JP2002514388A - Retroviral vectors containing modified envelope escort proteins - Google Patents

Abstract

  (57) [Summary] A retroviral vector having a first retroviral envelope protein and at least one modified retroviral envelope protein, wherein the first envelope protein comprises: (i) a receptor binding region, (ii) a hypervariable polyproline region, and (iii) ) A surface protein comprising a body protein, wherein the modified retroviral envelope protein has (i) a receptor binding region, (ii) a hypervariable polyproline region, and (iii) a body protein (body protein), wherein said modified retrovirus envelope protein has at least 90% of the amino acid residues of the receptor binding region of the surface protein of said modified retrovirus envelope protein removed, and is combined with a non-retrovirus protein or peptide. Wherein said retrovirus is modified to be substituted. Scan vector.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention is directed to "targeted" binding to a desired target molecule.
Retrovirus vector. More specifically, the present invention relates to retroviral vectors having a first envelope protein and at least one modified envelope protein. The first envelope protein includes a receptor binding domain, a hypervariable polyproline domain, and a surface protein including a body protein.
The at least one modified envelope protein is a modified retroviral envelope protein in which at least 90% of the amino acid residues in the receptor binding region of the envelope protein have been removed and replaced with a non-retroviral peptide. The non-retroviral peptide can be a ligand that binds to the desired target molecule. As used herein, the term "target molecule" refers to a molecule to which the ligand can bind. Non-limiting examples of such molecules include cell receptors, extracellular components such as extracellular matrix components, and antibodies.

[0002]Background of the Invention  Retroviral vector particles carry polynucleotides in cells, such as eukaryotic cells.
Is a useful agent for introducing

[0003] That is, retroviral vector particles have been used to introduce polynucleotides into cells for the purpose of gene therapy. One method involves harvesting cells from a patient, introducing the desired polynucleotide into the cells using retroviral vector particles, and returning the modified or treated cells to the patient for therapeutic purposes. Alternatively, the retroviral vector particles are administered to the patient in vivo, thereby transducing the patient's cells with the retroviral vector particles in vivo.

In many gene therapy protocols, it is desirable to target retroviral vector particles to infect a specific cell population in vivo or in vitro. Under these circumstances, the wide host range of typical retroviruses poses a significant problem. The key determinant of the viral host range is the "envelope" involved in binding to receptors on the surface of infected cells
Or "env protein (encoded by env gene)". If it is possible to purify the desired target cells before and after transduction, such purification requires undesirable cell manipulations, and it is difficult to purify suitable target cells, or In situations where the percentage of the target cells in the mixed cell population is low or variable, such purification is problematic. Accordingly, it would be advantageous to be able to use retroviral vector particles that can infect a particular type of mammalian cell.

[0005] Retroviral vectors with modified envelopes have been created. However, such vectors are generally less infectious than wild-type retroviral vectors or retroviral vectors that have a foreign gene but have an unmodified envelope.

Previously, attempts to insert large, complex, bulky polypeptides such as single chain antibodies, polypeptide ligands, or complement regulatory proteins,
This has been hampered by the low degree of expression, incorporation, folding and / or presentation of the chimeric env protein. The present invention relates to the basic framework of the env protein of a virus such as a retrovirus (ie, the N-terminal signal peptide, the N-terminus, the surface (SU) C-terminus, and the transmembrane:
(TM) region), incorporate a large polypeptide, express it, and assemble it (asse
mbly) to provide a “modified env protein”. Such a modification
The env protein lacks much of the receptor binding domain. Hereinafter, such a protein is referred to as “escort protein”. In order for the escort protein to acquire infectivity, coexpression with the wild-type env protein is always required. The escort protein gains function, i.e., target cells specific for the virus, or IgG, exposed collagen or EC.
A targeting motif is provided that directs or guides a target ligand such as M.

[0007] To avoid doubt, the following terms are defined. "Retroviral vector particles" are infectious virions derived from a retrovirus. "Retrovirus vector plasmid vector" refers to retrovirus D
It is a non-infectious plasmid containing NA and can be used as a vector for transfecting target cells. "Retroviral DNA" is DNA transcribed from retroviral RNA by reverse transcriptase
It is.

[0008]Summary of the Invention  Accordingly, it is an object of the present invention to provide "targeting"
 (targeted), while retaining the infectivity of the wild-type retrovirus
To provide a viral vector. That is, the present invention relates to the first envelope protein.
Retroviral vector having white matter and at least one modified envelope protein
To provide The first envelope protein contains a receptor binding region, a hypervariable polypeptide.
It contains a phosphorus protein and a surface protein consisting of a body protein. This
Una envelope protein an unmodified or wild-type envelope protein?
Or a receptor wherein a small peptide or ligand is inserted at the N-terminus
The N-terminus may be modified without removing any portion of the binding region. Less
And one modified envelope protein is a receptor binding region of the envelope protein.
At least a major portion of is removed, such as a ligand that binds to the desired target molecule,
It has been modified to replace a non-retroviral protein or peptide.

[0009]Disclosure of the invention  According to the present invention, as described in detail below, the modified retroviral envelope
Protein is provided. In general, prior to the modification, such modified retro
The viral envelope comprises a receptor binding region, a hypervariable polyproline region, and a body.
Includes surface proteins, including proteins (body proteins). Modified retrovirus envelope
Loop proteins have fewer amino acid residues in the receptor binding region of the envelope protein.
Both 90% are removed and replaced with non-retroviral proteins or peptides.
Has been qualified. Such modified retroviral envelope proteins are generally
Typically included in retroviral vectors. According to one embodiment, the retrovirus
Vectors contain such modified retroviral envelope proteins as well as receptor
Retro with unmodified binding region, hypervariable polyproline region, and body protein
It has a viral envelope protein.

That is, according to another aspect of the present invention, there is provided a retroviral vector having a first retroviral envelope protein and at least one modified retroviral envelope protein. The first envelope protein includes a surface protein. Surface proteins include (i) a receptor binding region, (ii) a hypervariable polyproline region or "hinge" region, and (iii) a body protein. Prior to modification, such modified retroviral envelope proteins comprise (i) a receptor binding region, (ii) a hypervariable polyproline or "hinge" region, and (iii) a body protein (body prot).
ein). The modified retrovirus envelope protein is
At least 90% of the amino acid residues in the receptor binding region of the surface protein of the modified retroviral envelope protein are removed and replaced with a non-retroviral protein or peptide, eg, a ligand that binds to the desired target molecule. Has been qualified.

In one embodiment, at least 92% of the amino acid residues of the receptor binding region of the surface protein of the modified retroviral envelope protein are removed, eg, a non-retroviral protein, such as a ligand, that binds to a desired target molecule. Or it is substituted with a peptide. In another embodiment, all of the amino acid residues in the receptor binding region of the surface protein of the modified retroviral envelope protein have been removed and replaced with a non-retroviral protein or peptide.

