or 2 NTA molecules and/or 3 NTA molecules and/or 4 NTA molecules and/or 5 NTA molecules and/or 6 NTA molecules and/or, while engineering several histidine residues in the binding site of the envelope polypeptide, or fragment thereof. The ligand-NTA molecule will then be able to bind to the envelope upon addition of Ni ²⁺ ions. The whole complex is able to target the virus towards the desired protein expressed on the surface of a target cell, for example a GPCR or a transporter.

The present invention is not limited to NTA or its derivatives such as commercially available [(1S)-λ/-(5-amino-1-carboxypentyl)iminodiacetic acid; NTA-NH ₂ ]), but any adaptor molecule may be used. Other adaptor molecules are for example but not restricted to DNA, or small peptides.

In one embodiment of the present invention, said receptor binding region is a combination of multiple receptor binding peptides. In another embodiment, said receptor binding region comprises 1 receptor binding peptide, and/or 2 receptor binding peptides, and/or 3 receptor binding peptides, and/or 4 receptor binding peptides, and/or 5 receptor binding peptides, and/or 6 receptor binding peptides. The multiple receptor binding peptides may either be identical peptides or a combination of different peptides.

One preferred embodiment of the present invention is the SL3-2 envelope polypeptide in which ligands are inserted for the Tachykinin NK1 receptor for which many non-

peptide ligands are known. Several ligands described in (Quartara and Maggi, 1997) contain amid-bonds. The NH ₂ group of these bonds is replaced with that Of NTA-NH ₂

In yet another embodiment of the present invention relates to an indirect targeting of a protein expressed on the surface of a cell, by inserting a tetracystein tag into the viral envelope. The tetracysteine tag is inserted into the SL3-2 envelope protein at amino acid position 165 as described in example 6. The tetracystein tag comprises the motif CCXXCC, where C is cystein and X is any amino acid. In one preferred embodiment the motif comprises CCPGCC, where P is proline and G is glycin. The tetracystein tag may also comprise amino acids linking the CCXXCC motif to the viral envelope sequence in order to achieve optimal effect of subsequent binding to a ligand for the tag.

In one embodiment the ligand for tetracystein tag may be a biarsenical reagent. In one embodiment upon binding to the tetracystein tag the biarsenical reagent converts into a fluorescent state. However, in another embodiment the biarsenical reagent is not fluorescent upon binding to the tetracysteine tag.. Nonlimiting examples of biarsenical reagents are ReAsH reagent ™(lnvitrogen), ReAsH-EDT ₂ ™ (Invitrogen), or FIAsH- EDT ₂ ™(lnvitrogen).The biarsenical reagent may be linked to a ligand for a desired protein expressed on the surface of a cell.

The ligand which may be fitted with a biarsenical reagent may thus be any ligand. In one embodiment the ligand is selected from the group consisting of ligands for monoamine transporters. In one embodiment the ligand is RTI-55 (3 beta-(4- iodophenyl)tropan-2 beta-carboxylic acid methyl ester). However, the present invention is not limited to any ligand.

The protein expressed on the surface of a target cell may be any protein for example the receptors as listed elsewhere herein. However, the listed examples are not meant to be limiting present invention. In one embodiment the receptor is a monoamine transporter. The receptor may be selected from the group consisting of SERT (Serotonin transporter), DAT (Dopamine transporter) and NET (norepinephrine transporter). In one embodiment the receptor is selected from the group consisting of hSERT (human Serotonin transporter), hDAT (human Dopamine transporter), and hNET (human norepinephrine transporter). In another embodiment the receptor is

hSERT (human Serotonin transproter), hDAT (human Dopamine transporter), or hNET (human norepinephrine transporter).

The protein expressed on the surface of a target cell may be an ion channel protein, or a symporter.

Example 6 describes embodiments involving non-peptide ligands.

Proteins on the surface of a target cell, to which ligands of the present invention bind The receptors to which the ligands of the present invention bind are any surface protein of any type in which the insertion of peptides or non-peptide molecules into the viral envelope protein may act directly as a ligand for a specific receptor. However, the inserted peptide, or non-peptide molecules may bind to a label present on a ligand for a specific receptor, thus targeting the specific receptor in an indirect fashion.

In one embodiment the present invention relates to g-protein-coupled receptors. (GPCRs) are a protein family of transmembrane receptors. The GPCRs are the largest protein family known, involved in all types of stimulus response pathways. GPCRs are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which together form a bundle of antiparallel alpha (.alpha.) helices. GPCRs range in size from under 400 to over 1000 amino acids (Strosberg, A. D. (1991) Eur. J. Biochem. 196:1-10; Coughlin, S. R. (1994) Curr. Opin. Cell Biol. 6:191-197). The amino-terminus of a GPCR is extracellular, is of variable length, and is often glycosylated. The carboxy-terminus is cytoplasmic and generally phosphorylated. Extracellular loops alternate with intracellular loops and link the transmembrane domains. Cysteine disulfide bridges linking the second and third extracellular loops may interact with agonists and antagonists. The most conserved domains of GPCRs are the transmembrane domains and the first two cytoplasmic loops. The transmembrane domains account, in part, for structural and functional features of the receptor. In most cases, the bundle of a helices forms a ligand-binding pocket. The extracellular N-tβrminal segment, or one or more of the three extracellular loops, may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor. In turn, the large, third intracellular loop of the activated receptor interacts with a heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular

signaling activities, including the activation of second messengers such as cyclic AMP (cAMP), phospholipase C, and inositol triphosphate, and the interaction of the activated GPCR with ion channel proteins. (See, e.g., Watson, S. and S. Arkinstall (1994) The G- protein Linked Receptor Facts Book, Academic Press, San Diego Calif., pp. 2-6; Bolander, F. F. (1994) Molecular Endocrinology, Academic Press, San Diego Calif., pp. 162-176; Baldwin, J. M. (1994) Curr. Opin. Cell Biol. 6:180-190.) GPCRs can be divided into three major subfamilies: the rhodopsin-like, secretin-like, and metabotropic glutamate receptor subfamilies. Members of these GPCR subfamilies share similar functions and the characteristic seven transmembrane structure, but have divergent amino acid sequences. The largest family consists of the rhodopsin-like GPCRs, which transmit diverse extracellular signals including hormones, neurotransmitters, and light. Rhodopsin is a photosensitive GPCR found in animal retinas. In vertebrates, rhodopsin molecules are embedded in membranous stacks found in photoreceptor (rod) cells. Each rhodopsin molecule responds to a photon of light by triggering a decrease in cGMP levels which leads to the closure of plasma membrane sodium channels. In this manner, a visual signal is converted to a neural impulse. Other rhodopsin-like GPCRs are directly involved in responding to neurotransmitters. These GPCRs include the receptors for adrenaline (adrenergic receptors), acetylcholine (muscarinic receptors), adenosine, galanin, and glutamate (N- methyl-D-aspartate/NMDA receptors). (Reviewed in Watson, S. and S. Arkinstall

(1994) The G-Protein Linked Receptor Facts Book, Academic Press, San Diego Calif., pp. 7-9, 19-22, 32-35, 130-131 , 214-216, 221-222; Habert-Ortoli, E. et al. (1994) Proc. Natl. Acad. Sci. USA 91 :9780-9783.)

The galanin receptors mediate the activity of the neuroendocrine peptide galanin, which inhibits secretion of insulin, acetylcholine, serotonin and noradrenaline, and stimulates prolactin and growth hormone release. Galanin receptors are involved in feeding disorders, pain, depression, and Alzheimer's disease (Kask, K. et al. (1997) Life Sci. 60:1523-1533). Other nervous system rhodopsin-like GPCRs include a growing family of receptors for lysophosphatidic acid and other lysσphosphoϋpids, which appear to have roles in development and neuropathology (Chun, J. et al. (1999) Cell Biochem. Biophys. 30:213-242).

The largest subfamily of GPCRs, the olfactory receptors, are also members of the rhodopsin-like GPCR family. These receptors function by transducing odorant signals.

Numerous distinct olfactory receptors are required to distinguish different odors. Each olfactory sensory neuron expresses only one type of olfactory receptor, and distinct spatial zones of neurons expressing distinct receptors are found in nasal passages. For example, the RA1 c receptor which was isolated from a rat brain library, has been shown to be limited in expression to very distinct regions of the brain and a defined zone of the olfactory epithelium (Raming, K et al. (1998) Receptors Channels 6:141- 151 ). However, the expression of olfactory-like receptors is not confined to olfactory tissues. For example, three rat genes encoding olfactory-like receptors having typical GPCR characteristics showed expression patterns not only in taste and olfactory tissue, but also in male reproductive tissue (Thomas; M. B. et al. (1996) Gene 178:1-5).

Members of the secretin-like GPCR subfamily have as their ligands peptide hormones such as secretin, calcitonin, glucagon, growth hormone-releasing hormone, parathyroid hormone, and vasoactive intestinal peptide. For example, the secretin receptor responds to secretin, a peptide hormone that stimulates the secretion of enzymes and ions in the pancreas and small intestine (Watson, supra, pp. 278-283). Secretin receptors are about 450 amino acids in length and are found in the plasma membrane of gastrointestinal cells. Binding of secretin to its receptor stimulates the production of cAMP. Examples of secretin-like GPCRs implicated in inflammation and the immune response include the EGF module-containing, mucin-like hormone receptor (Emr1) and CD97 receptor 7 proteins. These GPCRs are members of the recently characterized EGF- TM7 receptors subfamily. These seven transmembrane hormone receptors exist as heterodimers in vivo and contain between three and seven potential calcium-binding EGF-like motifs. CD97 is predominantly expressed in leukocytes and is markedly upregulated on activated B and T cells (McKnight, A. J. and S. Gordon (1998) J. Leukoc. Biol. 63:271-280).

The third GPCR subfamily is the metabotropic glutamate receptor family. Glutamate is the major excitatory neurotransmitter in the central nervous system. The metabotropic glutamate receptors modulate the activity of intracellular effectors, and are involved in long-term potentiation (Watson, supra, p. 130). The Ca.sup.2+-sensing receptor, which senses changes in the extracellular concentration of calcium ions, has a large extracellular domain including clusters of acidic amino acids which may be involved in calcium binding. The metabotropic glutamate receptor family also includes pheromone

receptors, the GABA.sub.B receptors, and the taste receptors.

Other subfamilies of GPCRs include two groups of chemoreceptor genes found in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae, which are distantly related to the mammalian olfactory receptor genes. The yeast pheromone receptors STE2 and STE3, involved in the response to mating factors on the cell membrane, have their own seven-transmembrane signature, as do the cAMP receptors from the slime mold Dictyostelium discoideum, which are thought to regulate the aggregation of individual cells and control the expression of numerous developmentally-regulated genes.

In another embodiment of the present invention relates to transporters, for example monoamine transporters. Monoamine transporters are a protein family of integral membrane transporters that are involved in transporting for example neurotransmitters in or out of a cell, for example removing neurotransmitters from the extracellular fluid. The present invention relates to group of monoamine transporters consisting of the serotonin transporter (SERT) ₁ the dopamine transporter (DAT) and the norepinephrine transporter (NET). In one embodiment the invention relates to the monoamine transporters of human origin, and thus the receptors may be selected from the group consisting of hSERT, hDAT and hNET. In another embodiment the receptor is hSERT, hDAT, or hNET.

In one embodiment of the present invention the chimeric viral envelope polypeptide, said receptor binding region, ligand or polypeptide sequence of a ligand binding region is selected from the group consisting of g-protein-coupled receptors. Thus, the receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof may be selected from the group of ligands consisting of apelin, substance P, neurokinin A, neurokinin B. In one embodiment the ligand is apelin. In another embodiment the ligand is neurokinin A. In a third embodiment the ligand is neurokinin B. In yet another embodiment the ligand is substance P.

However, the receptor binding region, ligand or polypeptide sequence of a iigand binding region or a fragment or homologue thereof binding GPRCs may be selected from the group of GPRCs consisting of

Rhodopsin receptor, alfa^ adrenergic receptor, beta-adrenergic receptor, beta ₂ adrenergic receptor, dopamine D ₁ receptor, dopamine D ₂ receptor, dopamine D ₃ receptor, dopamine D ₄ receptor, dopamine D ₅ receptor, serotonin 5HTi _B receptor, serotonin 5HT _1D receptor, serotonin 5HT _2A receptor, serotonin 5HT _2C receptor, serotonin 5HT ₆ receptor, histamine Hi receptor, histamine H ₂ receptor, histamine H ₃ receptor, cysteinyl leukotriene receptor, CySLT ₁ receptor, CysLT ₂ receptor, angiotensin Il type 1 receptor, endothelin A receptor, endothelin B receptor, luteinizing hormone receptor, follicle stimulating hormone (FSH) receptor, melanocortin MC1 R receptor, melanocortin MC4R receptor, adenocorticotropic hormone receptor (ACTHR), gonadotropin releasing hormone (GnH) receptor, parathyroid hormone receptor (PTHR1 ), thyrotropin receptor (TSHR), vasopressin V2 receptor (AV2), mu-opioid receptor (MOR), delta-opioid receptor (DOR), orexin 2 receptor, chemokine CCR2 receptor, chemokine CCR3 receptor, chemokine CCR5 receptor, chemokine receptor CX3CR1 receptor, thromboxane A2 receptor, and Ca-sensing receptor.

In another embodiment the receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof binding GPRCs may be selected from the group of GPRCs consisting of Rhodopsin receptor, alfa _2A adrenergic receptor, beta-iadrenergic receptor, beta ₂ adrenergic receptor, dopamine D ₁ receptor, dopamine D ₂ receptor, dopamine D ₃ receptor, dopamine D ₄ receptor, dopamine D ₅ receptor, serotonin 5HT _1B receptor, serotonin 5HT _1D receptor, serotonin 5HT _2A receptor, serotonin 5HT _2C receptor, serotonin 5HT ₆ receptor, histamine H-i receptor, histamine H ₂ receptor, histamine H ₃ receptor.

