CA2133411A1 - Gene therapy using targeted viral vectors - Google Patents

Gene therapy using targeted viral vectors

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Publication number
CA2133411A1
CA2133411A1 CA 2133411 CA2133411A CA2133411A1 CA 2133411 A1 CA2133411 A1 CA 2133411A1 CA 2133411 CA2133411 CA 2133411 CA 2133411 A CA2133411 A CA 2133411A CA 2133411 A1 CA2133411 A1 CA 2133411A1
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virus
envelope
method
protein
hybrid
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CA 2133411
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French (fr)
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Alexander T. YOUNG
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Alexander T. YOUNG
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Priority to US862,795 priority
Priority to US4074893A priority
Priority to US08/040,748 priority
Application filed by Alexander T. YOUNG filed Critical Alexander T. YOUNG
Publication of CA2133411A1 publication Critical patent/CA2133411A1/en
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    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00Reverse Transcribing RNA Viruses
    • C12N2740/00011Reverse Transcribing RNA Viruses
    • C12N2740/10011Retroviridae
    • C12N2740/13011Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00Reverse Transcribing RNA Viruses
    • C12N2740/00011Reverse Transcribing RNA Viruses
    • C12N2740/10011Retroviridae
    • C12N2740/13011Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041Use of virus, viral particle or viral elements as a vector
    • C12N2740/13045Special targeting system for viral vectors
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00Vectors comprising a targeting moiety
    • C12N2810/50Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/854Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from hormones
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00Vectors comprising a targeting moiety
    • C12N2810/50Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/855Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from receptors; from cell surface antigens; from cell surface determinants
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    • C12N2810/00Vectors comprising a targeting moiety
    • C12N2810/50Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/855Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from receptors; from cell surface antigens; from cell surface determinants
    • C12N2810/856Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from receptors; from cell surface antigens; from cell surface determinants from integrins
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00Vectors comprising a targeting moiety
    • C12N2810/50Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Abstract

A general method for delivering genes to specific target cells in vivo is described. Enveloped viruses are genetically engineered to infect specific target cells by replacing the cell surface receptor recognition domain of viral envelope proteins with ligands that direct the binding and fusion of these viruses to specific cell surface molecules.

Description

WO93/20221 . 913 3 g 11 PCT/US93/02957 J'~ . .