In yet another embodiment, at least 90% of the amino acid residues in the receptor binding region of the surface protein of the modified retroviral envelope protein have been removed and replaced with a non-retroviral protein or peptide, further comprising the modified retroviral envelope. At least a portion of the amino acid residues in the hypervariable polyproline region of the surface protein of the envelope protein has been removed and replaced with a non-retroviral protein or peptide. In one embodiment, all of the amino acid residues in the hypervariable polyproline region of the surface protein of the modified retrovirus envelope protein have been removed.

In yet another embodiment, before modification, the receptor binding region of the modified retroviral envelope protein has the sequence (SEQ ID NO: 1). In the modified retroviral envelope protein, amino acid residues 19 to 229 of the sequence (SEQ ID NO: 1) have been removed and replaced with a non-retroviral protein or peptide. In another embodiment, at least a portion of amino acid residues 19 to 229 of the sequence (SEQ ID NO: 1) and at least a portion of the amino acid residues of the hypervariable polyproline region of the surface protein of the modified retroviral envelope protein are removed. Replaced by a retroviral protein or peptide.

In general, a retroviral envelope protein comprises a surface (SU) domain or protein and a transmembrane (TM) domain or protein. In general, a surface protein, in the direction from its N-terminus to its C-terminus, sequentially comprises (i) a receptor binding domain, (ii) a hypervariable polyproline or "hinge" domain, and (iii) a transmembrane ( TM) Contains the body protein that is associated with the domain.

[0015] The first retrovirus envelope protein includes a surface (SU) domain and a transmembrane (TM) domain. Generally, such envelope proteins have no non-viral peptides. The first retrovirus envelope protein retains wild-type infectivity. In one embodiment, the first retroviral envelope protein has regions of different tropism. For example, in one embodiment, the first retroviral envelope protein comprises (i) an ecotropic receptor binding region, (ii) an amphotropic hypervariable polyproline region, and (iii) an ecotropic body protein. , Including surface proteins. "Amphotropic" means that it is capable of infecting both murine (mud) cells and other mammalian cells, including humans. “Allotrophic” means that it can infect only mouse (germ) cells.

As previously described, a modified retroviral envelope protein is a modified retroviral envelope protein that has been modified such that at least 90% of the amino acid residues in the receptor binding region of the surface protein have been removed and replaced with a non-retroviral protein or peptide. Retroviral envelope protein. The sequence (SEQ ID NO: 1) is the receptor binding region of the allotrope envelope of Moloney murine leukemia virus. The present inventors have maintained the wild-type infectivity, a first retrovirus envelope protein comprising a receptor binding region, an unmodified hypervariable polyproline region, and an unmodified body protein, and a surface protein. By constructing a retroviral vector comprising a modified retroviral envelope protein in which at least 90% of the amino acid residues of the receptor binding region have been removed and replaced with a non-retroviral protein or peptide, the modified retroviral envelope protein is For example, it has been found that one or more functions, such as targeting the retrovirus vector to a desired target cell, function as an “escort protein” that adds to the retrovirus vector. While such a retrovirus vector has the additional function, it retains the sensitivity of the retrovirus wild-type.

In one embodiment, the modified retroviral envelope protein may be an envelope having different directional regions, at least prior to modification of the receptor binding region, such as to include a non-retroviral protein or peptide. For example, the modified retrovirus envelope protein is a Moloney murine leukemia virus envelope protein comprising a surface protein (gp70 protein) having an ecotropic portion and an amphotropic portion and / or an xenotropic portion.

[0018] In another embodiment, the modified retroviral envelope protein comprises, prior to its modification, (i) an ecotropic receptor binding region, ie, a sequence (SEQ ID NO: 1), A hypervariable polyproline region, ie, the sequence (SEQ ID NO: 2); and (iii)
It has a gp70 protein containing an allotrophic body protein. At least 90% of the amino acid residues in the allotropic receptor binding region (SEQ ID NO: 1) have been removed and replaced with a non-retroviral protein or peptide. In yet another embodiment, at least a portion of the amphotropic hypervariable polyproline region (SEQ ID NO: 2) has also been removed. In one embodiment, amino acid residues 1-35 of the sequence (SEQ ID NO: 2) have been removed. In another embodiment, amino acid residues 1-48 of the sequence (SEQ ID NO: 2) have been removed. In a more specific example, all 60 amino acid residues of the sequence (SEQ ID NO: 2) have been removed.

In a preferred embodiment, the retroviral vector particle comprises a first retroviral envelope protein and a modified retroviral envelope protein. The first retrovirus envelope protein includes a receptor binding region, a hypervariable polyproline region, and a body protein, as described above. In the modified retroviral envelope protein, as described above, the non-retroviral protein or peptide is a ligand that binds to a desired target cell.

In one embodiment, the ligand comprises a binding region that binds to a receptor located on the desired cell type. Non-limiting examples of such ligands include antibodies such as single chain antibodies, monoclonal antibodies, and polyclonal antibodies and fragments thereof. Such antibodies include, for example, antibodies that bind to erb-B2, such as the e23 antibody, and fragments or portions thereof, such as antibodies that bind to a receptor such as the CD4 receptor on T cells, transferrin receptors. Binding antibody, human leukocyte antigen (HLA
), Antibodies that bind to carcinoembryonic antigens, antibodies to placental alkaline phosphatase found in testis and ovarian cancer cells, antibodies to high molecular weight melanoma-associated antigens, polymorphic epithelium found in ovarian cancer cells Antibody to mucin, antibody to β-human chorionic gonadotropin, antibody to CD20 antigen of B lymphoma cells, antibody to α-phytoprotein, antibody to prostate specific antigen, CD3
CKT-3 antibody that binds to T lymphocyte surface antigen, antibody that binds to B lymphocyte surface antigen, EGFR (c that is found in glioma cells, B lymphoma cells and breast cancer cells)
-Erb-B1 or c-erb-B2) -binding antibody, interleukin-2
Receptor binding anti-tac monoclonal antibody, anti-transferrin monoclonal antibody, monoclonal antibody to gp95 / gp97 found in melanoma cells, monoclonal antibody to p-glycoprotein, cluster-1 antigen (N-CAM) found in small cell lung cancer , Cluster-w4, cluster-5A, or cluster-6 (LeY), monoclonal antibody to fetal alkaline phosphatase, CA found in lung and ovarian cancer cells
-125, a monoclonal antibody against epithelial specific antigen (ESA) found in lung and ovarian cancer cells, C found in B lymphoma cells
Monoclonal antibodies against D19, CD22 and CD37, monoclonal antibody against 250 kDa proteoglycan found in melanoma cells, monoclonal antibody against p55 protein found in breast cancer cells, monoclonal antibody against TCR-IgH fusion protein found in childhood T-cell leukemia cells, T cells Antibodies to receptors, antibodies to cancer-specific antigens on B lymphoma cells, antibodies to organ cell surface markers, anti-HIV antibodies such as anti-HIV gp120-specific immunoglobulins, and anti-red blood cell antibodies are included.