In yet another embodiment the receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof binding GPRCs may be selected from the group of GPRCs consisting of cysteinyl leukotriene receptor, CySLT ₁ receptor, CysLT ₂ receptor, angiotensin Il type 1 receptor, endothelin A receptor, endothelin B receptor, luteinizing hormone receptor, follicle stimulating hormone (FSH) receptor, melanocortin MC1 R receptor, melanocortin MC4R receptor, adenocorticotropic hormone receptor (ACTHR), gonadotropin releasing hormone (GnH) receptor, parathyroid hormone receptor (PTHR1), thyrotropin receptor (TSHR), vasopressin V2 receptor (AV2), mu-opioid receptor (MOR), delta-opioid receptor (DOR), orexin 2 receptor, chemokine CCR2 receptor, chemokine CCR3 receptor,

chemokine CCR5 receptor, chemokine receptor CX3CR1 receptor, thromboxane A2 receptor, and Ca-sensing receptor.

In a further another embodiment the receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof binding

GPRCs may be selected from the group of GPRCs consisting of dopamine D ₁ receptor, dopamine D ₂ receptor, dopamine D ₃ receptor, dopamine D ₄ receptor, dopamine D ₅ receptor, serotonin 5HT _1B receptor, serotonin 5HTi _D receptor, serotonin 5HT _2A receptor, serotonin 5HT _2C receptor, serotonin 5HT ₆ receptor, histamine H ₁ receptor, histamine H ₂ receptor, histamine H ₃ receptor, cysteinyl leukotriene receptor, CySLT ₁ receptor, CysLT ₂ receptor, angiotensin Il type 1 receptor, endothelin A receptor, endothelin B receptor, luteinizing hormone receptor, follicle stimulating hormone (FSH) receptor, melanocortin MC1 R receptor, melanocortin MC4R receptor.

In yet a further embodiment the receptor binding region, ligand or polypeptide sequence of a ligand binding region or a fragment or homologue thereof binding GPRCs may be selected from the group of GPRCs consisting of melanocortin MC1 R receptor, melanocortin MC4R receptor, adenocorticotropic hormone receptor (ACTHR), gonadotropin releasing hormone (GnH) receptor, parathyroid hormone receptor (PTHR1), thyrotropin receptor (TSHR), vasopressin V2 receptor (AV2), mu-opioid receptor (MOR), delta-opioid receptor (DOR), orexin 2 receptor, chemokine CCR2 receptor, chemokine CCR3 receptor, chemokine CCR5 receptor, chemokine receptor CX3CR1 receptor, thromboxane A2 receptor, and Ca-sensing receptor.

In yet another embodiment the protein expressed on the surface of a target cell which is bound by the chimeric envelope according to the present invention is a ion channel. Ion channels are pore-forming proteins that help to establish and control the small voltage gradient, exisisting across the plasma membrane of all living by allowing the flow of ions down their electrochemical gradient. They are present in the membranes that surround all biological cells. Non limiting examples of ion channels are voltage- gated sodium channels, voltage-gated calcium channels, potassium channels, calcium- activated potassium channels, inward-rectifier potassium channels, two-pore-domain potassium channels, chloride channels, transient receptor potential channels, cyclic nucleotide-gated channels, hyperpolaπ ^' zation-activated, cyclic nucleotide-gated channels, light-gated channels, ligand-gated channels (LGICs) (Examples of LGICs

include the cation-permeable "nicotinic" Acetylcholine receptor, ionotropic glutamate- gated receptors and ATP-gated P2X receptors, and the anion-permeable y- aminobutyric acid-gated GABA _A receptor).

Non-limiting examples of voltage-gated sodium channels ar Navi .1, Na _v 1.2, Nav1.3, Nav1 A, Nav1.5, Na _v 1.6, Na _v 1 .7, Na _v 1.8, orNavi .9

Voltage-dependent calcium channels (VDCC) are a group of voltage-gated ion channels found in excitable cells, for example neurons, glial cells, muscle cells, etc. with a permeability to the ion Ca ²⁺ , which plays a role in the membrane potential. VDCCs are involved in the release of neurotransmitters and hormones, muscular contraction, excitability of neurons and gene expression. Non-limiting examples of voltage-gated calcium channels are α 1 subunit pores of the L, N and T type, β subunit proteins, α2δ subunits.or y subunits.

Potassium channels form potassium-selective pores that span cell membranes and are found in most cells, controlling cell function. In excitable cells such as neurons, they shape action potentials and set the resting membrane potential. Non-limiting examples of potassium channels are voltage-gated potassium channels, calcium-activated potassium channels (BK channels, also called MaxiK or slo1 channels), inward-rectifier potassium channels, and two-pore-domain potassium channels.

In yet a further embodiment the protein expressed on the surface of a target cell which is bound by the chimeric envelope according to the present invention is a symporter. A symporter, also known as a cotransporter, is an integral membrane protein that is involved in secondary active transport. It works by binding to two molecules at a time and using the gradient of one solute's concentration to force the other molecule against its gradient. Non limiting examples of symporters are the sodium-iodide symporter, H+- Pi symporter, sodium-chloride symporter.

Linker sequence The polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region may optionally comprise one or more flexible linkers. By linker sequence is meant flexible linker sequence(s) s) of one or more amino acid residues as known by one skilled in the art - for example 2-30 amino acid residues, such as 2-20 amino acid residues, such as 2-10 amino acid residues. The linker

sequences are preferably placed at the N-terminal and/or C-terminal of the polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region. It is understood that the linker sequence(s) link the envelope polypeptide and the polypeptide sequence of a receptor binding region, ligand or polypeptide sequence of a ligand binding region. The linker(s) is/are preferably positioned whereby such linkers increase rotational flexibility and/or minimize steric hindrance of the modified envelope polypeptide. Thus, in one embodiment of the present invention, a linker sequence is positioned at each end of the second polypeptide sequence that is to say at either end of the second polypeptide sequence. Any suitable linker sequence known to one skilled in the art can be used: examples of suitable linker sequences include, but are not restricted to, linkers described by Argos et al., 1990 (Argos, 1990). One preferred linker sequence has the polypeptide sequence SGGSG.

Agents

The chimeric viral envelope polypeptide or virus particle may further comprise an agent for delivery to a target cell. Such an agent may be selected from the group consisting of therapeutic agents, a gene or gene product, a diagnostic label, a label for bioimaging, enzymes for activating prodrugs, a si-RNA or a toxic agent. It is understood that also a virus particle comprising the chimeric envelope polypeptide is also an embodiment of the present invention.

In one embodiment of the present invention the agent may be comprised in the chimeric viral envelope. Other embodiments are that the agent is packaged into the virus particle during the budding of the virus particle from the host cell. Packaging of said agent may be obtained by operatively associating said agent with a packaging sequence directing the packacking of the agent into a virus particle.

Hemi fusion Hemi-fusion is the process where only the outer leaflets of the two-lipid-bilayer membranes of a virus particle and a target cell for the virus particle are fused.

Hemi-fusion can be thought of as an intermediate in the normal fusion process.

A technology to lock enveloped particles at the hemi-fused stage by the mutation of a critical histidine residue has been described (Zavorotinskaya T, et al. 2004).

Hemi-fusion may also provide innovative means for the delivery of cargo to the plasma membrane. Hemi-fused particles are expected to be in a locked state on the membrane as a result of the limited diffusion of integral membrane proteins that span both bi- layers. It is conceivable that the hemi-fused stage will only be reached following a very accurate interaction with the receptor at physiological temperature, which suggests that this way of labelling live cells could be very specific as well as stable.

One embodiment of the present invention therefore comprises the chimeric envelope polypeptide, polynucleotide, vectors, and virus particles in which the amino acid sequence or nucleotide sequence has been altered to yield chimeric envelope polypeptides or virus particles capable of hemi-fusion.

A person skilled in the art will know to alter the critical histidine residue of the envelope polypeptide. For example envelope mutants are arrested at at hemifusion by a single amino acid mutation (hisδ) as shown in (Zavorotinskaya T, et a/. 2004).

Methods using any of the aspects of the present invention

Any of the polynucleotide, vectors and/or envelope polypeptides provided herein can be used in the methods as described below. These polynucleotides, vectors and/or envelope polypeptides are herein below described as "constructs", by which it is meant that any of the polynucleotides, vectors and/or envelope polypeptides can advantageously be used. Preferably, said construct is a retroviral particle as described herein.

One could envisage the virus particles or chimeric envelope polypeptides or homologues or fragments thereof to be used as devices mediating receptor-dependent fusion or hemi fusion of biological membranes. In the field of bioimaging the present invention presents novel means for the detection and imaging of specific cell surface proteins on cells by receptor-dependent attachment of labelled viral particles or labelled chimeric envelope polypeptides. The attachment may subsequently be followed by membrane hemi fusion as described elsewhere herein. For example membrane association of drugs may be studied using the methods described herein. The methods may also be used for studies of drug target validation.

Also prodrug activating agents such as for examples enzymes or other activating agents may be studied in the methods of the present invention or employed in therapeutic methods.

Thus, one aspect of the present invention pertains to a method for targeting an agent to a G-protein coupled receptor, comprising the steps of: providing the chimeric envelope polypeptide; causing said chimeric envelope polypeptide to contact a target cell wherein said target cell comprises a receptor for the ligand of said chimeric envelope polypeptide. Furthermore, another aspect is a method for specifically tethering a chimeric retroviral envelope polypeptide to a specific cell type, comprising the steps of: providing an virus particle expressing a chimeric retroviral envelope polypeptide, said envelope polypeptide comprising a ligand capable of binding said specific cell-type; allowing said chimeric envelope polypeptide to specifically contact a cell of said specific cell type; allowing the outer membrane of the virus to undergo a hemi-fusion process with the outer membrane of said cell.

However, yet another aspect relates to a method for labelling one or more object of interest on a cell, comprising: providing the labelled vector, polypeptide or recombinant retrovirus ; allowing said labelled vector, polypeptide or recombinant retrovirus to contact said object of interest on said cell. One embodiment of this aspect further comprises the step of evaluating the presence of said object(s) of interest on said cell, preferably by taking an image of at least part of the cell. A person skilled in the art will be familiar with the type of imaging possible and the labels used for such imaging.

Thus, a further aspect relates to a method for quantifying the amount or number of an object of interest in a biological specimen, such as a cell, said method comprising the steps of: providing the labelled vector, chimeric envelope polypeptide or virus particle allowing said labelled vector, chimeric envelope polypeptide or virus particle to contact said object of interest on said cell.

Yet a further aspect relates to a method for screening for and analysis of drugs that target the envelope-receptor interaction, comprising the virus particle.

An example wherein the process of hemi-fusion in relation to the embodiments and aspects of the present invention is employed is shown in example 4. It is appreciated that the methods employing hemi-fusion is not limited to the particular example.

Another aspect of the present invention relates to targeting and penetration of virus receptor bearing cells by nanoparticles coated with said chimeric envelope proteins as disclosed herein.

In another aspect of the present invention, application of virus with liposome allow for delivery of a range of compounds directly to the cytoplasm, thus bypassing the hostile endosomal pathway. In one embodiment, said compounds include DNA and/or RNA, including mRNA, siRNA, miRNA, and tRNA. In another embodiment, said compounds include chemically modified nucleic acids, nucleic acids analogues such as peptide nucleic acids (PNA) and linked nucleic acids (LNA). In yet another embodiment, said compounds include drugs and/or nano-particles and/or fluorescence markers. In a further embodiment, said compounds include lipophile compounds or compounds joined to a lipophilic group to the plasma membrane for purposes of imaging (e.g. fluourescence markers), induction of apoptosis, antigens to activate the immune system.

Another aspect of the present invention pertains to selectively killing cells by receptor- dependent induction of syncytia. In one embodiment, the formation of syncytia is induced by the chimeric viral envelope peptide disclosed herein. In one embodiment, the induction of syncytia is directed towards tumor cells. In another embodiment, the induction of syncytia is directed towards membranes of parasites, including malaria. In another embodiment, the induction of syncytia is directed towards bacterial cells, including E. coli and/or salmonella.

Embodiments relating to gene therapy The recombinant virus particle can further comprise an agent for delivery to the target cell, optionally selected from the group consisting of a therapeutic agent or a gene or gene product, which agent is optionally operatively associated with a retroviral packaging sequence. Thus, the virus particle can in one embodiment comprise at least one heterologous gene to be expressed in the host after a gene therapy procedure. Said heterelogous gene to be expressed can for example comprise the polynucleotide

encoding the chimeric envelope polypeptide according to the present invention. The agent is preferably a therapeutic agent, such as a polynucleotide sequence.

The polynucleotide encoding the therapeutic agent is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma Virus (RSV) promoter; the histone promoter; the pollll promoter, the .beta.- actin promoter; inducible promoters, such as the MMTV promoter, the metallothionein promoter, heat shock promoters; adenovirus promoters; the albumin promoter; the ApoAI promoter; B19 parvovirus promoters; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex Virus thymidine kinase promoter; retroviral LTRS; human growth hormone promoters, and the MxIFN inducible promoter. The promoter also may be the native promoter which controls the polynucleotide encoding the therapeutic agent. It is to be understood, however, that the scope of the present invention is not to be limited to specific foreign genes or promoters.

The polynucleotides encoding the modified envelope polypeptide and the therapeutic agent may be placed into an appropriate vector by genetic engineering techniques known to those skilled in the art. When the modified vector is a retroviral vector particle, the polynucleotides encoding the modified envelope polypeptide and the therapeutic agent can e.g. be placed into an appropriate retroviral plasmid vector.

In one embodiment, the retroviral plasmid vector may be derived from Moloney Murine Leukemia Virus and is of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al., J. Virol., Vol. 61 , pgs. 1639-1649 (1987) and Miller, et al., Biotechniques, Vol. 7, pgs 980-990 (1989). Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon. The term "mutated" as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.

In another embodiment, the retroviral plasmid vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs. Preferred cloning sites are selected from the group consisting of Notl, SnaBI,

Sail, and Xhol. In a preferred embodiment, the retroviral plasmid vector includes each of these cloning sites. Such vectors are further described in U.S. patent application Ser. No. 08/340,805, filed Nov. 17, 1994, and in PCT Application No. W091 /10728, published JuI. 25, 1991 , and incorporated herein by reference in their entireties.