GENE THERAPY USTNG TARGETED VIRAL VECTORS
Backaround of the Invention The invention relates to gene therapy methods.
Gene therapy is an approach to treating a broad range of diseases by delivering therapeutic genes directly into the human body. Diseases that can potentially be cured by gene therapy include 1) diseases associated with the aging population such as cancer, 10 heart disease, Alzheimer' 8 disease, high blood pressure, atherosclerosis and arthritis; 2) viral infectious diseases such as acquired immune deficiency syndrome (AIDS) and herpes; and 3) inherited diseases such as diabetes, hemophilia, cystic fibrosis, and muscular 15 dystrophy.
Current methods of delivery of new genetic information into cells in vitro include cell fusion, chromosome-mediated insertion, microcell-mediated gene transfer, liposome DNA carriers, spheroplast fusion, DNA-20 mediated gene transfer, microinjection, infection withrecombinant RNA viruses, and infection with recombinant DNA viruses (Martin, J.C., 1984, Mol. C~ll Biochem. 59:3-10). These techniques are not generally applicable, howevsr, for use in animals or humans because of low 25 efficiency, instability of introduced genes, introduction of extraneous or undesirable genetic information, and lack of target specificity.
In one particular example, a favored approach for human gene therapy involves the transplantation of 30 genetically-altQred cells into patients (Rosenberg, et al., 1988, New Eng J Med~cine 323:570-578). This approach requires the surgical removal of cells from each patient to isolate target cells from nontarget cells.
Genes are introduced into these cells via viral vectors 35 or other maans, followed by transplantation of the W093/20221 ~13 3 ~1 1 i i; . ' .~ PCT/US93/02g57 genetically-altered cells back into the patient.
Although this approach is useful for purposes such as enzyme replacement therapy (for example, for transplantation into a patient of cells that secrete a 5 hormone that diseased cells can no longer secrete), transplantation strategies are less likely to be suitable for treating diseases such as cystic fibrosis or cancer, where the diseased cells themselves must be corrected.
Other problems commonly encountered with this approach lO include technical problems, including inefficient transduction of stem cells, low expression of the transgene, and growth of cells in tissue culture which may select for cells that are predisposed to cancer.
Finally, inappropriate expression of transplanted genes ~5 in nontarget oells may actually be harmful to patients.
An alternative approach to gene therapy involves the direct delivery of genes to target tissue in situ.
Two methods for in s~tu delivery of genes have been developed: biolistic transfer and double balloon 20 catheterization. Biolistic transfer of genes involves shooting DNA-~oated platinum or gold micropro~ectiles directly into target tissue. Biolistic transfer has been successful in the transient expression of genes in the ear, skin and surgically-exposed liver of live mice ~Johnston, S.A., l990, Natur~ ~46:776-777; Williams, R.S., et al., l99l, Proc Na~l Acad Sci USA 88:2726-2730).
Double balloon catheterization transduces genes into cells within a defined arterial wall segment. In this approach a double balloon catheter is inserted into an 30 artery until the end of the catheter is located within the target area. Inflation of two balloons at the end of the catheter creates an enclosed space into which retrovirus or DNA-loaded liposomes are infused. This ~ method has been successful in the transient expression of `~ 35 ~-galactosidase genes within a defined segment of the wo g3,2022. 2 1 3 3 4 1 1 ; ~ , PCT/US93/02gS7 ileofemoral artery of pigs (Nabel E.G., et al., 19~0, Science 249:1285-1288). Both biolistic transfer and double balloon catheterization however, althouqh locally specific, may be nonspecific in the individual cells that 5 they transduce within the target area, creating a problem of inappropriate gene regulation if the transgene is expressed in nontarget cells. Moreover, neither biolistic transfer nor double balloon catheterization have been shown to be effective for the treatment of - 10 tissue occupying large volumes such as lungs, muscles, tumors, or cells of the systemic circulation since the majority of the cells would be inaccessible for in situ gene transfer.
A third approach to gene therapy is the delivery 15 of genes to cells in vivo. This approach involves the introduction of viral vectors directly into patients by in~ection, spray or other means. Different species of viruses are engineered to deliver genes to the cells that the viruses normally infect. Adenovirus, for example, 20 which normally infects lung cells, has been developed as a vector to target genes to lung cells (Rosen~ield, et al., 1992, Cell 68 143-155). Most viral vectors, however, are single purpose vectors since they can only deliver genes to certain cells. Because the target cell 25 specificity of viral vectors i8 re~tricted to the normal tropisms of the viruses, viral vectors are generally limited in that they either infect too broad a range of cell types, or they do not infect certain types of cells at all.
Liposomes have been designed to deliver genes or drugs to specific target cells ~n v~vo. By chemically con~ugating antibodies or ligands to liposomes, liposomes have been targeted to specific cells. With this method, antisense env RNA has been delivered to human 35 immunodeficiency virus (HIV)-infected lymphocytes using W093/20221 ~ 1334 ~ PCT/US93/02ff~

anti-CD3-conjugated liposomes (Renneisen, K ., et al., 1990, J Biol Chem 265:16337-16342); chloramphenicol transacetylase (CAT) genes have been delivered to H2Kk positive lymphomas in H2Kk-negative nude mice using anti-5 H2Kk-conjugated liposomes (Wang, C. et al., 1987, Proc Natl Acad Sci USA 84:7851-7855); and xanthine guanine phosphoribosyltransferase (XGPRT) genes have been delivered to immunoglobulin-coated cells using ~ staphy}ococcus protein A-conjugated liposomes (Machy P., ;~ 10 et al., 1988, Proc Natl Acad sci USA 85:8027-8031). The major drawback to this technology can be the expense of mass producing ligand-con~ugated liposomes.
Wu et al. report a method to target naked DNA to specific cells. Asialoglycoprotein-DNA complexes are 15 targeted to hepatocytes expressing the asialoglycoprotein ;receptor (Wu G.Y., et al., 1991, Biotherapy 3:87-95) .
Similar to the problem encountered with immunotoxins, however, this ~trategy generally limits delivery of DNA
to cells expressing receptors that are capable of DNA-: `~
20 internalization.
Antisense DNA technology is a method ror inhibiting the expression of specific genes with complementary DNA (Moffat, 1991, Science ~:510-511).
Although antisense DNA is specific in the genes that it 25 affects, it is nonæpecific in the types of cells that it gets into. This can create problems ~n vivo because it is desirable that endogenous genes in normal cells remain unaffected by antisense DNA (e.g., protooncogenes).
Moreover, the cost of manufacturing and administering 30 antisense DNA may be high because the phosphate moieties of antisense DNA must be chemically mod~fied to allow passage through tbe plasma membrane, a process which entails expensive organic chemistry. Millimolar concentrations of antisense DNA are required to be 35 effective, posing problems of potential toxicity in vivo.