Other ligands include cytokines. Such cytokines include, for example, interleukin-1α, interleukin-1β,
And interleukins from interleukin-2 to interleukin-14, epidermal growth factor (EGF), TGF-α, TGF-β, fibroblast growth factor (
FGF), keratinocyte growth factor (KGF), PDGF-A, PDGF-B,
Growth factors, such as PD-ECGF, IGF-I, IGF-II, and nerve growth factor (NGF), which bind to the NGF receptor on nerve cells, stimulators such as GM-CSF, G-CSF and M-CSF Interferon such as leukemia inhibitory factor (LIF), interferon-α, interferon-β, and interferon-γ, inhibin A, inhibin B, chemotactic factor, α-type intercrine cytokine, and β-type intercline cytokine. is there.

Further, for example, the following ligands can be used in the present invention. Vascular endothelial growth factor (VEGF), melanoma stimulating hormone that binds to MSH receptor on melanoma cells, integrin V found on human histiocytic lymphoma cells
Sequence binding to LA3, VLA4 and VLA5: Cys-Gln-Ala-Gly-Thr-Phe-Ala-
Polypeptide F having Leu-Arg-Gly-Asp-Asn-Pro-Gln-Gly-Cys (SEQ.ID NO.5)
LA16, are sequences that bind to the integrin alpha _v beta ₃ found on melanoma cells: Gly-Glu-Arg-Gly -Asp-Gly-Ser-Phe-Phe-Ala-Phe-Arg-Ser-Pro-Phe ( SE
Q.IDNO.6) polypeptide structure, erythropoietin binding to erythropoietin receptor, adherins, selectins, CD34 binding to CD34 receptor on hematopoietic stem cells, promyeloblast leukemia cells CD33, stem cell factor, asialoglycoproteins, such as asialoorosomucoid, asialofetin and α-1 acid glycoprotein, which bind to asialoglycoprotein receptor of hepatocytes, insulin, glucagon, on hematopoietic stem cells Receptor binding gastrin polypeptides, C-kit ligands, tumor necrosis factors (TNFs) such as TNF-α and TNF-β, ApoB binding to LDL receptor on hepatocytes, LRP receptor on hepatocytes Binds to α-2-macroglobulin, which binds to the mannose receptor of macrophages Mannose-containing peptides, ELAM of activated endothelial cells
Sialyl-Lewis-X-antigen-containing peptide that binds to the -1 receptor, CD40 ligand that binds to the CD40 receptor on B lymphocytes, LFA-1 (CD11
b / CD18) receptor or Mac-1 (CD11a / CD1) on macrophages
8) ICAM-1, binding to receptor, c-fms of spleen and bone marrow macrophages
M-CSF binding to the receptor, VLA-4 binding to the VCAM-1 receptor of activated endothelial cells, LFA-1 binding to the ICAM-1 receptor of activated endothelial cells, CD4 receptor of T helper cells HIV gp120 and class II MHC antigens that bind to the LDL receptor binding region of the apolipoprotein E (ApoE) molecule.
However, it should be understood that the scope of the present invention is not limited to any particular ligand.

[0023] In one embodiment, the ligand is a single chain antibody. In another embodiment, the ligand comprises a binding domain that binds to an extracellular matrix component. As used herein, "extracellular matrix component" refers to a molecule that occupies the extracellular space of a tissue. Such extracellular matrix components include, for example, collagen (including collagen types I and IV), laminin, fibronectin, elastin, glycosaminoglycans, proteoglycans, and arginine-glycine-aspartic acid (
There are sequences that bind to fibronectin, such as the RGD) sequence. Binding regions that bind to extracellular matrix components and may be included in the target polypeptide include, for example, polypeptide domains that are functional domains in Willebrand factor or derivatives thereof, such polypeptides Domains bind to collagen. In one embodiment, the binding region has the structural formula: Trp-Arg-Glu-Pro-Ser-Phe-Met-
It is a polypeptide having Ala-Leu-Ser (SEQ. ID NO. 7).

Other binding regions that bind to extracellular matrix components and can be included in the second retrovirus envelope include, for example, an arginine-glycine-aspartate (RGD) sequence that binds fibronectin, and binds fibronectin There is a polypeptide having the structural formula: Gly-Gly-Trp-Ser-His-Trp (SEQ. ID NO. 8).

In addition to the binding region, the ligand further has a linker sequence consisting of one or more amino acid residues located at the N-terminus or C-terminus of the binding region; Is increased and / or steric hindrance is minimized.

In another embodiment, the ligand is a peptide or protein that binds to the antibody. Such peptides or proteins include, for example, the IgG binding domain of protein A,
There are synthetic IgG binding domains such as protein ZZ and protein G. However, it should be understood that the scope of the present invention is not limited to any particular ligand, binding region, or target molecule to which the ligand binds.

According to another aspect of the present invention, a modified retroviral envelope polypeptide (
That is, a modified polynucleotide encoding the modified retrovirus envelope or escort protein) is provided. Retroviral envelope polypeptides include a receptor binding region. In the modified polynucleotide, a polynucleotide encoding at least 90% of the amino acid residues in the receptor binding region is removed;
For example, as described above, it has been replaced with a polynucleotide encoding a non-retroviral protein or peptide, such as a ligand that binds to the desired target molecule.

In one embodiment, prior to modification, the polynucleotide encoding the receptor binding region encodes the sequence (SEQ ID NO: 1). In the modified polynucleotide, the sequence (
The polynucleotide containing the codon encoding amino acid residues 19 to 229 of SEQ ID NO: 1) has been removed and replaced with the polynucleotide encoding the ligand. In another embodiment, the polynucleotide encoding at least a portion of the hypervariable polyproline region is similarly removed. In one embodiment, the hypervariable polyproline region has the sequence (SEQ ID NO: 2). The receptor binding region having the sequence (SEQ ID NO: 1) is encoded by a polynucleotide having the sequence (SEQ ID NO: 3) or a degenerate derivative or analog thereof. The hypervariable polyproline region having the sequence (SEQ ID NO: 2) is encoded by a polynucleotide having the sequence (SEQ ID NO: 4) or a degenerate derivative or analog thereof.

As used herein, the term “derivative or analog” refers to the polypeptide (S
The polynucleotides encoding EQ ID NO: 1) and (SEQ ID NO: 2) may have different sequences from the polynucleotides (SEQ ID NO: 3) and (SEQ ID NO: 4), It still means that they encode the same polypeptide. Such differences in polynucleotide sequence are, for example, due to the degeneracy of the genetic code. Depending on the polynucleotide encoding the ligand (SEQ ID NO: 2) or (SEQ ID NO: 2)
Prior to modifying 4), (SEQ ID NO: 2) or (SEQ ID NO: 4) has been modified such that one or more codons encodes an amino acid residue different from the unmodified sequence Cases are also within the scope of the invention. Such modifications may facilitate insertion of a polynucleotide encoding the ligand.