When a retroviral plasmid vector including such cloning sites is employed, there may also be provided a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of Notl, SnaBI, Sail, and Xhol located on the retroviral plasmid vector. The shuttle cloning vector also includes at least one desired polynucleotide encoding a therapeutic agent which is capable of being transferred from the shuttle cloning vector to the retroviral plasmid vector.

The shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.

The shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems. The shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria. Thus, for example, the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.

The retroviral plasmid vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et a!., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., ceWular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and .beta.-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

Cell lines and animal models

In another embodiment, there is provided a packaging cell which includes a nucleic acid sequence encoding a modified chimeric envelope in accordance with the invention, and which may further include nucleic acid sequences encoding the gag and pol proteins. A producer cell for generating viral particles which includes a modified envelope in accordance with the invention is produced by introducing into such packaging cell either a retroviral vector particle or a retroviral plasmid vector, in each case including a polynucleotide encoding a therapeutic agent. The producer cell line thus generates infectious retroviral particles including the modified chimeric envelope and the polynucleotide encoding the therapeutic agent.

Thus, in another aspect of the present invention is provided a cell transfected with the vector according to the present invention. In one preferred embodiment, said cell is comprised in a stable cell line comprising such cells. The present invention further relates to "packaging cell lines" for producing the vectors of the present invention.

MLV based packaging cells are widespread tools for research. Packaging cells based on ecotropic viruses have the advantage of being harmless to humans and are used in bio- safety level 1 laboratories.

One embodiment of the present invention relates to a packaging cell construct comprising the vector comprising a nucleic acid coding for a polypeptide envelope as described in the present application, and optionally a non-viral or viral promoter and poly-adenylation signals.

Another embodiment of the present invention relates to use of any of the vectors according to the present invention for the generation of a packaging cell.

The packaging cell line can for example be engineered to produce the viral Gag, Pol and Env proteins from constructs that lack the packaging signal (to prevent them from being taken up by budding virions). Thus, when a vector is inserted into a packaging cell line, it will be packaged into budding virions and can be transferred into target cells. Representative examples of packaging cell lines include, but are not limited to, the PE501 and PA317 cell lines disclosed in Miller, et al., Biotechniques, Vol. 7 pgs. 980- 990 (1989).

In one embodiment, the packaging cell line is a "pre-packaging" cell line which includes polynucleotides encoding the gag and pol retroviral proteins, but not the envelope, or env, protein. Examples of such "pre-packaging" cell lines include, but are not limited to, GP8 cells, GPL cells, and GPNZ cells as described in Morgan, et al., J. Virol., Vol. 67, No. 8, pgs. 4712-4721 (August 1993). Such cell lines, upon transduction with the retroviral plasmid vector, generates infectious retroviral particles including the modified, or chimeric, envelope and a polynucleotide encoding the therapeutic agent.

In another embodiment, a retroviral plasmid vector which includes a polynucleotide encoding a modified polynucleotide encoding a modified envelope polypeptide in accordance with the invention and a polynucleotide encoding a therapeutic agent is used to transduce a packaging cell line including nucleic acid sequences encoding the gag, pol, and wild-type (i.e., unmodified) env retroviral proteins. Examples of such packaging cell lines include, but are not limited to, the PE501 , PA317 (ATCC No. CRL 9078), .psi.-2, .psi.-AM, PA12, T19-14X, VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1 , pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, and use of liposomes, such as hereinabove described, and CaPO.sub.4 precipitation. Such producer cells generate infectious retroviral vector particles which include the modified envelope, the wild-type retroviral envelope, a polynucleotide encoding the modified, or chimeric, envelope, and a polynucleotide encoding a therapeutic agent.

In another preferred embodiment, said cell is comprised in an animal model using methods known to one skilled in the art. Said model is preferably a mouse,

Antibodies

The present invention further relates to an antibody capable of specifically binding one of the molecules provided in the present invention, such as a chimeric envelope polypeptide according to the present invention, and/or a retroviral particle expressing said chimeric envelope polypeptide.

It is understood that the term "antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab').sub.2, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind GCREC polypeptides

can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.

Therapeutic methods using any of the aspects of the present invention Any of the polynucleotide, vectors and/or envelope polypeptides provided herein can be used in therapeutic methods and/or prophylaxis of disease, such as viral disease. These polynucleotides, vectors and/or envelope polypeptides are herein below described as "constructs", by which it is meant that any of the polynucleotides, vectors and/or envelope polypeptides can advantageously be used. Preferably, said construct is a retroviral particle as described herein.

Thus, the present invention relates in one embodiment to a therapeutic method for treatment of an individual in need thereof, said method comprising administering the chimeric viral envelope polypeptide. Similarly embodiments for a therapeutic method for treatment of an individual in need thereof, said method comprising administering the vector or a virus particle.

Therefore, the present invention relates to a method for delivering an agent to a mammalian target cell in an individual in need thereof, comprising the steps of: (i) providing the chimeric envelope polypeptide or virus particle,(ii) causing said chimeric envelope polypeptide or virus particle to contact a target cell population of said individual's cells, wherein said target cell comprises a receptor for the ligand of said chimeric envelope polypeptide, (iii) allowing the virus particle to bind said receptor.

The binding of said chimeric viral envelope polypeptide or virus particle displaying said chimeric viral envelope polypeptide to the cognate receptor may result in blockage of the binding of other ligands for the cognate receptor which may in some diseases be of relevance in terms of treatment.

Likewise one embodiment of the present invention relates to the binding of said chimeric viral envelope polypeptide or virus particle displaying said chimeric viral envelope polypeptide to the cognate receptor may block binding of other viral molecules to the cell bound by said construct. Without being bound by theory this may be achieved by the mechanism of viral interference or superinfection.

The binding can also lead to prevention and/or reduction of syncitial formation between another, pathogenic virus and the cell bound by the construct of the present invention.

Another effect of the binding can be reduction in the expression level of the bound viral receptor (such as a viral co-receptor, such as the CXCR4 co-receptor) on the surface of the cell bound by said construct.

In one embodiment, the bound construct (preferably a virus particle) is taken up into the cell which it has specifically bound. An agent for delivery into the target cell may further be comprised in the chimeric viral envelope polypeptide, or virus particle. Such an agent may be an si-RNA molecule directed against a gene expression product of interest in the treatment of an individual.

Similarly, the agent may be a polynucleotide which will have a therapeutic effect when delivered to a target cell. The polynucleotide may be a heterologous gene. The polynucleotide affects the signalling pathway of the target cell. However, a person skilled in the art will appreciate that a number of therapeutic genes may be delivered to the target cell in the treatment of a number of diseases.

In one embodiment of the above method, an agent is delivered to said cell by said construct (preferably a virus particle). Said agent can for example be an anti-viral drug or a polynucleotide.

Where said agent is a polynucleotide, said method is advantageous to use for gene therapy. The polynucleotide introduced into the cell by said gene therapy method can for example be the polynucleotide according to the present invention encoding the chimeric envelope polypeptides described herein, however said polynucleotide can in equally be another anti-viral polynucleotide, such as encoding a polypeptide with antiviral activity. The term "introducing" as used herein encompasses a variety of methods of transferring polynucleotides into a cell, such methods including transformation, transduction, transfection, and transinfection.

Thus, retroviral vector particles of the present invention can be used for introducing polynucleotides into cells for gene therapy purposes. In one approach, cells are obtained from a patient, and retroviral vector particles are used to introduce a desired polynucleotide into the cells, and such modified cells are returned to the patient with the engineered cells for a therapeutic purpose. In another approach, retroviral vector particles may be administered to the patient in viva, whereby the retroviral vector particles transduce cells of the patient in vivo.

Methods for in vivo and ex vivo gene therapy are well known in the art, such as for example described in e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764; Friedmann, 1989, Science, 244:1275-1281 ; Mulligan, 1993, Science, 260:926-932, R. Crystal, 1995, Science 270:404-410, each of which are incorporated herein by reference in their entirety). An increasing number of these methods are currently being applied in human clinical trials (Morgan, R., 1993, BioPharm, 6(1):32-35; see also The Development of Human Gene Therapy, Theodore Friedmann, Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. ISBN 0-87969-528-5, which is incorporated herein by reference in its entirety).

It may be preferable to remove the majority of a target cell population prior to therapy, for example surgically.

In fact, any of the therapies described herein can be in vivo or ex vivo, that is to say that said contacting occurs outside the individual, and then the target population is returned to the individual's body.

Said specific cell type can for example be T cells, a cell expressing CXCR5 or CXCR4, or macrophage cells.

In another embodiment of the present invention is provided a method for treatment or prevention of a viral disease, comprising administering a construct as disclosed herein to an individual in need thereof. Said construct is preferably the viral envelope polypeptide as disclosed herein, or the recombinant virus particle as disclosed herein. Said viral disease is preferably selected from HIV (for example, HIV-1 or HIV-1)

Target cell populations for any of the therapeutic methods of the present invention In one embodiment of the present invention the target cell population are cells that express receptors or transporters on their surface, for example G-protein-coupled receptors on the ceil surface and/or monoamine transporters. In one embodiment, said cell is a Keratinizing epithelial cell, such as selected from the group consisting of:

Epidermal keratinocyte (differentiating epidermal cell), Epidermal basal cell (stem cell), Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell), Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell , Cuticular hair root sheath cell , Hair root sheath cell of Huxley's layer,Hair root sheath cell of Henle's layer, External hair root sheath cell, Hair matrix cell (stem cell).

In another embodiment, said cell is a wet stratified barrier epithelial cell, such as selected from the group consisting of: Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina Urinary epithelium cell (lining urinary bladder and urinary ducts)

In another embodiment, said cell is a Exocrine secretory epithelial cell, such as selected from the group consisting of:

Salivary gland mucous cell (polysaccharide-rich secretion, Salivary gland serous cell (glycoprotein enzyme-rich secretion), Von Ebner's gland cell in tongue (washes taste buds) , Mammary gland cell (milk secretion) , Lacrimal gland cell (tear secretion) Ceruminous gland cell in ear (wax secretion) , Eccrine sweat gland dark cell

(glycoprotein secretion) , Eccrine sweat gland clear cell (small molecule secretion) Apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive), Gland of Moll cell in eyelid (specialized sweat gland) , Sebaceous gland cell (lipid-rich sebum secretion), Bowman's gland cell in nose (washes olfactory epithelium) , Brunner's gland cell in duodenum (enzymes and alkaline mucus)

Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm) , Prostate gland cell (secretes seminal fluid components) , bulbourethral gland cell (mucus secretion) , Bartholin's gland cell (vaginal lubricant secretion) , Gland of Littre cell (mucus secretion) , Uterus endometrium cell (carbohydrate secretion) , Isolated goblet cell of respiratory and digestive tracts (mucus

secretion) .Stomach lining mucous cell (mucus secretion) .Gastric gland zymogenic cell (pepsinogen secretion) .Gastric gland oxyntic cell (hydrochloric acid secretion) Pancreatic acinar cell (bicarbonate and digestive enzyme secretion) .Paneth cell of small intestine (lysozyme secretion) Jype Il pneumocyte of lung (surfactant secretion) Clara cell of lung.

In another embodiment, said cell is a Hormone secreting cell, such as selected from the group consisting of:

Anterior pituitary cells Somatotropes

Lactotropes

Thyrotropes

Gonadotropes

Corticotropes Intermediate pituitary cell, secreting melanocyte-stimulating hormone

Magnocellular neurosecretory cells secreting oxytocin secreting vasopressin

Gut and respiratory tract cells secreting serotonin secreting endorphin secreting somatostatin secreting gastrin secreting secretin secreting cholecystokinin secreting insulin secreting glucagon secreting bombesin

Thyroid gland cells thyroid epithelial cell parafollicular cell

Parathyroid gland cells

Parathyroid chief cell oxyphil cell

Adrenal gland cells chromaffin cells

secreting steroid hormones (mineralcorticoids and gluco corticoids) Leydig cell of testes secreting testosterone Theca interna cell of ovarian follicle secreting estrogen Corpus luteum cell of ruptured ovarian follicle secreting progesterone Kidney juxtaglomerular apparatus cell (renin secretion) Macula densa cell of kidney Peripolar cell of kidney Mesangial cell of kidney

In another embodiment, said cell is a cell of the Gut, Exocrine Glands and Urogenital

Tract, such as selected from the group consisting of:

Intestinal brush border cell (with microvilli)

Exocrine gland striated duct cell

Gall bladder epithelial cell Kidney proximal tubule brush border cell

Kidney distal tubule cell

Ductulus efferens nonciliated cell

Epididymal principal cell

Epididymal basal cell

In another embodiment, said cell is a Metabolism and storage cell, such as selected from the group consisting of:

Hepatocyte (liver cell)

White fat cell Brown fat cell

Liver lipocyte

In another embodiment, said cell is a cell of the Lung, Gut, Exocrine Glands or

Urogenital Tract, such as selected from the group consisting of:

Type I pneumocyte (lining air space of lung) Pancreatic duct cell (centroacinar cell)

Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.)

Kidney glomerulus parietal cell

Kidney glomerulus podocyte

Loop of Henle thin segment cell (in kidney) Kidney collecting duct cell

Duct cell (of seminal vesicle, prostate gland, etc.)