WO93/20221 ~ 3 34fll~ PCT/US93/02g5 Human gene therapy is therefore limited by.the available technology for gene delivery. Transplantation strategies, which require surgery, limit gene therapy to an expensive service industry for a small number of 5 diseases. Targeting of genes in s~tu through local transduction is generally not precise enough. Viral vectors limit the delivery of genes ~n vivo to cells that the viruses normally infect. Liposome technologies may be infeasible because of the expense of production.
lO Simple ligand-DNA complexes will not introduce genes into cells unless the receptors, against which the ligan~s are direct~d, internalize. Acoordingly, currently available gene delivery systems impose severe limitations on the spectrum of diseases that can be treated by gene therapy.
Summary of the Invention The invention features a method for expressing a nucleic acid of intere~t in a heterologous host cell.
The method involves providing a virus whose genome comprises i) the nucleic acid of ~nterest, and ii) a ; 20 hybrid envelope gene. The hybrid gene encodes an ;~ envelope fragment ~oined to a targeting ligand, whereby the envelope fragment does not facilitate recognition or binding of its normal host cell but does facilitate efficient incorporation of the virus into a mature viral 25 particle, and whereby the targeting ligand facilitates targeting ~nd binding of the mature viral particle to the surface of the heterologous host cell. The method also involves administering the virus so as to permit viral infection of the cell.
By "efficient incorporation", is meant that the hybrid envelope protein ~g incorporated into a mature viral particle at least 25% a8 frequently as the corresponding wild-type envelope protein is incorporated into a mature viral particle.

,_,, !
WO93/20221 rcT/usg3/o29s7 ~1334~11; `

In another aspect the invention features a virus, the genome of which encodes a hybrid envelope protein, wherein the hybrid protein comprises an envelope fragment joined in frame to a targeting ligand, whereby the s envelope fragment does not facilitate recognition or binding of its normal host cell but which does facilitate efficient incorporation of the hybrid envelope protein into a mature viral particle and whereby the non-viral protein facilitates targeting and binding of the mature 10 viral particle to the surface of a cell not normally infected by the virus.
In a third aspect the invention features a method for~delivering a nucleic acid of interest to a heterologous host cell. The method involves providing a 15 virus that~comprises i) the nucleic acid of interest, and ~-~ ii) a hybrid envelope gene, the hybrid gene encoding an envelope fragment joined to a targeting ligand, whereby the envelope fragment does not facilitate recognition or binding to its normal host cell but does facilitate 20 efficient incorporation of the virus into a m~ture viral particle, and whereby the targeting ligand faailitates targeting and binding of the mature viral particle to the surface of the heterologous host cell. The method also involves administering the virus so as to permit viral 25 infection of the cell.
In various preferred embodiments the virus is an enveloped virus, preferably a Herpesviridae, a Paramyxoviridae, or a Retroviridae, most preferably a Moloney murine leukemia virus, or the viru~ may also 30 preferably be a Hepadnaviridae, a Poxviridae, or an Iridoviridae. Similarly tbe virus may be a Togaviridae, a Flaviviridae, a Coronaviridae, a Rhabodoviridae, a Filoviridae, an Orthomyxoviridae, a BunyaviridaQ, or an Arenaviridae, or any other, yet unclassified, enveloped 35 virus.

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.