The above polynucleotide can be constructed by genetic engineering techniques known to those skilled in the art. For example, a first expression plasmid containing a polynucleotide encoding the unmodified envelope protein can be constructed. The plasmid may be a polynucleotide encoding at least 90% of the amino acid residues of the receptor binding region,
In some embodiments, the polynucleotide encoding at least a portion of the hypervariable polyproline region is further modified such that the polynucleotide is removed and replaced with a polynucleotide encoding a ligand. The polynucleotide encoding the ligand may be contained within the second expression plasmid or may be present as a naked polynucleotide sequence. The polynucleotide encoding the ligand, or a plasmid containing such a polynucleotide, is cut at an appropriate restriction site and cloned into a first expression plasmid that has also been cut at the appropriate restriction site. The resulting expression plasmid contains a polynucleotide containing the modified retroviral envelope protein. Such a plasmid also contains a polynucleotide encoding the minimal signal peptide of the retroviral envelope protein. "Minimal signal peptide" means the signal peptide and cleavage site.

As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including ribonucleotides and deoxyribonucleotides. The term further refers to single- and double-stranded DNA, and single- and double-stranded RNs.
A. The term also includes modified polynucleotides such as methylated or capped polynucleotides.

In a preferred embodiment, the retroviral vector particles having the first envelope protein and the modified envelope protein of the present invention are heterologous (expressed in a desired cell).
(Foreign) polypeptides. In one embodiment, the heterologous polypeptide is a therapeutic agent. The term "therapeutic agent" is used in a general sense and includes therapeutic agents, prophylactic agents, and replacement agents. However, it is to be understood that the scope of the present invention is not limited to any particular therapeutic agent.

The polynucleotide encoding the therapeutic agent is under the control of a suitable promoter. However, it should be understood that the scope of the present invention is not limited to any particular exogenous gene or promoter. A polynucleotide encoding a therapeutic agent can be incorporated into a suitable retroviral plasmid vector by genetic engineering techniques known to those skilled in the art.

In one embodiment, the retroviral plasmid vector is derived from Moloney murine leukemia virus and is derived from Bender, et al., J. Virol. , Vol . 61, pgs. 1639-1649 (1987).
And the LN series of vectors described in detail in Miller, et al., Biotechniques , Vol. 7, pgs. 980-990 (1989). Such vectors have a packaging signal portion from mouse sarcoma virus and a mutated gag start codon. As used herein, the term "mutation" means that the gag initiation codon has been deleted or altered so that the gag protein or a fragment or fragment thereof is not expressed.

In another embodiment, the retroviral plasmid vector comprises at least four cloning or restriction enzyme sites, wherein at least two of the sites have an average frequency of occurrence in eukaryotic genes of less than once per 10,000 base pairs. That is, the restriction enzyme product has an average DNA size of at least 10,000 base pairs. Suitable cloning sites are selected from the group consisting of NotI, SnaBI, SalI, and Xhol. In a preferred embodiment, the retroviral plasmid vector contains each of these cloning sites. Such vectors are described in detail in U.S. Patent No. 5,672,510, the contents of which are incorporated herein.

When a retroviral plasmid vector containing such a cloning site is used, NotI, Sn located on the retroviral plasmid vector
A shuttle cloning vector containing at least two cloning sites compatible with at least two cloning sites selected from the group consisting of aBI, SalI, and XhoI can also be used. The shuttle cloning vector further includes at least one desired polynucleotide encoding a therapeutic agent capable of being transferred from the shuttle cloning vector to the retroviral plasmid vector.

The shuttle cloning vector can be constructed by ligating one or more linkers containing cloning or restriction sites to an underlying “backbone” vector or fragment. The cloning site may include a compatible or complementary cloning site as described above. A gene and / or a promoter having an end corresponding to a restriction enzyme site of the shuttle cloning vector can be ligated to the shuttle cloning vector by a method known to those skilled in the art.

[0038] The shuttle cloning vector can also be used to amplify DNA sequences in prokaryotic cell lines. The shuttle cloning vector is prepared from a plasmid commonly used in prokaryotic cell lines, especially bacteria. That is, for example, it is derived from a plasmid such as pBR322 and pUC18.

The retroviral plasmid vector contains one or more promoters for the genes contained in the vector. Suitable promoters include, for example, retroviral LTR, SV40 promoter, and Miller, et al.
l., Biotechniques , Vol. 7, No. 9, 980-990 (1989), or the human cytomegalovirus (CMV) promoter, or any other promoter (
For example, cell promoters such as eukaryotic cell promoters such as histone, pol III, and β-actin promoters. Other viral promoters that can be used include, for example, the adenovirus promoter, the TK promoter, and the B19 parvovirus promoter. Suitable promoters can be readily selected by one of skill in the art based on the techniques described herein contained herein.

In one embodiment, the polynucleotide encoding the modified retroviral envelope protein is contained in another expression vehicle, such as an expression plasmid. Alternatively, a polynucleotide encoding the modified retroviral envelope protein is contained in a retroviral plasmid vector for transducing and expressing the modified retroviral envelope protein in a producer cell line.

In one embodiment, a retroviral plasmid vector containing a polynucleotide encoding a therapeutic agent, and an expression vehicle containing a polynucleotide encoding a modified retroviral envelope protein are prepared using gag, pol, and wild-type (unmodified). 3.) Transducing the packaging cell line containing the nucleic acid sequence encoding the env retroviral protein. An example of such a packaging cell line is PE5
01, PA317 (ATCC No. CRL9078), Ψ-2, Ψ-AM, PA-12, T19
-14X, VT-19-17-H2, $ CRE, $ CRIP, GP + E-86,
GP-envAm12 and Miller, Human Gene Therapy , Vol. 1, pgs. 5-14 (1
990). Vectors can transduce the packaging cell line by any method known in the art. Such methods include, for example, electroporation, use of liposomes, CaPO4 precipitation, and the like. Such a producer cell line produces an infectious retroviral vector particle comprising a first unmodified wild-type retroviral envelope protein, a modified retroviral envelope protein, and a polynucleotide encoding a therapeutic agent.

In another embodiment, a polynucleotide encoding a gag and pol protein, a polynucleotide encoding a first retroviral envelope protein that does not contain a non-retroviral peptide (eg, a wild-type retroviral envelope protein), and a modified retrovirus A packaging cell containing a polynucleotide encoding a viral envelope protein is provided. Producer cells that produce the retroviral vector particles containing the first and modified retroviral envelope proteins of the present invention may comprise, in such packaging cells, retroviral vector particles or retroviral plasmids containing a polynucleotide encoding a therapeutic agent. It can be prepared by introducing a vector. Producer cells prepared in this way produce infectious retroviral vector particles comprising a first retroviral envelope protein, a modified retroviral envelope protein, and a polynucleotide encoding a therapeutic agent.