In another embodiment, said cell is an epithelial cell lining a closed internal body cavity, such as selected from the group consisting of: Blood vessel and lymphatic vascular endothelial fenestrated cell

Blood vessel and lymphatic vascular endothelial continuous cell

Blood vessel and lymphatic vascular endothelial splenic cell

Synovia) cell (lining joint cavities, hyaluronic acid secretion)

Serosal cell (lining peritoneal, pleural, and pericardial cavities) Squamous cell (lining perilymphatic space of ear)

Squamous cell (lining endolymphatic space of ear)

Columnar cell of endolymphatic sac with microvilli (lining endolymphatic space of ear)

Columnar cell of endolymphatic sac without microvilli (lining endolymphatic space of ear) Dark cell (lining endolymphatic space of ear)

Vestibular membrane cell (lining endolymphatic space of ear)

Stria vascularis basal cell (lining endolymphatic space of ear)

Stria vascularis marginal cell (lining endolymphatic space of ear)

Cell of Claudius (lining endolymphatic space of ear) Cell of Boettcher (lining endolymphatic space of ear)

Choroid plexus cell (cerebrospinal fluid secretion)

Pia-arachnoid squamous eel)

Pigmented ciliary epithelium cell of eye

Nonpigmented ciliary epithelium cell of eye Corneal endothelial cell

In another embodiment, said cell is a Ciliated cell with propulsive function, such as selected from the group consisting of:

Respiratory tract ciliated cell Oviduct ciliated cell (in female)

Uterine endometrial ciliated cell (in female)

Rete testis cilated cell (in male)

Ductulus efferens ciliated cell (in male)

Ciliated ependymal cell of central nervous system (lining brain cavities)

In another embodiment, said cell is an Extracellular matrix secretion cell, such as selected from the group consisting of:

Ameloblast epithelial cell (tooth enamel secretion)

Planum semilunatum epithelial cell of vestibular apparatus of ear (proteoglycan secretion)

Organ of Corti interdental epithelial cell (secreting tectorial membrane covering hair cells)

Loose connective tissue fibroblasts

Corneal fibroblasts Tendon fibroblasts

Bone marrow reticular tissue fibroblasts

Other nonepithelial fibroblasts

Pericyte

Nucleus pulposus cell of intervertebral disc Cementoblast/cementocyte (tooth root bonelike cementum secretion)

Odontoblast/odontocyte (tooth dentin secretion)

Hyaline cartilage chondrocyte

Fibrocartilage chondrocyte

Elastic cartilage chondrocyte Osteoblast/osteocyte

Osteoprogenitor cell (stem cell of osteoblasts)

Hyalocyte of vitreous body of eye

Stellate cell of perilymphatic space of ear

In another embodiment, said cell is a Contractile cell, such as selected from the group consisting of:

Red skeletal muscle cell (slow)

White skeletal muscle cell (fast)

Intermediate skeletal muscle cell nuclear bag cell of Muscle spindle nuclear chain cell of Muscle spindle

Satellite cell (stem cell)

Ordinary heart muscle cell

Nodal heart muscle cell Purkinje fiber cell

Smooth muscle cell (various types)

Myoepithelial cell of iris

Myoepithelial cell of exocrine glands

Red Blood Cell

In another embodiment, said cell is a Blood or immune system cell, such as selected from the group consisting of:

Erythrocyte (red blood cell)

Megakaryocyte (platelet precursor) Monocyte

Connective tissue macrophage (various types)

Epidermal Langerhans cell

Osteoclast (in bone)

Dendritic cell (in lymphoid tissues) Microglial cell (in central nervous system)

Neutrophil granulocyte

Eosinophil granulocyte

Basophil granulocyte

Mast cell Helper T cell

Suppressor T cell

Cytotoxic T cell

B cells

Natural killer cell Reticulocyte

Stem cells and committed progenitors for the blood and immune system (various types)

In another embodiment, said cell is a Sensory transducer cell, such as selected from the group consisting of: Auditory inner hair cell of organ of Corti

Auditory outer hair cell of organ of Corti

Basal cell of olfactory epithelium (stem cell for olfactory neurons)

Cold-sensitive primary sensory neurons

Heat-sensitive primary sensory neurons Merkel cell of epidermis (touch sensor)

Olfactory receptor neuron

Pain-sensitive primary sensory neurons (various types) Photoreceptor rod cell of eye Photoreceptor blue-sensitive cone cell of eye Photoreceptor green-sensitive cone cell of eye Photoreceptor red-sensitive cone cell of eye Proprioceptive primary sensory neurons (various types) Touch-sensitive primary sensory neurons (various types) Type I carotid body cell (blood pH sensor) Type Il carotid body cell (blood pH sensor)

Type I hair cell of vestibular apparatus of ear (acceleration and gravity) Type Il hair cell of vestibular apparatus of ear (acceleration and gravity) Type I taste bud cell

In another embodiment, said cell is an Autonomic neuron cell, such as selected from the group consisting of:

Cholinergic neural cell (various types)

Adrenergic neural cell (various types)

Peptidergic neural cell (various types)

In another embodiment, said cell is a sense organ or peripheral neuron supporting cell, such as selected from the group consisting of:

Inner pillar cell of organ of Corti

Outer pillar cell of organ of Corti Inner phalangeal cell of organ of Corti

Outer phalangeal cell of organ of Corti

Border cell of organ of Corti

Hensen cell of organ of Corti

Vestibular apparatus supporting cell Type I taste bud supporting cell

Olfactory epithelium supporting cell

Schwann cell

Satellite cell (encapsulating peripheral nerve cell bodies)

Enteric glial cell

In another embodiment, said cell is a Central nervous system neuron or glial cell, such as selected from the group consisting of: Astrocyte (various types)

Neuron cells (large variety of types, still poorly classified) Oligodendrocyte Spindle neuron

In another embodiment, said cell is a lens cell, such as selected from the group consisting of: Anterior lens epithelial cell

Crystallin-containing lens fiber cell is in body when heart is breathing hard

In another embodiment, said cell is a pigment cell, such as selected from the group consisting of: Melanocyte Retinal pigmented epithelial cell

In another embodiment, said cell is a Germ cell, such as selected from the group consisting of:

Oogonium/Oocyte

Spermatid

Spermatocyte

Spermatogonium cell (stem cell for spermatocyte) Spermatozoon

In another embodiment, said cell is a nurse cell, such as selected from the group consisting of: Ovarian follicle cell Sertoli cell (in testis) Thymus epithelial cell

In another embodiment of the therapeutic method of the present invention, the target cell population comprises or consists of T cells. In another preferred embodiment of the therapeutic method of the present invention, said target population comprises or

consists of cells expressing CXCR5 or CXCR4. In another embodiment of the therapeutic method of the present invention, said target population comprises or consists of macrophage cells.

Further example of cells which may be targeted for binding, infection or transduction with the chimeric envelope polypeptides or vector particles of the present invention include, but are not limited to, T cell, endothelial cells, tumor cells, chondrocytes, fibroblasts and fibroelastic cells of connective tissues; osteocytes and osteoblasts in bone; endothelial and smooth muscle cells of the vasculature; epithelial and subepithelial cells of the gastrointestinal and respiratory tracts; vascular cells, connective tissue cells, and hepatocytes of a fibrotic liver, the reparative mononuclear and granulocytic infiltrates of inflamed tissues, liver cells, T-cells, lymphocytes, endothelial cells, T4 helper cells, or macrophages.

In another embodiment, the receptor binding region is a hepatitis B virus surface protein binding region, and the target cell is e.g. a liver cell.

Medicament, pharmaceutical formulations, administration and dosages In another aspect of the present invention is further disclosed a medicament or pharmaceutical formulation comprising any of the constructs described herein, such as a chimeric envelope polypeptide or viral particle as disclosed herein.

The constructs of the present invention may be directly administered to a desired target cell ex vivo, and such cells may then be administered to a patient as part of a gene therapy procedure.

Although the chimeric polypeptides and/or vector particles may be administered directly to a target cell, they may also be engineered such that they are resistant to inactivation by human serum, and thus may be administered to a patient by (e.g. intravenous) injection, and travel directly to a desired target cell or tissue without being inactivated by human serum.

The vector particles may be concentrated from dilute vector stocks in vitro by contacting a dilute vector stock with an extracellular matrix component to which the modified viral surface protein will bind. Such binding enables one to obtain a

concentrated stock of the vector particles.

In addition, the modified viral surface proteins of the present invention may be employed to form proteoliposomes; i.e., the modified viral surface protein forms a portion of the liposome wall. Such proteoliposomes may be employed for gene transfer or for drug delivery to cells located at a site of an exposed extracellular matrix component.

Any of the constructs disclosed herein may be administered to a host in an amount effective to produce a therapeutic effect in the host. The host may be a mammalian host, which may be a human or non-human primate host. The exact dosage which may be administered is dependent upon a variety of factors, including the age, sex, and weight of the patient, the cells which are to be transduced, the therapeutic agent which is to be administered, and the severity of the disorder to be treated.

The vector particles may be administered systemically, such as, for example, by intravenous, intracolonic, intratracheal, intraperitoneal, intranasal, intravascular, intrathecal, intraarterial, intracranial, intramarrow, intrapleural, intradermal, subcutaneous, intramuscular, intraocular, intraosseous and/or intrasynovial administration. The vector particles also may be administered topically.

Examples

Example 1 Sequence identity: The percent sequence identity between the protein sequences of gammaretroviruses when compared with the SL3-2 envelope protein sequence of gammaretroviruses was calculated using the VECTOR NTI computer program:

Wherein RBD is the receptor binding domain. The RBD domain can be defined as the domain corresponding to the polypeptide domain that by itself is able to bind to the receptor. In the present invention the RBD of the envelope polypeptide is preferably defined as the region delineated by the first amino acid of the envelope polypeptide and the amino acid preceeding the proline rich region (PPR).

The amino acid sequence of SL3-2 is identical to that of SEQ ID NO. 2. The amino acid sequence of MCF247, NZB-9-1, MoMLV and 4070A can be obtained from the NCBI databank at the link http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search& ;DB=nucleotide or from the original scientific journals: MCF247 (Kelly et al. 1983; Khan 1984), MoMLV (Shinnick et al. 1981; Miller and Verma 1984), 4070A (Ott et al. 1990), NZB-9-1 (O'Neill et al. 1985).

Example 2: Flow Cytometry to detect CXCR4 CSE

Method: Flow cytometric analysis was carried out of Cell Surface Expression (CSE) of CXCR4 of cells transduced with vectors comprising the chimeric envelope polypeptide of the present invention. D17 CD4 CXCR4 cells were transduced with a vector expressing SL3-2 envelope with V3 loop inserted at positions indicated.

The transductions were done by co transfecting Moloney gagpol 2ug, VSV-G expressing vector 2ug and a mini-virus expressing the SL3-2 (V3 construct 6 ug) in 293T cells using calcium phosphate transfection protocol (see detailed protocol in Bahrami et al., "Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors", Gene. 2003 Oct 2;315:51-61). The virus containing supernatant was subsequently used to transduce the D17 CXCR4 CD4 cells.

Six chimeric variants have been tested (sequences used shown in SEQ ID NO: 3-8 and 42-47). In all panels the control D17 CD4 CXCR4 cells with SL3-3 envelope only (without the V3 insert) was tested against the same envelope comprising a V3 loop variant.

Protocol for flow cytometry: • Harvest cells ( by nice trypzination) and spin down gently (1200 rpm, 5 min)

• Keep cells on ice in all subsequent steps.

• Wash once in wash buffer (2% Serum, 98% PBS -cold) =>2% Wash

• Resuspend in small amount of wash buffer: 0,1-1 ml (>10 ⁷ cells/ml)

• Count the total number of cells • Dilute the suspension appropriate (wash buffer) and transfer 5*10 ^{5 "} 10 ⁶ cells in 100 μl volume to Flow tubes

• Add 5 μl primary IgG (AbCam, UK) diluted in 100 μl PBS with 10% FBS

• Vortex and incubate 45 min at 4 ⁰ C in dark (ie. Fridge)

• Wash cells twice in 1 ml wash buffer (1200 rpm, 5 min) • Pour of buffer

• Add 5 μl secondary Ab (goat anti Mouse IgG - PE/FITCH conjugated) diluted in 100 μl PBS with 10% FBS and vortex briefly

• Incubate 45 min at 4 ⁰ C in dark (ie. Fridge)

• Wash cells twice in 1 ml wash buffer (1200 rpm, 5 min) • After final spin, resuspend cells in 500 μl 1% formaldehyde in PBS, pH 7,4

• Store samples in the fridge until Flow analysis

Remember to include uninfected control cells, and make the staining of these cells with and without primary Ig. That is minimum number of samples are 4 (test, pos. control, 2 neg. control)

Conclusions: Results are shown in figure 6. The results show that the V3 constructs are able to significantly inhibit binding of the anti CXCR-4 antibody to the CXCR4 receptor significantly. The effect is a result of the V3 domain insertion in the SL3-2 backbone as the wild-type SL3-2 control does not affect the expression levels of CXCR-4.

Example 3a: Syncytia Assay

Protocol:

293T cells are cultured in DMEM media with 10% Foetal Bovine Serum and 1% penicillin/streptomycin. D17 CD4 CXCR4 cells are cultured in α-MEM media with 10% Foetal Bovine Serum and 1% penicillin/streptomycin.

Day 1 : 293T cells are seeded at 1*10 ⁴ cells/cm ² in 6-well plate (Nunc, Roskilde)

Day 2: Fresh media is added to 293T three hours prior to transfection. 293T cells are transfected with an HIV-1 Envelope expressing constructs (see sequence information below) via CaPO ₄ -precipitation method.

Day 3: Media is replenished on 293T cells and D17 CD4 CXCR4 cells are co- cultured in the 6-well plate at a density of 2*10 ⁴ cells/cm ² .

Day 4: Syncytia formation is determined visually and pictures are taken of the plaques formed. Nuclei are counted.

Results: The capability of performing cell-cell fusion mediated by the interaction of the HIV-1 envelope protein and the CD4/CXCR4 receptors utilising 293T and D17 cells is seen. The egfp expression localises to the syncytia formation. Control transfection (egfp expression plasmid without HIV-1) yields no syncytia.

This is shown in figures 8-9.

Conclusions: The comparison of control D17 CD4 CXCR4 cells to the D17 CD4 CXCR4 cells transduced with SL3-3 envelope with V3 loop inserted reveal a trend toward smaller syncytia (as determined by number of nuclei pr syncytia). That is fewer cells have the ability to undergo membrane fusion as mediated by envelope receptor interaction.