W093/20221 ~i 3 3 ~ ; PCT/US93/02gS7 In other various preferred embodiments the nucleic acid of interest may include, without limitation, an antisense oncogene; a tumor suppressor gene, e.g., a gene encoding p53, or a gene encoding retinoblastoma protein 5 Rb; a toxin gene, e.g., a diphtheria toxin gene; or a gene encoding a cytokine, e.g., a tumor necrosis factor, or an interferon. The nucleic acid of interest may be either DNA or RNA, e.g., antisense DNA, or antisense RNA, or a nucleic acid encoding an antisense RNA. The nucleic lO acid of interest may also be a gene invoking intracellular immunity, or a nucleic acid therapeutic for an inherited disease, e.g., an insulin gene, or a cystic fibrosis transmembrane regulator gene. A "gene that invokes intracellular immunity" is a gene that confers a lS dominant negati~e re~istant phenotype to the cell it is in.~thereby protecting the cell against an invading aqent.
The heterologous host cell may be a cell that has acquired mutations that re~ult in a disea~e ~tate, 20 preferably a cancer cell, e.g., a colon cancer cell. The ` heterologous host cell may be a cell infected ~ith a second virus, e.g., a human immunodeficiency virus (HIV), a cell infected with an organism, or an infectious agent such as a bacterium or parasite. The infectiouæ agent 25 may be either unicellular or multicellular. The heterologous host cell may also be a cell affected by a hereditary disease, e.g., a pancreatic beta cell, or a ~` lung cell.
The targeting ligand, in additional various 30 preferred embodiments, may include a protein, preferably a hormone, or an immunoglobulin, more preferably an anti-tumor associated antigen-specific immunoglobulin, most preferably an anti-carcinoembryonic antigen-specific ` immunoglobulin, or an anti-HIVgpl20 antigen-specific 35 immùnoglobulin. The targeting ligand may also be a WO93/20221 Z 1 3 3 ~ PCT/US93/02957 carbohydrate, or a lipid. The hybrid envelope fragment may consist of a receptor binding domain, an oligomerization domain, a transmembrane domain, a virus budding domain, sorting signals, a signal sequence, and 5 preferably a fusion domain. In some cases the fusion activity of the envelope fragment may be performed by a second protein. The second protein would therefore direct fusion of the virus with the membrane of the targeted cell.
The mode of admini~tration may include, but is not limited to, l) direct injection of the purified virus; or 2) implanting a container enclosing the virus into a patient. When the virus is administered inside a container, the virus is preferably inside a packaging ~lS cell. A "packaging cell" is a cell that supplies viral ;~proteins necessary for production of viral vectors. By "container" is meant a virus permeable enclosure containing virus, or containing packaging cells with virus therein.
"Normal host cell" as used herein, is a cell type commonly infected by the naturally occurring ~irus. In contrast, the term "heterologous host cell" or a "targeted cell", as used herein, refers to a cell that is' recognized as a function of the targeting ligand portion 25 of the hybrid envelope protein, but i8 not recognized as a function of the envelope portion of the hybrid envelope protein. By "targeting ligand" is meant a molecule that has binding affinity for a moleculQ on tbe surface of a desired targeted cell. A "hybrid envelope protein", as 30 used herein, is a protein that includes a portion of a viral envelope protein ~or a biologically active analog thereof) covalently linked to a targeting ligand. For example, by a "hybrid immunoglobulin-env protein" is meant a portion of an immunoglobulin covalently linked to 35 a portion of an envelope protein. A "hybrid envelope W O 93/20221 ~ 1 3 3 4 1 1 " , `, PC~r/US93/02957 gene" i~ a nucleic acid that provides genetic instructions for a hybrid envelope protein. By "hybrid anti-carcinoembryonic antigen-specific immunoglobulin" is meant a hybrid immunoglobulin-env protein that 5 specifically binds to a carcinoembryonic antigen.
The term "fragment", as applied to an envelope protein fragment, includes some but not all of the envelope protein. A fragment will ordinarily be at least about about 20 amino acids, typically at least about 30 10 amino acids, usually at least about 40 contiguous amino acids, preferably at least about 50 amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length. Fragments of an envelope protein can be generated by methods known to those skilled in the 15 art (e.g., those described herein~.