An infectious retroviral vector particle comprising a first retroviral envelope protein, a modified retroviral envelope protein, and a polynucleotide encoding a therapeutic agent is administered to a host to express the therapeutic agent in the host. In one embodiment, an infectious retroviral vector particle is administered to a host in an amount effective to produce a therapeutic effect in the host. The host can be a mammal, such as a human or a non-human primate. In a preferred embodiment, the retroviral vector particles are administered to a host to target the desired cells in vivo. After being administered to the host,
The retroviral vector particles are delivered to and transduced to the desired target cells, and the transduced cells express the therapeutic agent in vivo. When the modified retroviral envelope protein contains a ligand that binds to the antibody, the retroviral vector particle binds to the antibody via the ligand after administration to the host. Thereafter, the retroviral vector particles and the bound antibody are carried to a target cell having a receptor that binds to the antibody and transduced into the cell. The exact dosage of the retroviral vector particles is determined by various factors, including, for example, age, gender, patient weight, target cells to be transduced, therapeutic agent to be administered, and the extent of the disease being treated. .

The retroviral vector particles may be, for example, intravenous, intraperitoneal, colon, trachea, intranasal, intravascular, intrathecal, intraarterial, intracranial, intramedullary, intravesical, intrapleural, intradermal. It can be administered systemically by subcutaneous, intramuscular, intraocular, intraosseous and intrasynovial administration. The retroviral vector particles can also be administered locally.

The cells transduced by the retroviral vector particles of the present invention include, for example, primary nucleated blood cells, primary primary cancer cells, endothelial cells, epithelial cells, vascular cells,
Primary cells such as keratinocytes, stem cells, hepatocytes, chondrocytes, connective tissue cells, fibroblasts and fibrous elastic cells of connective tissue, mesenchymal cells, mesothelial cells, and parenchymal cells, vascular smooth muscle Cells, hematopoietic stem cells, T lymphocytes, B lymphocytes, neutrophils, macrophages, platelets, erythrocytes, repairable mononuclear granule infiltrates of inflammatory tissues, nerve cells, brain cells, muscle cells, bone cells and bone in bone Mention may be made of blasts, lung cells, pancreatic cells, epithelial and subepithelial cells of the gastrointestinal and respiratory tracts, as well as malignant and benign tumor cells. The particular cell to be transduced is determined by the disease or disorder to be treated and the ligand contained in the second retroviral envelope protein. The scope of the present invention is:
It should be understood that it is not limited to any particular target cell.

The diseases or diseases treated by the retroviral vector particles of the present invention include, for example, severe combined immunodeficiency caused by adenosine deaminase deficiency, sickle cell anemia, thalassemia, hemophilia A and B, obesity Pulmonary emphysema caused by α-1-antitrypsin deficiency, Alzheimer's disease, AIDS,
Chronic granulomatosis, Gauche cell disease, Rensch-Nyhan syndrome, muscular dystrophy including Duchenne muscular dystrophy, parkinsonism, cystic fibrosis, phenylketonuria, hypercholesterolemia, and, for example, cholesterol metabolism disorders and immune system defects Growth disorders and heart diseases caused by the above, and other cardiovascular diseases and the like.

When the modified retroviral envelope protein of the retroviral vector particle of the present invention contains a ligand that binds to an extracellular matrix component, a disease associated with the extracellular matrix component exposing such a retroviral vector particle or Can be used to treat disease. Such diseases or conditions include, for example, cardiovascular disease, cirrhosis, connective tissue disorders (including those associated with ligaments, tendons, cartilage) and vascular diseases associated with collagen exposure. The retroviral vector particles, in addition to suppressing the proliferative and fibrotic responses associated with neointima formation, restore endothelial cell function and carry therapeutic genes to combat thrombosis Used for It can also be used in the treatment of vascular injuries, ulcer injuries, areas of inflammation, such as laser injured sites such as the eye, surgical sites, arthritic joints, scars and keloids. The retroviral vector particles can also be used for wound healing.

Further, the retroviral vector particles of the present invention, wherein the modified retroviral envelope protein comprises a ligand that binds to an extracellular matrix component, can be used to treat tumors, including benign and malignant tumors. Without wishing to be bound by any theory, it is believed that when a tumor enters a normal tissue or organ, it secretes enzymes such as collagenases or metalloproteinases that expose extracellular matrix components. By targeting the retroviral vector particles to such exposed extracellular matrix components, the retroviral vector particles are enriched in extracellular matrix components near the tumor, and as a result, the retroviral vector Particles can become infected. Such tumors include, for example, sarcomas such as cancer, chondrosarcoma, osteosarcoma, and fibrosarcoma, and brain tumors. For example, a modified retroviral envelope protein that includes a ligand that binds to an extracellular matrix component located at a tumor site as described above, and, for example, a negative selectable marker or a “herpes simplex virus thymidine kinase (TK) gene, such as a gene. A retroviral vector particle containing a polynucleotide encoding the "suicide" gene is administered to the patient and the tumor is transduced with the retroviral vector particle. After being transduced, a reactive agent or prodrug such as ganciclovir or acyclovir is administered to the patient to kill the transduced tumor cells. It is to be understood that the scope of the present invention is not limited to any particular disease or condition.

[0049] Retroviral vector particles containing a first retroviral envelope protein, a modified retroviral envelope protein, and a polynucleotide encoding a therapeutic agent can be used as part of an animal model for studying the effects of gene therapy treatments. It can also be administered to animals in vivo. By providing different doses of the retroviral vector particles to different animals of the same species, the target cells of the animals can be transduced. The animal is then evaluated for in vivo expression of the desired therapeutic agent in the animal. From the data obtained by such evaluation, the amount of retroviral vector particles to be administered to a human patient can be determined.

The retroviral vector particles of the present invention can also be used for in vitro transduction of desired target cells contained in cultured cells containing a cell mixture. After transduction of the target cells in vitro, the target cells produce the therapeutic agent or protein in vitro. The therapeutic agent or protein can be obtained from cell culture by methods known to those skilled in the art.

The retroviral vector particles of the present invention can also be used to transduce cells in vitro for the purpose of studying the mechanisms of cell genetic engineering in vitro.

Further, “escort proteins” that form a modified retrovirus envelope protein can be used to form proteoliposomes. That is, the escort protein can form part of the liposome wall. Such proteoliposomes can be used for drug delivery to desired target cells.

In another embodiment, the retroviral vector particle has one or more additional modified retroviral envelope proteins in addition to the first retroviral envelope protein and the modified retroviral envelope protein, Non-retroviral proteins or peptides that replace amino acid residues removed from the unmodified envelope protein provide additional functions to the retroviral vector particle. Such functions include, for example, complement regulation or complement resistance, resistance to humoral or cellular immune responses, and retroviruses such that more target cells are infected by the retroviral vector particles. Stimulation of proliferation of cells infected by vector particles can be mentioned. Such proteins or peptides included in additional retroviral envelope proteins include, for example, CD55
, CD46 and CD59, complement regulatory or complement resistant proteins, TG
Immunosuppressive drugs such as F-β1 and interleukin-10, and
Mention may be made of growth factors and cytokines such as GF, IGF, VEGF and interleukins. Such additional modified retroviral envelope proteins can generally be produced by transducing packaging cells with a polynucleotide encoding such modified envelope proteins, as described above. In this way, retroviral vector particles targeted to specific cells and having additional functions as described above can be constructed.