Sequence information of vector sequence used:

EgfpHIVMo (derived from a mouse Virus (MLV) containing elements from the virus for gene transcription, together with an egfp marker gene - an IRES element that is needed for bicistronic RNA translation - and the HIV-1 envelope:- tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtca cagcttgtctgtaagcggat gccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctgg cttaactatgcggcatca gagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaagg agaaaataccgcatcagg cgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcg ctattacgccagctggcgaa agggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacg ttgtaaaacgacggccagt gaattctaccttacgtttccccgaccagagctgatgttctcagaaaaacaagaacaagga agtacagagaggctggaa agtaccgggactagggccaaacaggatatctgtggtcaagcactagggccccggcccagg gccaagaacagatggt ccccagaaacagagaggctggaaagtaccgggactagggccaaacaggatatctgtggtc aagcactagggccccg gcccagggccaagaacagatggtccccagaaatagctaaaacaacaacagtttcaagaga cccagaaactgtctca aggttccccagatgaccggggatcaaccccaagcctcatttaaactaaccaatcagctcg cttctcgcttctgtacccgcg

cttattgctgcccagctctataaaaagggtaagaaccccacactcggcgcgccagtc ctccgatagactgagtcgcccg ggtacccgtgtatccaataaagccttttgctgttgcatccgaatcgtggtctcgctgatc cttgggagggtctcctcagagtga ttgactgcccagcctgggggtctttcatttgggggctcgtccgggatttggagacccccg cccagggaccaccgacccac cgtcgggaggtaagctggccagcgatcgttttgtctccgtctctgtctttgtgcgtgtgt gtgtgtgtgccggcatctactttttgc gcctgcgtctgattctgtactagttagctaactagatctgtatctggcggctccgtggaa gaactgacgagttcgtattcccga ccgcagccctgggagacgtctcagaggcatcgggggggggatccagagctcgagatggtg agcaagggcgaggag ctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaag ttcagcgtgtccggcgag ggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaag ctgcccgtgccctggccc accctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatg aagcagcacgacttcttca agtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggca actacaagacccgcgcc gaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttc aaggaggacggcaaca tcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgaca agcagaagaacggcatc aaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccac taccagcagaacaccc ccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccc tgagcaaagaccccaac gagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggc atggacgagctgtacaagt aaagcggccgtacgcgttgatcagttaacgaattcgaagggtcccaggcctcggagatct gggcccatgcggccgccc cctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgtta ttttccaccatattgccgtcttttgg caatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttc ccctctcgccaaaggaatgca aggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaac gtctgtagcgaccctttgcag gcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataaga tacacctgcaaaggcg gcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcc tcaagcgtattcaacaaggg gctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcac atgctttacatgtgtttagtcg aggttaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacac gataataccatgagagca gaagacagtggcaatgagagtgaaggagaaatatcagcacttgtggagatgggggtggag atggggcaccatgctcc ttgggatgttgatgatctgtagtgctacagaaaaattgtgggtcacagtctattatgggg tacctgtgtggaaggaagcaac caccactctattttgtgcatcagatgctaaagcatatgatacagaggtacataatgtttg ggccacacatgcctgtgtaccca cagaccccaacccacaagaagtagtattggtaaatgtgacagaaaattttaacatgtgga aaaatgacatggtagaac agatgcatgaggatataatcagtttatgggatcaaagcctaaagccatgtgtaaaattaa ccccactctgtgttagtttaaa gtgcactgatttgaagaatgatactaataccaatagtagtagcgggagaatgataatgga gaaaggagagataaaaaa ctgctctttcaatatcagcacaagcataagaggtaaggtgcagaaagaatatgcattttt ttataaacttgatataataccaa tagataatgatactaccagctataagttgacaagttgtaacacctcagtcattacacagg cctgtccaaaggtatcctttga gccaattcccatacattattgtgccccggctggttttgcgattctaaaatgtaataataa gacgttcaatggaacaggaccat gtacaaatgtcagcacagtacaatgtacacatggaattaggccagtagtatcaactcaac tgctgttaaatggcagtctag cagaagaagaggtagtaattagatctgtcaatttcacggacaatgctaaaaccataatag tacagctgaacacatctgta gaaattaattgtacaagacccaacaacaatacaagaaaaagaatccgtatccagagagga ccagggagagcatttgt

tacaataggaaaaataggaaatatgagacaagcacattgtaacattagtagagcaaa atggaataacactttaaaaca gatagctagcaaattaagagaacaatttggaaataataaaacaataatctttaagcaatc ctcaggaggggacccaga aattgtaacgcacagttttaattgtggaggggaatttttctactgtaattcaacacaact gtttaatagtacttggtttaatagtac ttggagtactgaagggtcaaataacactgaaggaagtgacacaatcaccctcccatgcag aataaaacaaattataaa catgtggcagaaagtaggaaaagcaatgtatgcccctcccatcagtggacaaattagatg ttcatcaaatattacagggc tgctattaacaagagatggtggtaatagcaacaatgagtccgagatcttcagacctggag gaggagatatgagggaca attggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcac ccaccaaggcaaagagaa gagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgg gagcagcaggaagcactat gggcgcagcctcaatgacgctgacggtacaggccagacaattattgtctggtatagtgca gcagcagaacaatttgctg agggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctc caggcaagaatcctggctg tggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactca tttgcaccactgctgtgccttg gaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatgga gtgggacagagaaattaac aattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaat gaacaagaattattggaat tagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataa aattattcataatgatagtaggag gcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagg gatattaaccattatcgttcttaagac aatagaagattgtaaatcacgtgaataaaagattttattcagtttacagaaagagggggg aatgaaagaccccttcataa ggcttagccagctaactgcagtaacgccattttgcaaggcatgggaaaataccagagctg atgttctcagaaaaacaag aacaaggaagtacagagaggctggaaagtaccgggactagggccaaacaggatatctgtg gtcaagcactagggcc ccggcccagggccaagaacagatggtccccagaaacagagaggctggaaagtaccgggac tagggccaaacagg atatctgtggtcaagcactagggccccggcccagggccaagaacagatggtccccagaaa tagctaaaacaacaac agtttcaagagacccagaaactgtctcaaggttccccagatgaccggggatcaaccccaa gcctcatttaaactaacca atcagctcgcttctcgcttctgtacccgcgcttattgctgcccagctctataaaaagggt aagaaccccacactcggcgcg ccagtcctccgatagactgagtcgcccgggtacccgtgtatccaataaagccttttgctg ttgcatccgaatcgtggtctcgc tgatccttgggagggtctcctcctctgtcggtcgacctgcaggcatgcaagcttggcgta atcatggtcatagctgtttcctgt gtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaa agcctggggtgcctaatga gtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctg tcgtgccagctgcattaatgaat cggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcac tgactcgctgcgctcggtcgtt cggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatca ggggataacgcaggaaa gaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggc gtttttccataggctcc gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacag gactataaagatacca ggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgg atacctgtccgcctttctcccttcg ggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgtt cgctccaagctgggctgtgtgca cgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaa cccggtaagacacgacttatc gccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctac agagttcttgaagtggtgg cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagtt accttcggaaaaagagttggt

agctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaag cagcagattacgcgcagaaaaaa aggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaa ctcacgttaagggattttggtca tgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaat caatctaaagtatatatgagtaaa cttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctat ttcgttcatccatagttgcctgactc cccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatg ataccgcgagacccacgct caccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtg gtcctgcaactttatccg cctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaata gtttgcgcaacgttgttgccattg ctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttccc aacgatcaaggcgagttacatga tcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagt aagttggccgcagtgttatcact catggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttc tgtgactggtgagtactcaaccaag tcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggat aataccgcgccacatagcag aactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatctt accgctgttgagatccagttcg atgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttct gggtgagcaaaaacaggaaggc aaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcc tttttcaatattattgaagca tttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaac aaataggggttccgcgcacatttc cccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaa ataggcgtatcacgaggccc tttcgtc

Example 3b:

Fig 10 shows further results of Syncitia assays. The arrows indicate multi-nuclei cell syncytia. NIH cells stably expressing the HIV-1 envelope protein were co-cultured with different HeLa target cells (HeLa expresses CXCR-4 endogenously). Top left HeLa cells lacking the CD4 receptor. Bottom left HeLa cells with CD4. Middle, HeLa CD4 cells transfected with Moloney MLV envelope. Right, HeLa CD4 cells transfected with two variants of chimeric Moloney/V3 envelopes. We note a reduction in syncytia formation in the presence of V3 envelope, in particular in the upper right panel.

Example 4: Generating the envelope protein of SL3-2

The envelope protein of SL3-2 was taken from genomic DNA of NIH 3T3 cells infected with SL3-2 virus. PCR was used to amplify the envelope. The upstream primer was chosen to match a conserved sequence upstream of the splice acceptor site among different MLV strains. The downstream primer was designed according to the known

sequence of SL3-2 LTR (Dai etal., 1990). The amplified PCR fragment was subsequently cloned into the mini- virus to replace the original Akv envelope. The new construct was designated NeoSL3- 2mo. Three clones were chosen for sequencing.

One of the clones contained a frameshift mutation and was not infectious. This SL3-2 envelope shows a 92% homology on nucleotide level with the envelope protein of MCF-247 polytropic MLV. The latter has a wide host range and is able to infect cells from many species (Rein 1982), (Hartley et al., 1977), (Chattopadhyay etal., 1982), whereas the original reports claimed that SL3-2 has the same host range as the ecotropic viruses (Pedersen et al., 1981 ), (Rein et al., 1984).

Cloning of SL3-2 envelope The envelope of SL3-2 was amplified by the following PCR from the genomic DNA of infected NIH 3T3 cells.

PCR conditions: ¹ ] 0, uL 10 x buffer, O.δmM dNTP (0.2mM of each nucleotide), 0.25pM of each primer and 2.625 units of enzyme (Expand High Fidelity PCR System (Roche)).

Using primers: 204820: CTCTCCAAGCTCACTTACAGGCCCTC 205585: TGCGGCCGCGTCGACTGGCTAAGCCTTATGAA95 C : 2 min. , 45 x(95 C : 1 min,60 C : 30 sec.,73 C : 4 min.),73 C : 5 min.

Example 5: sequence alignment of various envelope polypeptides, showing homology Sequence alignments were undertaken for a variety of homologous viral envelope polypeptides, see Figures 5a and 5b.

For figure 5b, all of the sequences except that of SL3-2 were downloaded from NCBI databank at the following World Wide Web address: http://www.ncbi.nlm.nih.gov/entrez/guery .fcgi?CMD=search&DB=nucleotide

Information on the aligned sequences is as follows: 1. SL3-2: (SEQ ID NO:2)

2. MCF 247:

DEFINITION Mink cell focus-forming 247 MuLV env gene, 3' end and LTR. KeIIy ₁ M., Holland, C.A., Lung.M.L, Chattopadhyay,S.K., Lowy,D.R. and Hopkins.N.H.

TITLE Nucleotide sequence of the 3' end of MCF 247 murine leukemia virus JOURNAL J. Virol. 45, 291-298 (1983)

DEFINITION Mink cell-focus forming 247 murine leukemia provirus pol gene, 3 ¹ end and env gene, 5'end. AUTHORS Khan.A.S.

TITLE Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses JOURNAL J. Virol. 50, 864-871 (1984)

3. MCF CI-3:

DEFINITION MCF-MuLV proviral DNA (isolated from in vitro-transformed mink lung epithelial C3H/MCA 5 cells), clone pCI-3.

AUTHORS Mark.G.E. and Rapp.U.R.

TITLE Envelope gene sequence of two in vitro-generated mink cell focus-forming murine leukemia viruses which contain the entire gp70 sequence of the endogenous nonecotropic parent JOURNAL J. Virol. 49, 530-539 (1984) ,

4. ERV-1:

DEFINITION Murine leukemia virus erv1 envelope protein gene, complete cds. AUTHORS Liu,S., Li ₁ H., Barker.C. and Cloyd.M. TITLE Envelope sequences of ecotropic recombinant virus JOURNAL Unpublished

5. Friend MCF:

AUTHORS Adachi,A., Sakai.K., Kitamura,N., Nakanishi.S., Niwa.O., Matsuyama,M. and Ishimoto.A.

TITLE Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA JOURNAL J. Virol. 50 (3), 813-821 (1984)

6. Friend SFV:

AUTHORS Hoatlin.M.E., Gomez-Lucia.E., LiIIy ₁ F., Beckstead.J.H. and Kabat.D. TITLE Direct Submission

JOURNAL Submitted (15-OCT-1997) Biochemistry and Molecular Biology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Way, Portland, OR 97201 , USA

7. Invitro MCF:

AUTHORS Mark.G.E. and Rapp.U.R.

TITLE Envelope gene sequence of two in vitro-generated mink cell focus-forming murine leukemia viruses which contain the entire gp70 sequence of the endogenous nonecotropic parent JOURNAL J. Virol. 49 (2), 530-539 (1984)

8. MCF 1223: TITLE Direct Submission

JOURNAL Submitted (17-AUG-1994) Elisabeth J.A. Sijts, Immunohematology and Blood Bank, University Hospital Leiden, Leiden, Zuid-Holland, 2300 RC, NetherlandsCOMMENT MCF1233 is a type I env recombinant MuLV, composed of an ecotropic backbone with acquired polytropic sequences in the 3' pol-5' env region. The U3 LTR contains multiple mutations, distinguishing this sequence from ecotropic U3 sequences. Boundaries of repeat regions and protein-encoding regions, annotated in the feature table, are based on alignment with the sequence of the ecotropic Akv MuLV (Herr, W., J. Virol. 49, 471-478 (1984)).

9. MLV DBA/2:

AUTHORS Grohmann.U., Puccetti.P., Belladonna, M. L., Fallarino.F., Bianchi.R., Binaglia.L., Sagakuchi.K., Mage.M.G., Appella,E. and Fioretti.M.C. TITLE Multiple point mutations in an endogenous retroviral gene confer high immunogenicity on a drug- treated murine tumor JOURNAL J. Immunol. 154 (9), 4630-4641 (1995)

10. Mo-MCF:

AUTHORS Bosselman,R.A., van Straaten,F., van Beveren,C.P., VermaJ.M. and

Vogt.M.