In one preferred embodiment, the retroviral vector particle of the present invention comprises, in addition to the first retroviral envelope protein, a first modified retroviral envelope protein and a second modified retroviral envelope protein as described above. Have. In the first modified retroviral envelope protein, the non-retroviral protein or peptide is a ligand that binds to a desired target cell, and in the second modified retroviral envelope protein, the non-retroviral protein or peptide is a complement regulatory protein. It is. Such retroviral vector particles are administered to a host, the retroviral vector particles are targeted to desired cells, retain infectivity of the wild-type retrovirus, and are resistant to complement.

[0055]

【Example】

 This will be described in detail in the following examples of the present invention. However, the following examples are not intended to limit the scope of the invention.
It is not intended to limit the enclosure in any way.Example 1 Construction of retrovirus vector having escort protein binding to collagen  An orifice encoding a collagen binding region with a strategic linker
Gonucleotides were synthesized. Encodes a collagen-binding region with a linker
The polypeptide is as follows. GHMWREPFMALSGAS (SEQ ID NO: 9) USC Microchemical Core Facility
Use as a deoxyoligonucleotide to encode the above polypeptide
Oligonucleotides were synthesized.

[0056]

Embedded image

The tandem synthetic oligonucleotide was annealed by heating at 95 ° C. and gradually cooling to room temperature. G25 column (5 Prime ^R 3 Prime, Inc., Boulder,
Colorado) to separate the DNA double strand into single strands. Agarose gel was used to confirm the purity and conformation of the synthetic oridonucleotide insert.

The insert was inserted into three single (unique) restriction sites (AvrII (codon 1 of the hinge region).
Position), PstI (position of codon 35 in hinge region), and StuI (position of codon 48 in hinge region)) and NgoMI restriction site (position of codon 60 in hinge region)
Amphotropic hypervariable polyproline with hinge region or hinge region (SEQ ID NO: 2) (ect
ropic) was cloned into a CEE (autotrophic) -delta hinge env construct (Wu, et al., J. Virol ., July 1998, p5383-5391) which had been modified by substitution. The vector was cut with the following restriction enzymes to obtain each construct. BstEII
AvrII insert; BstII to PstI; BstEII to StuI; BstEII to NgoMI;
And Stul insert. The linearized vector was confirmed by restriction enzyme analysis on agarose gel, purified by the Gene Clean method (Bio 101, Visat, California), and then each insert having a collagen binding region and T4 DNA ligase (New England
(Biolabs, Bever, Massachusetts) for 3 hours at room temperature or overnight at 4 ° C.

After ligation, the various plasmid DNA constructs were used to transform E. coli XL1 blue strains and cultured on LB agar plates under ampicillin selection. QIA Prep Minipr
Plasmid DNA was extracted from selected transformed clones using ep Kits (Qiagen, Valencia, California). Each construct was digested with the appropriate restriction enzymes described above and each insert was analyzed and confirmed. Restriction enzyme analysis: T7 sequence restriction sequence kit
(Amersham life Science, Inc., Cleveland, Ohio) using direct DNA sequence analysis.

The N-terminal 18 amino acid residues of the receptor binding region of the allotrope envelope,
A plasmid having a collagen binding region, a hypervariable polyproline region of an amphotropic envelope protein, the remaining C-terminus of a surface protein, and a coding sequence for a modified retroviral envelope protein having a transmembrane protein is described herein. Hereinafter, it may be referred to as “pESCORT”.

[0061] The retroviral vector that has a escort proteins, Soneoka, et al., Nuc leic Acids Research, Vol.23, with improved pgs.628-633 (1995), wild-type (amphotropic or ecotropic) Envelope Were assembled using a four plasmid transient transfection system such that was co-expressed. The four plasmids used were (i) pHIT112; (ii) pHIT60; (iii) one of the pESCORT plasmids; (iv)
pCAE or pCEE (Morgan et l., J. Virol ., Vol. 67, No. 8, pgs. 4712-4721 (Aug
ust 1993)). Plasmid pHIT60 is from Dr. Oxford (UK)
A retroviral gag- gene, provided by Paula Cannon, having the SV40 origin of replication and under the control of the cytomegalovirus (CMV) promoter.
Contains the pol gene. Plasmid pHIT112 is from USC Gene Therapy Laboratori
e, provided by Ling Li of Los Angels, California, Hybrid C
It contains the LacZ gene under the control of the MV-LTR promoter and the neomycin resistance gene under the control of the SV40 promoter. Each plasmid 10
293T expressing SV40 large T antigen by the calcium phosphate method.
Cells (Pear, et al., Proc. Nat.Acad . Sci. , Vol . 90, pgs . 8392-8396 (Septem
ber 1993)). Thereafter, the producer cells were treated with 10 mM sodium butyrate for 8-12 hours, and 24 hours after infection,
The retrovirus supernatant was collected. Next, the retroviral vector supernatant was
i) Modified ELISA (Hall, et al., Human Gene Therapy , Vol. 8, pgs. 2183-219)
2 (1997)), and (ii) N
Infectivity by expression of β-galactosidase in IH3T3 cells (Hall, et a
l., 1997).

In the above ELISA assay, 50 ml of vector supernatant was injected into each collagen-coated microtiter well, allowed to bind for 20 minutes, washed with 1 × PBS, and incubated with the primary antibody diluted 1: 1,000-fold for 4 hours at room temperature. did. Biotinylated goat antibody to rat IgG was added and streptavidin horseradish peroxidase conjugate was added. Microtiter plates were enhanced with nickel chloride using diaminobenzidine (DAB) as a color source.

Vital titers were measured and quantified by expression of the β-galactosidase reporter gene. Briefly, 2.5 × 10 ⁴ NIH3T3 cells were seeded on each of the 6-well plates before transduction. 8 mg / m medium
It was replaced with 1 ml of a serial dilution of the surviving supernatant containing 1 polybrene and left for 2 hours.
1 ml of fresh D10 was added to the medium and maintained overnight at 37 ° C. and 5% CO ₂ . The medium was replaced with fresh D10 and the culture was maintained for another 24 hours. NIH3T
48 hours after transduction of 3 cells, the expression of β-galactosidase in each culture was evaluated by X-gal staining.

In another test, 1.5 ml of vector supernatant or buffer was incubated at 37 ° C. in a 6-well plate with collagen islets in cloning rings and washed twice with 1 × PBS. Then, DMEM containing 8 mg / ml polybrene
The 1x10 ⁵ cells of NIH3T3 cells suspended in -10% FBS medium was added to each well. The culture was incubated at 37 ° C. overnight and the polybrene-free D1
The medium was replaced with medium 0 and maintained at 37 ° C. for an additional 24 hours, and then evaluated by X-gal staining.