TITLE Analysis of the env gene of a molecularly cloned and biologically active moloney mink cell focus-forming proviral DNA JOURNAL J. Virol. 44, 19-31 (1982)

11. Ns-6(186) MCF:

AUTHORS Chattopadhyay.S.K., Baroudy.B.M., Holmes.K.L, Fredrickson.T.N., Lander,M.R., Morse,H.C. III. and Hartley, J. W.

TITLE Biologic and molecular genetic characteristics of a unique MCF virus that is highly leukemogenic in ecotropic virus-negative mice JOURNAL Virology 168 (1), 90-100 (1989)

12. Rauscher sfv:

AUTHORS Bestwick,R.K., Boswell.B.A. and Kabat.D.

TITLE Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat JOURNAL J. Virol. 51 (3), 695-705 (1984)

13. Endogenous from 129 GIX+ mice: AUTHORS Levy,D.E., Lemer,R.A. and Wilson.M.C. TITLE Normal expression of polymorphic endogenous retroviral RNA containing segments identical to mink cell focus-forming virus JOURNAL J. Virol. 56 (3), 691-700 (1985)

14. Ampho-MCF: DEFENTION: Murine leukemia virus Mo Ampho MCF recombinant gPrδO envelope polyprotein (env) gene, complete cds.

AUTHORS Vanin,E.F., Kaloss.M., Broscius.C. and Nienhuis.A.W.

TITLE Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis

JOURNAL J. Virol. 68 (7), 4241-4250 (1994)

15. MCF (Ter-Grigorov):

AUTHORS Ter-Grigorov, V.S., KrifuksA, Liubashevsky.E., Nyska.A., Trainin.Z. and Toder.V.

TITLE A new transmissible AIDS-like disease in mice induced by alloimmune stimuli JOURNAL Nat. Med. 3 (1), 37-41 (1997)

16. MCF (Broscius): AUTHORS Purcell.D.F.J., Broscius, CM., Vanin.E.F., Buckler.C.E., Nienhuis.A.W. and Martin, M.A.

TITLE Direct Submission

JOURNAL Submitted (23-SEP-1995) Damian F.J. Purcell, AIDS Cellular Biology Unit, Macfarlane Burnet Centre for Medical Research, Yarra Bend Road, PO Box 254, Fairfield, Victoria 3078, Australia

17. Friend MCF #2:

AUTHORS Koch.W., Zimmermann.WA, Oliff.A. and Friedrich,R.W. TITLE Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences JOURNAL J. Virol. 49, 828-840 (1984)

18. R-XC-:

AUTHORS Vogt,M., Haggblom.C., Swift,S. and Haas.M. TITLE Specific sequences of the env gene determine the host range of two XC- negative viruses of the Rauscher virus complex JOURNAL Virology 154 (2), 420-424 (1986)

19. Xeno R-MCF-1 : AUTHORS Vogt.M., Haggblom.C., Swift.S. and Haas,M.

TITLE Specific sequences of the env gene determine the host range of two XC- negative viruses of the Rauscher virus complex JOURNAL Virology 154 (2), 420-424 (1986)

20. DG-75 Xeno:

DEFINITION DG-75 Murine leukemia virus, complete genome.

AUTHORS Raisch,K.P., Pizzato.M., Sun,H.-Y., Takeuchi.Y., Cashdollar.LW. and

Grossberg,S.E.

TITLE Molecular cloning, complete sequence, and biological characterization of a xenotropic murine leukemia virus constitutively released from the human B- lymphoblastoid cell line, DG-75 JOURNAL Unpublished

21. Xeno NZB-9-1 :

DEFINITION Murine leukemia virus NZB-9-1 xenotropic proviral DNA, pol and env genes, and 5' leader sequences.

AUTHORS O'Neill, R.R., Buckler,C.E., Theodore.T.S., Martin, M.A. and Repaske.R. TITLE Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus JOURNAL J. Virol. 53, 100-106 (1985)

22. Xeno CWM-S-5-X: AUTHORS MasseyAC Coppola,M.A. and Thomas,C.Y.

TITLE Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1 -related xenotropic viruses from CWD mice JOURNAL J. Virol. 64 (11), 5491-5499 (1990)

23. Xeno Bxv-1 related:

AUTHORS Massey.A.C, Coppola, M.A. and Thomas,C.Y.

TITLE Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1 -related xenotropic viruses from CWD mice

JOURNAL J. Virol. 64 (11), 5491-5499 (1990)

24. 4070A:

DEFENITION 4070A Amphotropic Murine leukemia virus envelope gene, complete cds.

AUTHORS Ott.D.E., Friedrich,R. and Rein.A. TITLE Sequence analysis of amphotropic and 10A1 murine leukemia viruses: Close relationship to mink cell focus-inducing viruses

JOURNAL J. Virol. 64, 757-766 (1990)

25. 10A1: AUTHORS Vaillancourt,P. and GrafskyλJ-

TITLE Direct Submission

JOURNAL Submitted (09-JAN-2001 ) Technical Services, Stratagene, 11011 N. Torrey

Pines Rd, La JoIIa, CA 92037, USA

26. Akv:

DEFINITION AKV murine leukemia virus, complete provira) genome. AUTHORS Etzerodt M., Mikkelsen T., Pedersen FS., Kjeldgaard NO., and Jorgensen P.

TITLE The nucleotide sequence of the Akv murine leukemia virus genome. JOURNAL Virology. 1984 Apr 15; 134(1): 196-207.

27. SL3-3:

DEFINITION Murine leukemia virus SL3-3, complete genome. AUTHORS LundAH. and Pedersen.F.S. TITLE The nucleotide sequence of the high-leukemogenic murine retrovirus SL3-3 reveals a patch of mink cell focus forming-like sequences upstream of the ecotropic envelope gene. Brief report JOURNAL Arch. Virol. 144 (11), 2207-2212 (1999)

28. Friend: DEFINITION Friend murine leukemia virus, complete genome.

AUTHORS Masuda.M., Remington, M. P., Hoffman, P. M. and Ruscetti.S.K.

TITLE Molecular characterization of a neuropathogenic and nonerythroleukemigenic variant of Friend murine leukemia virus PVC211

JOURNAL J. Virol. 66, 2798-2806 (1992)

29. Moloney:

AUTHORS Shinnick.T.M., Lerner.R.A. and Sutcliffe.J.G.

TITLE Nucleotide sequence of Moloney murine leukaemia virus

JOURNAL Nature 293 (5833), 543-548 (1981)

AUTHORS MillerAD. and Verma,I.M.

TITLE Two base changes restore infectivity to a noninfectious molecular clone of

Moloney murine leukemia virus (pMLV-1 )

JOURNAL J. Virol. 49 (1), 214-222 (1984)

Examples 6-11 Constructs

The underlined sequence is inserted in between the designated amino acids in SL3-2 envelope. Apelin sequence is double underlined and the linker sequence is underlined with a broken line. Please see below for the insertion sites.

The given primers are used to make an overlap extension fragment (Jespersen et al., 1997) that was ligated into the Ncol og BstEII sites of the SL3-2 expression vector (Bahrami et al., 2004). SL3-2 Apelin @155 GNT QGIYQC .SQGSG ORPRLSHKGPMPF SGGSG CG PCYD

Primer ->

TCC GGT GGCAGT GGA Caq cqq ccc cqc etc tec cat aaq qqa ccc atq cct ttc AGC GGT

GGA TCT GGC TGT GGG CCC TGT TAT GAT TCC TCG GTG GTC

Primer <r

GGG CCG CTG TCC ACT GCC ACC GGA GCA CTG GTA GAT TCC TTG.AGT GTT

TCC TCG CTT AAG GGA

SL3-2 Apelin @165 Between amino acid no 144 (D) and 157 (G) in the FeLV-B amino acid sequence is inserted:

PCYDS SG.G _. S.G. ORPRLSHKGPMPF SGGSG- SSSAQGA

Primer forward TCC GGT GGC AGT GGA Gag egg ccc cgc etc tec cat aag gga ccc atg cct ttc AGC GGT GGA TCT GGC TCC AGT AGC GCC CAG GGT GCC ACA

Primer reverse: GGG CCG CTG TCC ACT GCC ACC GGA GGA ATC ATA ACA GGG GCC

CTG GCC

Transfection and transduction

Transfections were done by calcium phosphate precipitation method, described by Graham and van der Eb (Graham and van der Eb, 1973) leaving the calcium precipitate on the cells for 24 hours. 293T cells were transfected using 8 μg of envelope expression plasmid, 2μg gagpol expression plasmid (Morita et al., 2000) and 1 μg of an EGFP expressing plasmid. EGFP was included as a visual aid to determine the success of transfections. Medium on the transfected cells was renewed every day

until it was used for transduction, usually between 48 and 72 hours after transfection, depending on the number of green fluorescing cells.

The day before transduction, medium was changed from fetal calf serum to newborn calf serum containing medium. Semi-packaging cells (Bahrami et al., 2003) were seeded at concentration of 10 ⁴ cells/cm ² 24 hours before transduction. Supernatant from transfected cells was filtered (0.22 μ). The supernatant with 6 μg/mL polybrene was added to the target cells. 24 hours later, the infected cells were selected using medium described before containing 600μg/mL G418 for 10 days.

Example 6

Sequence identity between gamma retroviruses

The percent sequence identity between the protein sequences of gammaretroviruses when compared with the SL3-2 envelope protein sequence of gammaretroviruses was calculated using the VECTOR NTI computer program:

Name Tropism Percent sequence Percent sequence identity identity in envelope in RBD of envelope protein protein

FeLV-B Pit-1 receptor 60.2 52.3 but from cats

MoMLV Ecotropic 63.5 35.4

MCF247 Polytropic 92.7 92.0

NZB-9-1 Xenotropic 76.4 71.0

4070A Amphotropic 87.6 84.7

Where RBD is the receptor binding domain, corresponding to the receptor binding domain (RCB) of the envelope polypeptide is defined as the region delineated by the first amino acid of SEQ ID NO: 60 and the amino acid preceeding the proline rich region (PPR) corresponding to the amino acid number 114 of SEQ ID NO: 60.

The amino acid sequence of SL3-2 is identical to that of SEQ ID NO: 60.

The amino acid sequence of MCF247, NZB-9-1, MoMLV and 4070A can be obtained from the NCBI databank at the link http://www.ncbι.nlm.nih.qov/entrez/querv.fcαi?CMD=search&a mp;DB=nucleotide

or from the original scientific journals: MCF247 (Kelly et al. 1983; Khan 1984), MoMLV (Shinnick et al. 1981; Miller and Verma 1984), 4070A (Ott et al. 1990), NZB-9-1 (O'Neill et al. 1985).

Information on the definition and references regarding gammaretroviruses is found below.

1. SL3-2:

Sequence presented in this Ph.D. thesis.

2. MCF 247:

DEFINITION Mink cell focus-forming 247 MuLV env gene, 3' end and LTR. KeIIy ₁ M., Holland.CA, Lung.M.L., Chattopadhyay.S.K., Lowy,D.R. and Hopkins.N.H. TITLE Nucleotide sequence of the 3' end of MCF 247 murine leukemia virus JOURNAL J. Virol. 45, 291- 298 (1983)

DEFINITION Mink cell-focus forming 247 murine leukemia provirus pol gene, 3' end and env gene, 5'end. AUTHORS Khan.A.S.

TITLE Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses

JOURNAL J. Virol. 50, 864-871 (1984)

6. MCF CI-3:

DEFINITION MCF-MuLV proviral DNA (isolated from in vitro-transformed mink lung epithelial

C3H/MCA 5 cells), clone pCI-3.

AUTHORS Mark.G.E. and Rapp.U.R.

TITLE Envelope gene sequence of two in vitro-generated mink cell focus-forming murine leukemia viruses which contain the entire gp70 sequence of the endogenous nonecotropic parent

JOURNAL J. Virol. 49, 530-539 (1984)

7. ERV-1 :

DEFINITION Murine leukemia virus eπ/1 envelope protein gene, complete cds.

AUTHORS Liu.S., Li ₁ H., Barker.C. and Cloyd.M. TITLE Envelope sequences of ecotropic recombinant virus

JOURNAL Unpublished

8. Friend MCF:

AUTHORS AdachiA, Sakai.K., Kitamura.N., Nakanishi.S., Niwa.O., Matsuyama.M. and lshimoto,A.

TITLE Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA

JOURNAL J. Virol. 50 (3), 813-821 (1984)

θ.Friend SFV:

AUTHORS Hoatlin.M.E., Gomez-Lucia,E., Lilly,F., Beckstead.J.H. and Kabat.D. TITLE Direct Submission JOURNAL Submitted (15-OCT-1997) Biochemistry and Molecular Biology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Way, Portland, OR 97201, USA

7. Invitro MCF:

AUTHORS Mark.G.E. and Rapp.U.R.

TITLE Envelope gene sequence of two in vitro-generated mink cell focus-forming murine leukemia viruses which contain the entire gp70 sequence of the endogenous nonecotropic parent

JOURNAL J. Virol. 49 (2), 530-539 (1984)

8. MCF 1223:

TITLE Direct Submission

JOURNAL Submitted (17-AUG-1994) Elisabeth J.A. Sijts, lmmunohematology and Blood Bank, University Hospital Leiden, Leiden, Zuid-Holland, 2300 RC, NetherlandsCOMMENT MCF1233 is a type I env recombinant MuLV, composed of an ecotropic backbone with acquired polytropic sequences in the 3' Pols' env region. The U3 LTR contains multiple mutations, distinguishing this sequence from ecotropic U3 sequences. Boundaries of repeat regions and protein-encoding regions, annotated in the feature table, are based on alignment with the sequence of the ecotropic Akv MuLV (Herr, W., J. Virol. 49, 471-478 (1984)).

11. MLV DBA/2:

AUTHORS Grohmann.U., Puccetti.P., Belladonna.M.L., Fallarino.F., Bianchi.R., Binaglia.L., Sagakuchi.K.,

Mage.M.G., Appella.E. and Fioretti.M.C. TITLE Multiple point mutations in an endogenous retroviral gene confer high immunogenicity on a drug-treated murine tumor JOURNAL J. Immunol. 154 (9), 4630-4641 (1995)

12. Mo-MCF:

AUTHORS Bosselman,R.A., van Straaten.F., van Beveren.C.P., Verma.l.M. and Vogt.M. TITLE Analysis of the env gene of a molecularly cloned and biologically active moloney mink cell focus- forming proviral DNA JOURNAL J. Virol. 44, 19-31 (1982)

11. Ns-6(186) MCF:

AUTHORS Chattopadhyay.S.K., Baroudy.B.M., Holmes.K.L., Fredrickson.T.N., Lander.M.R., Morse.H.C. III. and Hartley.J.W.

TITLE Biologic and molecular genetic characteristics of a unique MCF vims that is highly leukemogenic in ecotropic virus-negative mice JOURNAL Virology 168 (1), 90-100 (1989)

12. Rauscher sfv:

AUTHORS Bestwick.R.K., Boswell.B.A. and Kabat.D. TITLE Molecular cloning of biologically active Raυscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat JOURNAL J. Virol. 51 (3), 695-705 (1984)

13. Endogenous from 129 GIX+ mice:

AUTHORS Levy.D.E., Lerner.R.A. and Wilson.M.C.

TITLE Normal expression of polymorphic endogenous retroviral RNA containing segments identical to mink cell focus-forming virus

JOURNAL J. Virol. 56 (3), 691-700 (1985)

30. Ampho-MCF:

DEFENTION: Murine leukemia virus Mo Ampho MCF recombinant gPrδO envelope polyprotein (env) gene, complete cds. AUTHORS Vanin.E.F., Kaloss.M., Broscius,C. and Nienhuis,A-W.

TITLE Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis JOURNAL J. Virol. 68 (7), 4241-4250 (1994)

31. MCF (Ter-Grigorov):

AUTHORS Ter-Grigorov,V.S., Krifuks.O., Liubashevsky.E., NyskaA, Trainin,Z. and Toder.V. TITLE A new transmissible AIDS-like disease in mice induced by alloimmune stimuli JOURNAL Nat. Med. 3 (1), 37-41 (1997)

32. MCF (Broscius):

AUTHORS Purcell.D.F.J., Broscius.C.M., Vanin.E.F., Buckler.C.E., Nienhuis.A.W. and Martin,M.A. TITLE Direct Submission JOURNAL Submitted (23-SEP-1995) Damian F.J. Purcell, AIDS Cellular Biology Unit, Macfarlane Burnet

Centre for Medical Research, Yarra Bend Road, PO Box 254, Fairfield, Victoria 3078, Australia

33. Friend MCF #2:

AUTHORS Koch.W., Zimmermann.WA, OKfF ₁ A. and Friedrich.R.W.

TITLE Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences JOURNAL J. Virol. 49, 828-840 (1984)

34. R-XC-:

AUTHORS Vogt,M., Haggblom.C, Swift,S. and Haas.M.

TITLE Specific sequences of the env gene determine the host range of two XC-negative viruses of the Rauscher virus complex

JOURNAL Virology 154 (2), 420-424 (1986)

35. Xeno R-MCF-1 :

AUTHORS Vogt.M., Haggblom,C, Swift,S. and Haas.M.

TITLE Specific sequences of the env gene determine the host range of two XC-negative viruses of the

Rauscher virus complex

JOURNAL Virology 154 (2), 420-424 (1986)

36. DG-75 Xeno:

DEFINITION DG-75 Murine leukemia virus, complete genome.

AUTHORS Raisch.K.P., Pizzato.M., Sun,H.-Y., Takeuchi.Y., Cashdollar.L.W. and Grossberg.S.E. TITLE Molecular cloning, complete sequence, and biological characterization of a xenotropic murine leukemia virus constitutively released from the human B-lymphoblastoid cell line, DG-75

JOURNAL Unpublished

37. Xeno NZB-9-1 :

DEFINITION Murine leukemia virus NZB-9-1 xenotropic proviral DNA, pol and env genes, and 5 ¹ leader sequences.

AUTHORS O'Neill.R.R., Buckler.C.E., Theodore.T.S., Martin, M.A. and Repaske.R. TITLE Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus

JOURNAL J. Virol. 53, 100-106 (1985)

38. Xeno CWM-S-5-X:

AUTHORS MasseyAC., Coppola,M.A. and Thomas.C.Y.

TITLE Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1 -related xenotropic viruses from CWD mice

JOURNAL J. Virol. 64 (11), 5491-5499 (1990)

39. Xeno Bxv-1 related:

AUTHORS Massey,A-C., Coppola.M.A. and Thomas.C.Y.

TITLE Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1- related xenotropic viruses from CWD mice JOURNAL J. Virol. 64 (11), 5491-5499 (1990)

40. 4070A:

DEFENITION 4070A Amphotropic Murine leukemia virus envelope gene, complete cds. AUTHORS Ott,D.E., Friedrich.R. and Rein.A.

TITLE Sequence analysis of amphotropic and 10A1 murine leukemia viruses: Close relationship to mink cell focus-inducing viruses JOURNAL J. Virol. 64, 757-766 (1990)

41. 10A1 :

AUTHORS Vaillancourt,P. and GrafskyAJ. TITLE Direct Submission

JOURNAL Submitted (09-JAN-2001) Technical Services, Stratagene, 11011 N. Torrey Pines Rd, La JoIIa, CA 92037, USA

42. Akv:

DEFINITION AKV murine leukemia virus, complete proviral genome. AUTHORS Etzerodt M., Mikkelsen T., Pedersen FS., Kjeldgaard NO., and Jorgensen P.

TITLE The nucleotide sequence of the Akv murine leukemia virus genome. JOURNAL Virology. 1984 Apr 15; 134(1): 196-207.

43. SL3-3:

DEFINITION Murine leukemia virus SL3-3, complete genome. AUTHORS Lund,A.H. and Pedersen, F.S.

TITLE The nucleotide sequence of the high-leukemogenic murine retrovirus SL3-3 reveals a patch of mink cell focus forming-like sequences upstream of the ecotropic envelope gene. Brief report JOURNAL Arch. Virol. 144 (11), 2207-2212 (1999)

44. Friend:

DEFINITION Friend murine leukemia virus, complete genome. AUTHORS Masuda.M., Remington, M.P., Hoffman, P.M. and Ruscetti.S.K.

TITLE Molecular characterization of a neuropathogenic and nonerythroleukemigenic variant of Friend murine leukemia virus PVC211 JOURNAL J. Virol. 66, 2798-2806 (1992)

Example 7

Targeting the APJ receptor The ability of redirecting the retroviral fusion machinery to a desired receptor would have wide biotechnological and potentially also nanotechnological applications. However, the regulatory mechanisms that interconnect receptor binding with fusion are poorly understood, which has made intelligent engineering of the envelope protein difficult. Many attempts at redirecting the receptor-specificity have found that incorporation of a ligand into the envelope protein may cause receptor-dependent binding without activation of the fusion machinery. Using the envelope protein of the SL3-2 murine leukemia virus isolate (Pedersen et al., 1981) as a backbone and insertion of the 13 amino acid peptide ligand apelin by structure based design, we achieved efficient membrane fusion in a manner dependent upon APJ, the receptor for apelin (Fan et al., 2003; Kawamata et al., 2001) (Fig. 12) . APJ belongs to the large family of G-protein-coupled receptors (GPCRs) involved in cell signaling with more than 1000 members in man (Puffer et al., 2000). All GPCRs are characterized by having 7 transmembrane helices (7-TM). Some of the receptors respond to peptide ligands such as apelin. All peptide-binding GPCRs have the organization shown in Fig. 13, where the ligand is known to bind to the N-terminal part, located extracellularly.

The results of titer experiments are shown below Titer experiment 1

The results of titer experiment 2 is shown in fig. 12.

The chimeric envelope peptides (exemplified by AP@155 RT and Gl, and AP@165 RT and Gl, respectively) mediate specific entry into D17 dog cells of an SL3-2 envelope virus engineered to harbor its cognate ligand at a critical position (see Fig. 14). The insertion site in AP@155 is suboptimal relative to AP@165. NIH cells are mouse cells

that can be infected by the wt SL3-2 as well as the three mutants. SL3-2GI is a mutant that infects human cells, but not dog cells.

Example 8 Targeting the Tachykinin NK1 receptor

The tachykinin NK1 receptor is well-characterized, and pharmacologically important. This receptor has several natural small-peptide ligands such as substance P (11 amino acids) , neurokinin A and neurokinin B (both 10 amino acids) (Quartara and Maggi, 1997). An expression vector for the human tachykinin NK1 receptor is available and the peptide motifs for its ligands will be engineered into the SL3-2 envelope as done for Apelin. The redirection of receptor specificity will be tested on D17 dog cells with or without the Tachykinin NK1 receptor and possibly other cell lines from various species if needed to reduce the background of receptor-independent infection. The system most optimal for receptor-dependent infection will be used to test the ability of soluble tachykinin NK1 ligands (peptides and synthetic non-peptide ligands) to inhibit infection in a competitive manner, parallel to what we have found for the apelin/APJ model (data not shown).

Example 9 Targeting receptor-dependent cell labelling by hemifusion

In the field of bioimaging, this work may suggest novel means for the detection and imaging of specific cell surface proteins on live cells by the receptor-dependent attachment of labelled viral particles to cell surfaces by membrane hemi-fusion, where only the outer leaflets of the two lipid-bilayer membranes are fused. This is an intermediate in the normal fusion process (see Fig. 15) Hemi-fusion may also provide innovative means for the delivery of cargo to the plasma membrane. Hemi-fused particles are expected to be in a locked state on the membrane as a result of the limited diffusion of integral membrane proteins that span both bi-layers. It is conceivable that the hemi-fused stage will only be reached following a very accurate interaction with the receptor at physiological temperature, which suggests that this way of labelling live cells could be very specific as well as stable. A technology to lock enveloped particles at the hemi-fused stage by the mutation of a critical histidine residue has been described (Zavorotinskaya T, et al. 2004). We will use fluorochrome- labelled virus particles with or without this mutation to study the attachment of virus particles to cells in vitro with respect to the sensitivity of detection, the specificity of

labeling, and the stability of the complex. The receptor-ligand panel to be used in the experiments include SL3-2 wild-type/Xpii receptor; Akv/mCAT receptor; SL3-2 apelin/APJ, and SL3-2 substanceP/Tachykinin NK1). Quantitative and qualitative readout of cell labeling will be done by flow cytometry and fluorescence microscopy, respectively.

Example 10

Targeting HIV coreceptors and obtaining viral interference

Among the murine γ-retroviruses a phenomenon termed receptor interference has been used to classify viruses based on their tropism (Sommerfelt et al. 1990). Upon infection the virus synthesize de novo envelope proteins for the production of new viral particles. Some of these envelope proteins will engage the receptor via an unknown mechanism and shield the receptor (see Fig. 16). This shielding prevents the recurrence of an infective event by an exogenous virus. In cell culture the interference is very effective in that complete block of infection can be observed.

HIV-1 is somewhat different with regard to receptor usage. For HIV-1 entry to occur a two-step binding mechanism is required. First the HIV-1 envelope protein binds the CD4 receptor (primary receptor) (Eckert et al 2001). This event initiates a conformational change that exposes a region termed V3 (Variable loop 3) which is responsible for a second interaction with a co-receptor (either CCR-5 or CXCR-4) (Huang et al 2005). This co-receptor interaction is absolutely required for infection to occur. In cell culture the same degree of receptor interference is not observed by HIV-1 infection, which may be due to the dual receptor requirement. We wish to take advantage of the ability of the γ-retroviral envelopes to confer superinfection resistance to block entry of HIV into CD4+ cells, a further development of the above mentioned targeting principle. The idea is to use an engineered SL3-2 envelope that contains the V3 region of HIV in place of the Apelin peptide (see Fig. 14) to block the HIV co-receptors CCR-5 and CXCR-4 and thereby preventing HIV infection. We have preliminary data suggesting that expression of the V3 region in the gamma-retroviral envelope can interfere with cell-cell fusion caused by the HIV- envelope (see Fig. 17).

Example 11

Construction of a chimeric SL3-2 envelope comprising a tetra cystein motif at amino acid position 165.

Insertion of a cloning linker at position 165 of SL3-2 envelope (neo SL3-2 link@165 mo):

The sequence TCA GGT GGC TCC GGA GGG TCT GGC TCG was inserted at position 165 in the SL3-2 env gene between sequences: CCC TGT TAT GAT TCC AGT AGC.

The linker sequence has three unique restriction sites :

CCC TGT TAT GAT TCC TCA GGT GGC TCC GGA GGG TCT GGC TCG AGT AGC

BspEI site: T ^λ CCGG ^λ A Xhol site: C ^λ TCGA ^λ G Bsu36l site: CC ^λ TNA ^λ GG

Insertion of any ligand sequence into the Xhol and Bsu36I site results in minimal alteration of the native SL3-2 sequence flanking the insert.

Insertion of any ligand sequence into the BSpEI site result in regeneration of the SGGSG linker on each side of the insert.

Cloning procedure:

Two PCR fragments were made using primers:

Fragment 1 :

TCA GGT GGC TCC GGA GGG TCT GGC TCG AGT AGC GCC CAG GGT GCC ACA CCG And

CGG GTC GGG AGG GGG GTA ACT

Fragment 2:

GCC AGA CCC TCC GGA GCC ACC TGA GGA ATC ATA ACA GGG GCC CTG GCC

And

TGA AAA ACA CGA TAA TAC CAT

And the plasmid NeoSL3-2mo as the template.

The fragments were coupled together in an overlap extension reaction. The resulting fragment was digested with BstEII and Ncol and ligeted into the BstEII, Ncol fragment of the plasmid NeoSL3-2mo.