FIG. 1 shows the retroviral vectors WT-CEE, BS-CEE. CE
E, BN-CEE. CEE and BA-CEE. It is the result of ELISA of CEE. The vector WT-CEE is a wild type vector having a homologous envelope protein. BS-CEE. CEE is a retroviral vector having a wild-type ecotropic envelope protein and an "escort protein" formed by inserting a collagen binding domain between the BstEII and StuI sites of the CEE (autotrophic) delta hinge construct. is there. BN-CEE. CEE is a retroviral vector having a wild-type allotropic envelope protein and an "escort protein" formed by inserting a collagen binding domain between the BstEII and NgoMI sites of the CEE (autotropic) -delta hinge construct. is there. BA-CEE. CEE is a wild-type allotrope envelope protein, and CEE
(Ecotropic)-a retroviral vector having an "escort protein" formed by inserting a collagen binding domain between the BstII and AvrII sites of the delta hinge construct.

As shown in FIG. 1, BS-CEE. CEE, BN-CEE. CEE and B
A-CEE. The CEE vector was bound to collagen coated wells. That is, it was confirmed that most of the receptor binding region of the envelope protein and part or all of the hypervariable polyproline region could be removed and replaced with the collagen binding domain.

[0067]Example 2 IgG binding of protein A-env escort protein  Protein A that binds IgG (Lowenadler, et al.,Gene, Vol.58, pgs.87
-97 (1987)) comprising a chimeric envelope protein comprising
Vector (i) pHIT60; (ii) pHIT112; and (iii) protein A.
Has been replaced by part of the rope, or protein A has been
Vector encoding chimeric envelope protein inserted between amino acid residues
And / or (iv) encoding a wild-type CEE or CAE envelope protein
It was constructed using the vector. These plasmids were described in Example 1 in 293T cells.
Cotransfect as described above, treat with sodium butyrate, and
A retroviral vector was produced. Retroviral vectors listed in Table 1 below
Created.

[0068]

[Table 1]

The mouse leukemia virus env protein (Evans, et al., J. Virol. , Vol . 64, No.
12, pgs. 6176-6183 (1997)), using the standard ELISA technique described in Hall, 1997 using the 83A25 rat monoclonal antibody against wells coated with purified human IgG (Gamma Immune N) instead of type I collagen. The binding affinities of protein A-retained virions for purified IgG were changed to wild-type CEE and C
The evaluation was made in comparison with the AE virion.

As shown in FIG. 2, the wild-type envelope protein and the “escort protein” in which a part of the envelope protein has been removed and replaced with protein A
The virions containing retained their binding to IgG upon washing with PBS. On the other hand, wild-type CEE and CAE virions were removed by washing.

[0071]Example 3 Construction of retrovirus vector having protein ZZ-containing escort protein
 Retroviral vector PABA. CAE and PABN. CAE in Example 2
It was constructed as described. Vector PZBA. CAE becomes “Escort protein”
, A protein of 116 amino acid residues that binds to IgG, protein ZZ (Nils
son, et al.,Protein Eng., Vol. 1, pgs. 107-113 (1987)) with BstEII and AvrII.
 PABA. Except that it was inserted between the sites. Same as CAE. These vectors
Has a viability titer comparable to that of the vector containing the wild-type envelope protein.
(PABA.CAE = 2 × 10⁶cfu / ml; PZBA. CAE =
2x10⁶cfu / ml; PABN. CAE = 1 × 10⁶cfu / ml; wild type
= 2x10⁶cfu / ml) and further to an IgG1-coated ELISA plate
Showed high affinity for The ELISA assay was performed according to the procedure described in Example 2.
Was.

Wells containing 5 × 10 ⁵ KSY1 Kaposi's sarcoma cells (Masood, et al., Proc. Natl. Acad . Sci ., Vol. 94, pgs. 979-984 (1994)) were filled with 1,000 ng or 5,000 ng.
KDR / Flk-1 antibody or polybrene. One well was not contacted with any of these. Cells contacted with the antibody are treated with a severity of infection (MOL) of 4
With a titer of 2 × 10 ⁶ cfu / ml in PABA. CAE or wild type C
Contacted with AE. These cells were stained with X-gal and blue colonies were counted. Table 2 shows the results.

[0073]

[Table 2]

In another experiment, 0 ng of wells containing 1 × 10 ⁵ KSY1 Kaposi's sarcoma cells
, 1,000 ng or 5,000 ng of KDR / Flk-1 antibody. Cells contacted with the antibody were purified using PABN.PET with a titer of 1 × 10 ⁶ cfu / ml at an infection severity (MOL) of 10. Contact with CAE or wild-type CAE. These cells were stained with X-gal and blue colonies were counted. Table 3 shows the results.

[0075]

[Table 3]

The above results show that the efficiency of transducing endothelial KSY1 Kaposi's sarcoma cells coated with the KDR / Flk-1 antibody was higher in the antibody-dependent and It shows that it increases with dependence. These data indicate that the chimeric virion molecule is "tethered" to the endothelial cell surface.
Indicates that transduction efficiency has increased. Based on the above results, such an IgG-targeted vector can be used to efficiently transfer genes to endothelial cell receptors in transplanted organs such as heart, kidney, lung, pancreas, and liver, and in transplanted vascular segments. A gene transfer vehicle can be provided.

All patent specifications, publications (including published patent application specifications), and accession numbers cited herein are identified by their respective patent specifications, publications, and accession numbers. All of their contents are specifically incorporated herein as specifically and individually indicated and cited.

However, it should be understood that the scope of the present invention is not limited to the specific examples described above. The invention may be practiced other than as specifically described and still fall within the scope of the invention as set forth in the appended claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1. Collagen-coated wells and WT-C, a retroviral vector
EE, BS-CEE. CEE, BN-CEE. CEE and BA-CEE. CEE
Shows the results of the ELISA assay in which was contacted.