The resulting envelope gene has the following sequence:

ATGgAAGGTCCAGCGTTCTCAAAACCCCTTAAAGATAAGATTAACCCGTGGGGCCCC CTAATAGTCCTGG GAATCTTAATGAGGGCAAGAGTATCAGTACAACATGACAGCCCTCATCAGGTCTTCAATG TTACTTGGAG AGTTACCAACTTAATGACAGGACAAACAGCTAATGCTACCTCCCTCCTGGGGACAATGAC CGATGCCTTT CCTAAACTGTACTTTGACTTGTGCGATTTAATAGGGGACGACTGGGATGAGACTGGACTC GGGTGTCGCA CTCCCGGGGGAAGAAAAAGGGCAAGAATATTTGACTTCTATGTTTGCCCCGGTCACACTG TGCTAGCAGG GTGTGGAGGGCCGAGAGAGGGCTACTGTGGCAAATGGGGATGTGAGAcCACTGGACAGGC ATACTGGAAG CCATCATCATCATGGGACCTAATTTCCCTTAAGCGAGGAAACACTCCTAAAGGCCAGGGC CCCTGTTATG ATTCC

TCA GGT GGC TCC GGA GGG TCT GGC TCG

AGTAGCGcCCAGGGTGCCACACCGGGGGGTCGATGCAACCCCCTAGTCCTAGAATTC ACTGACGCGGGTa aaagggccagctgggACGCCTCCAAAGCATGGGGACTAAGACTGTACCGATCCACAAGGA CCGACCCGGT GACCCGGTTCTCTTTGACCCGCCAGGTCCTCAATATAGGGCCCCGCGTCCCCATTGGGCC TAATCCCGTG ATCATTGACCAGTTACCCCCCTCCCGACCCGTGCAGATCATGCTCCCCAGGCCTCCTCAG CCTCCTCCAC CAGGCGCAGCCTCTACAGTCCCTGAGACTGCCCCACCTTCCCAACAACCTGGGACGGGAG ACAGGCTGCT AAACCTGGTAAATGGAGCCTACCAAGCTCTCAACCTCACCAGTCCTGACAAAACCCAA GAGTGCTGGTTGTGTCTGGTAGCGGGACCCCCCTACTACGAAGGGGTTGCCGTCCTAGGT ACTTATTCCA ACCATACCTCTGCCCCAGCTAACTGCTCCGTGGCCTCCCAACACAAGCTGACCCTGTCCG AAGTGACCGG ACAGGGACTCTGCGTAGGAGCAGTTCCCAAAACCCATCAGGCCCTGTGTAATACCACCCA GAAGACGAGC AACGGGTCCTACTATCTGGCTGCTCCCGCCGGGACCATTTGGGCTTGCAACACCGGGCTC ACTCCCTGCC TATCTACCACTGTGCTCGACCTCACCACCGATTACTGTGTCCTGGTTGAGCTCTGGCCAA AAGTGACCTA CCACTCCCCTGGTTATGTTTATGGCCAGTTTGAAGAAAAAACCAAATATAAAAGAGAACC CGTCTCACTA ACTCTGGCCCTACTATTAGGAGGACTCACTATGGGCGGAATTGCCGCCGGAGTGGGAACA GGGACTACCG CCCTAGTGGCCACTCAGCAGTTCCAACAACTCCAGGCTGCCATGCAGGATGACCTTAAAG AAGTTGAAAA GTCCATCACTAATCTAGAAAGATCTTTGACCTCCTTGTCCGAAGTAGTGTTACAGAATCG TAGAGGCCTA GATCTACTATTCCTAAAAGAGGGAGGTTTGTGTGCTGCCTTAAAAGAAGAATGCTGTTTC TATGCCGACC

ACACAGGATTGGTACGGGATAGCATGGCCAAACTTAGAGAAAGATTGAGTCAGAGAC AAAAACTCTTTGA ATCCCAACAAGGGTGGTTTGAAGGGCTGTTTAACAAGTCCCCTTGGTTCACCACCCTGAT ATCCACCATC ATGGGTCCCCTGATAATCCTCTTGTTAATTTTACTCTTTGGGCCTTGTATTCTCAATCAC CTGGTCCAGT TTATCAAAGACAGGGTTTCGGTAGTGCAGGCCCTGGTCCTGACTCAACAATATCATCAAC TTAAGACAAT AGAAGATTGTgAATCACGTGAATAA

Construction of the envelope genes containing the tetracystein motifs. Two constructs were made, one (neo SL3-2 tetC6@165mo) contained the SGGSG CCPGCC SGGSG and the other (neo SL3-2 tetC12@165mo) contained the sequence SGGSG HRWCCPGCCKTF SGGSG sequence at position 165 of SL3-2.

neoSL3-2 tetC6@165mo was made by annealing the following primers together and cloning them into the Xhol and Bsu36l site of the neo SL3-2 link@165 mo plasmid:

TCA GGT GGC TCC GGT TGTTGT CCAGGC TGC TGC AGT GGG GGC AGC GGC And TCG AGC CGC TGC CCC CAC TGC AGC AGC CTG GAC AAC AAC CGG AGC CAC C

neoSL3-2 tetC12@165mo was made by annealing the following primers together and cloning them into the Xhol and Bsu36l site of the neo SL3-2 link@165 mo plasmid:

TCA GGT GGC TCC GGT CAT AGA TGG TGT TGT CCA GGC TGC TGC AAG ACG TTC AGT GGG GGC AGC GGC

And

TCG AGC CGC TGC CCC CAC TGAACG TCTTGC AGC AGC CTG GAC AAC ACC ATC TAT GAC CGG AGC CAC C

The constructs have been tested on NIH3T3 murine cells. NeoSL3-2 tetC6@165mo infects these cells with wt efficiency, whereas neoSL3-2 tetC12@165mo has a titer of approximately two orders of magnitude lower.

The envelope genes have the following sequences:

neoSL3-2 tetC6@165mo:

ATGgAAGGTCCAGCGTTCTCAAAACCCCTTAAAGATAAGATTAACCCGTGGGGCCCC CTAATAGTCCTGG GAATCTTAATGAGGGCAAGAGTATCAGTACAACATGACAGCCCTCATCAGGTCTTCAATG TTACTTGGAG AGTTACCAACTTAATGACAGGACAAACAGCTAATGCTACCTCCCTCCTGGGGACAATGAC CGATGCCTTT CCTAAACTGTACTTTGACTTGTGCGATTTAATAGGGGACGACTGGGATGAGACTGGACTC GGGTGTCGCA CTCCCGGGGGAAGAAAAAGGGCAAGAATATTTGACTTCTATGTTTGCCCCGGTCACACTG TGCTAGCAGG GTGTGGAGGGCCGAGAGAGGGCTACTGTGGCAAATGGGGATGTGAGAcCACTGGACAGGC ATACTGGAAG CCATCATCATCATGGGACCTAATTTCCCTTAAGCGAGGAAACACTCCTAAAGGCCAGGGC CCCTGTTATG ATTCCTCAGGTGGCTCCGGTTGTTGTCCAGGCTGCTGCAGTGGGGGCAGCGGCTCGAGTA GCGCCCAGGG TGCCACACCGGGGGGTCGATGCAACCCCCTAGTCCTAGAATTCACTGACGCGGGTaaaag ggccagctgg gACGCCTCCAAAGCATGGGGACTAAGACTGTACCGATCCACAAGGACCGACCCGGTGACC CGGTTCTCTT TGACCCGCCAGGTCCTCAATATAGGGCCCCGCGTCCCCATTGGGCCTAATCCCGTGATCA TTGACCAGTT ACCCCCCTCCCGACCCGTGCAGATCATGCTCCCCAGGCCTCCTCAGCCTCCTCCACCAGG CGCAGCCTCT ACAGTCCCTGAGACTGCCCCACCTTCCCAACAACCTGGGACGGGAGACAGGCTGCTAAAC CTGGTAAATG GAGCCTACCAAGCTCTCAACCTCACCAGTCCTGACAAAACCCAAGAGTGCTGGTTGTGTC TGGTAGCGGG ACCCCCCTACTACGAAGGGGTTGCCGTCCTAGGTACTTATTCCAACCATACCTCTGCCCC AGCTAACTGC

TCCGTGGCCTCCCAΆCACAAGCTGACCCTGTCCGAAGTGACCGGACAGGGACTCTG CGTAGGAGCAGTTC CCAAAACCCATCAGGCCCTGTGTAATACCACCCAGAAGACGAGCAACGGGTCCTACTATC TGGCTGCTCC CGCCGGGACCATTTGGGCTTGCAACACCGGGCTCACTCCCTGCCTATCTACCACTGTGCT CGACCTCACC ACCGATTACTGTGTCCTGGTTGAGCTCTGGCCAAAAGTGACCTACCACTCCCCTGGTTAT GTTTATGGCC AGTTTGAAGAAAAAACCAAATATAAAAGAGAACCCGTCTCACTAACTCTGGCCCTACTAT TAGGAGGACT CACTATGGGCGGAATTGCCGCCGGAGTGGGAACAGGGACTACCGCCCTAGTGGCCACTCA GCAGTTCCAA CAACTCCAGGCTGCCATGCAGGATGACCTTAAAGAAGTTGAAAAGTCCATCACTAATCTA GAAAGATCTT TGACCTCCTTGTCCGAAGTAGTGTTACAGAATCGTAGAGGCCTAGATCTACTATTCCTAA AAGAGGGAGG TTTGTGTGCTGCCTTAAAAGAAGAATGCTGTTTCTATGCCGACCACACAGGATTGGTACG GGATAGCATG GCCAAACTTAGAGAAAGATTGAGTCAGAGACAAAAACTCTTTGAATCCCAACAAGGGTGG TTTGAAGGGC TGTTTAACAAGTCCCCTTGGTTCACCACCCTGATATCCACCATCATGGGTCCCCTGATAA TCCTCTTGTT AATTTTACTCTTTGGGCCTTGTATTCTCAATCACCTGGTCCAGTTTATCAAAGACAGGGT TTCGGTAGTG CAGGCCCTGGTCCTGACTCAACAATATCATCAACTTAAGACAATAGAAGATTGTGAATCA CGTGAATAA

neoSL3-2 tetC12@165mo:

ATGgAAGGTCCAGCGTTCTCAAAACCCCTTAAAGATAAGATTAACCCGTGGGGCC CCCTAATAGTCCTGGGAATCTTAATGAGGGCAAGAGTATCAGTACAACATGACAG CCCTCATCAGGTCTTCAATGTTACTTGGAGAGTTACCAACTTAATGACAGGACAAA CAGCTAATGCTACCTCCCTCCTGGGGACAATGACCGATGCCTTTCCTAAACTGTA CTTTGACTTGTGCGATTTAATAGGGGACGACTGGGATGAGACTGGACTCGGGTGT CGCACTCCCGGGGGAAGAAAAAGGGCAAGAATATTTGACTTCTATGTTTGCCCCG GTCACACTGTGCTAGCAGGGTGTGGAGGGCCGAGAGAGGGCTACTGTGGCAAAT GGGGATGTGAGAcCACTGGACAGGCATACTGGAAGCCATCATCATCATGGGACCT

AATTTCCCTTAAGCGAGGAAACACTCCTAAAGGCCAGGGCCCCTGTTATGATTCC TCAGGTGGCTCCGGTCATAGATGGTGTTGTCCAGGCTGCTGCAAGACGTTCAGTG GGGGCAGCGGCTCGAGTAGCGcCCAGGGTGCCACACCGGGGGGTCGATGCAAC CCCCTAGTCCTAGAATTCACTGACGCGGGTaaaagggccagctgggACGCCTCCAAAGC ATGGGGACTAAGACTGTACCGATCCACAAGGACCGACCCGGTGACCCGGTTCTC TTTGACCCGCCAGGTCCTCAATATAGGGCCCCGCGTCCCCATTGGGCCTAATCCC GTGATCATTGACCAGTTACCCCCCTCCCGACCCGTGCAGATCATGCTCCCCAGGC CTCCTCAGCCTCCTCCACCAGGCGCAGCCTCTACAGTCCCTGAGACTGCCCCAC CTTCCCAACAACCTGGGACGGGAGACAGGCTGCTAAACCTGGTAAATGGAGCCT ACCAAGCTCTCAACCTCACCAGTCCTGACAAAACCCAAGAGTGCTGGTTGTGTCT GGTAGCGGGACCCCCCTACTACGAAGGGGTTGCCGTCCTAGGTACTTATTCCAAC CATACCTCTGCCCCAGCTAACTGCTCCGTGGCCTCCCAACACAAGCTGACCCTGT CCGAAGTGACCGGACAGGGACTCTGCGTAGGAGCAGTTCCCAAAACCCATCAGG CCCTGTGTAATACCACCCAGAAGACGAGCAACGGGTCCTACTATCTGGCTGCTCC CGCCGGGACCATTTGGGCTTGCAACACCGGGCTCACTCCCTGCCTATCTACCACT GTGCTCGACCTCACCACCGATTACTGTGTCCTGGTTGAGCTCTGGCCAAAAGTGA CCTACCACTCCCCTGGTTATGTTTATGGCCAGTTTGAAGAAAAAACCAAATATAAA AGAGAACCCGTCTCACTAACTCTGGCCCTACTATTAGGAGGACTCACTATGGGCG GAATTGCCGCCGGAGTGGGAACAGGGACTACCGCCCTAGTGGCCACTCAGCAGT TCCAACAACTCCAGGCTGCCATGCAGGATGACCTTAAAGAAGTTGAAAAGTCCAT CACTAATCTAGAAAGATCTTTGACCTCCTTGTCCGAAGTAGTGTTACAGAATCGTA GAGGCCTAGATCTACTATTCCTAAAAGAGGGAGGTTTGTGTGCTGCCTTAAAAGA AGAATGCTGTTTCTATGCCGACCACACAGGATTGGTACGGGATAGCATGGCCAAA CTTAGAGAAAGATTGAGTCAGAGACAAAAACTCTTTGAATCCCAACAAGGGTGGTT TGAAGGGCTGTTTAACAAGTCCCCTTGGTTCACCACCCTGATATCCACCATCATG GGTCCCCTGATAATCCTCTTGTTAATTTTACTCTTTGGGCCTTGTATTCTCAATCAC CTGGTCCAGTTTATCAAAGACAGGGTTTCGGTAGTGCAGGCCCTGGTCCTGACTC AACAATATCATCAACTTAAGACAATAGAAGATTGTgAATCACGTGAATAA