FIG. 2 shows the results of an ELISA assay in which an IgG-coated well was contacted with a retroviral vector having an envelope escort protein containing protein A.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 9/00 A61P 13/00 13/00 25/00 25/00 25/16 25/16 25/26 25 / 26 31/18 31/18 35/00 35/00 37/02 37/02 43/00 111 43/00 111 C07K 14/15 C07K 14/15 19/00 19/00 C12N 7/04 C12N 7/04 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB , GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ , VN, YU, ZA, ZW (72) Inventor Gordon Ellinda Maria United States 91203 Glendale Suite 1803 Dove Rue, Pioneer Drive No. 345 (72) Inventor Anderson Davre French California 91108 San Marino Oxford Road 960 F-Term (reference) 4B024 CA05 DA02 EA02 GA11 HA12 HA17 4B065 AA90X AA93X AA97Y AB01 BA02 CA24 CA44 4C084 AA02 AA13 BA23 BA35 CA01 NA14 ZA161 ZAZZAZZZAZZZAZZZAZZZAZZAZAZAZAZAZAZAA ZA682 ZA701 ZA702 ZA751 ZA752 ZA811 ZA812 ZA891 ZA892 ZA941 ZA942 ZA961 ZA962 ZB071 ZB072 ZB081 ZB082 ZB111 ZB112 ZB211 ZB212 ZB261 ZB262 ZC331 ZC332 ZC551 ZC11 4H0A ABAA

Claims (22)

[Claims] 1. A retroviral vector having a first retroviral envelope protein and at least one modified retroviral envelope protein, wherein the first envelope protein comprises (i) a receptor binding region, and (ii) a hypervariable polyproline. Region, and (iii) a surface protein consisting of a body protein,
The modified retrovirus envelope protein comprises, prior to modification, (i) a receptor binding region, (ii) a hypervariable polyproline region, and (iii) a surface protein consisting of a body protein. The protein is characterized in that at least 90% of the amino acid residues in the receptor binding region of the surface protein of the modified retrovirus envelope protein have been removed and replaced with a non-retrovirus protein or peptide. , The retroviral vector. 2. The vector according to claim 1, wherein at least 92% of the amino acid residues in the receptor binding region of the surface protein of the modified retrovirus envelope protein have been removed and replaced with a non-retrovirus protein or peptide. 3. The vector according to claim 2, wherein all of the amino acid residues in the receptor binding region of the surface protein of the modified retrovirus envelope protein have been removed and replaced with a non-retrovirus protein or peptide. 4. At least 90% of the amino acid residues of the receptor binding region of the surface protein of the modified retrovirus envelope protein and at least 90% of the amino acid residues of the hypervariable polyproline region of the surface protein of the modified retrovirus envelope protein. Some have been removed and replaced with non-retroviral proteins or peptides,
The vector according to claim 1. 5. Prior to modification, the receptor binding region of the modified retrovirus envelope protein has the sequence (SEQ ID NO: 1), and in the modified retrovirus envelope protein, the amino acid of the sequence (SEQ ID NO: 1) Residues 19 to 229 are removed,
2. The vector according to claim 1, wherein said vector is substituted with a non-retroviral protein or peptide. 6. The amino acid residues 19 to 229 of the sequence (SEQ ID NO: 1) and at least a part of the amino acid residues of the hypervariable polyproline region of the surface protein of the modified retrovirus envelope protein are removed. The vector according to claim 5, wherein the vector is substituted with a retrovirus protein or peptide. 7. The hypervariable polyproline region of the surface protein of the modified retrovirus envelope protein has the sequence (SEQ ID NO: 2), amino acid residues 19 to 229 of the sequence (SEQ ID NO: 1), and the sequence The vector according to claim 6, wherein amino acid residues 1 to 35 of (SEQ ID NO: 2) have been removed and replaced with a non-retroviral protein or peptide. 8. The hypervariable polyproline region of the surface protein of the modified retrovirus envelope protein has the sequence (SEQ ID NO: 2), and amino acid residues 19 to 229 of the sequence (SEQ ID NO: 1) and the sequence (SEQ ID NO: 1). The vector according to claim 7, wherein amino acid residues 1 to 48 of SEQ ID NO: 2) have been removed and replaced with a non-retroviral protein or peptide. 9. The hypervariable polyproline region of the surface protein of the modified retrovirus envelope protein has the sequence (SEQ ID NO: 2), wherein amino acid residues 19 to 229 of the sequence (SEQ ID NO: 1) and the sequence 9. The vector of claim 8, wherein amino acid residues 1 to 60 of SEQ ID NO: 2) have been removed and replaced with a non-retroviral protein or peptide. 10. The vector of claim 1, comprising a first retroviral envelope protein and one modified retroviral envelope protein, wherein prior to modification, the modified retroviral envelope comprises (i) a receptor binding region, (ii)
A hypervariable polyproline region, and (iii) a surface protein comprising a body protein, wherein the modified retrovirus envelope protein comprises at least 90% of the amino acid residues in the receptor binding region of the surface protein of the envelope protein. Said vector has been removed and modified to replace a ligand that binds to the desired target molecule. 11. The vector according to claim 10, wherein the target molecule is an extracellular matrix component. 12. The vector according to claim 11, wherein the extracellular matrix component is collagen. 13. The vector according to any one of claims 1 to 9, wherein the non-retroviral protein or peptide is a complement regulatory protein. 14. The vector according to any one of claims 1 to 9, wherein the non-retroviral protein or peptide is selected from the group consisting of protein A, protein ZZ and VEGF. 15. The vector according to any one of claims 1 to 9, wherein the vector has a first modified retrovirus envelope protein and a second modified retrovirus envelope protein, and is in the first modified retrovirus envelope protein. A non-retroviral protein or peptide is a ligand that binds to a desired target molecule;
The above-described vector, wherein the non-retroviral protein or peptide in the second modified retrovirus envelope protein is a complement regulatory protein. 16. The vector according to any one of claims 1 to 15, further comprising a polynucleotide encoding a polypeptide that is heterologous to the retrovirus. 17. The vector according to any one of claims 1 to 15, wherein the first retrovirus envelope protein is a wild-type retrovirus envelope protein. 18. A modified polynucleotide encoding a modified retrovirus envelope protein, wherein prior to modification, the modified retrovirus envelope protein comprises: (i) a receptor binding region; (ii) a hypervariable polyproline region; (iii) a surface protein comprising a body protein, wherein in the modified polynucleotide, at least 90% of the amino acid residues in the receptor binding region of the surface protein are included.
Wherein said polynucleotide sequence has been removed and replaced with a polynucleotide sequence encoding a non-retroviral protein or peptide. (19) A method for expressing a heterologous polypeptide in a host, comprising administering the vector according to any one of (1) to (17) to the host. 20. A method for expressing a heterologous polypeptide in a cell, comprising administering the vector according to any one of claims 1 to 17 to the cell. 21. A modified retroviral envelope protein, wherein prior to modification, the modified retroviral envelope protein comprises: (i) a sequence (SEQ ID NO: 1)
(Ii) a hypervariable polyproline region having a sequence (SEQ ID NO: 2), and (iii) a surface protein consisting of a body protein.
The modified retroviral envelope protein has been modified such that at least 90% of the amino acid residues in the receptor binding region of the surface protein of the modified retroviral envelope protein have been removed and replaced with a non-retroviral protein or peptide. The modified retroviral envelope protein, wherein amino acid residues 1 to 35 of the sequence (SEQ ID NO: 2) have been removed and replaced with a non-retroviral protein or peptide. 22. The modified retrovirus envelope protein having a sequence (SE
22. The modified retroviral envelope protein of claim 21, wherein amino acid residues 19 to 229 of QID NO: 1) have been removed and replaced with a non-retroviral protein or peptide.