A biologically active fragment of a viral anvelope protein is one that possesses at least one of the following activities: a) it can bind to a cell membrane if given the appropriate targeting ligand; b) it can 20 enable fusion with a cell membrane; or c) it can enable incorporation of proteins into a mature viral-particle.
These three biological activities can be performed by the same envelope protein fragment, or by two separate envelope protein fragments. As stated above, the 25 envelope fragments of this invention do not facilitate recognition or binding of the virus' normal host cell.
This is accomplished by either destroying the activity of the normal receptor binding region by mutation, or by physically deleting it. A new recombinant receptor-30 binding region i8 added in its place. The ability of acandidate fragment to exhibit a biological activity of a viral envelope protein can be assessed by methods known to those skilled in the art.
The envelope fragment may include the amino acid 35 sequence of a naturally-ocurring viral envelope or may be WO93/20221 '~1 3 3 4 1 I PCT/US93/02957 a biologically-active analog thereo~. The biologLca activity of an envelope analog is assessed using the methods described herein for testing envelope fragments for activity.
S Applicants have provided an efficient and reliable means for ~pecifically delivering therapeutic genes or antisense nucleic acids to particular animal, plant or .
human cell types, or to cells of infectious agents.
Their method facilitates treatments for mutagenically 10 acquired, infectious, or inherited diseases, e.g., by either 1~ antagonizing the effect of an existing cellular ~; gene; 2) complementing the defect of an existing cellular gene; 3) destroying the target cells through the introduction of new genetic material; or 4) changing the 15 phenotype of the target cells through the introduction of ` new g-netic material. To specifically target cells for delivery, a hybrid envelope protein (e.g., an envelope-antibody or envelope-ligand hybrid) is utilized which directs specific interaction with a particular target 20 host cell. The viru~es itself, through its efficient internalization mechanisms, facilitates efficient uptake of the therapeutic gene. Such viral vectors are uniquely ~; adapted to deliver genes, RNA, or drugs to cell surface proteins that do not normally internalize.
Another advantage of this invention is that it overcomes the problem of gene regulation encountered with other methods of gene therapy. ~enetically-altered cells must not only synthesize the gene products at the right location, at the right time, and in the right amounts, 30 but must also be regul~ted in the ~me manner as the indigenous tis~ue. That is, the transduced cells must also have all the proper signal transduction mechanisms to respond to extracellular signals. This may be a problem in gene therapy for diabetes, for example, where 35 transplanting fibroblasts with insulin genes can be ' , W O 93/20221 ~ -~ 3 3 4 1 I PC~r~US93/02957 ineffective or even harmful. As fibroblasts do not contain the same receptors and signal transduction machinery as pancreatic beta cells, the insulin genes may be expressed differently. Targeting genes to the right 5 cells insures that the genes will be properly regulated.
In an additional aspect of the invention, a selection scheme is devised for creation of hybrid envelope protein-containing viruses. This strategy will be feasible for env proteins fused with immunoglobulins 10 or with any ligand that recognizes specific receptors on cells.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
I5 Detailed Description The drawings will first briefly be described.
Drawinas FIG. 1 is a representation of a scheme for constructing retroviral vector pLNCX*.
FIG. 2 is a representation of a scheme for constructing plasmid LNCenvpA.
FIG. 3 is a representation of a scheme for constructing plasmid LNC~nv.
FIG. 4 is a representation of a scheme for 25 constructing plasmid pUC Star-Sig.
FIG. 5 is a representation of a scheme for constructing plasmid LNC-Sig.
FIG. 6 is a representation of a scheme for ` constructing plasmid LNC-ant~CEA.
FIG. 7 is a representation of plasmids used in the construction of targeted viruses.
FIG. 8 is a representation of a strategy for generating targeted retroviruses involving construction of hybrid immunoglobulin-env genes in vitro.

2~33~1, , j WO93/20221 ~ ~, PCT/US93/02957 FIG. 9 is a representation of a strategy ~or generating targeted retroviruses involving generation of pooled virus constructions.
FIG. lo is a representation of a strategy for s generating targeted retroviruses involving selection and characterization of targeted virus.
FIG. 11 is a representation of a plasmid containing a targeted retroviral vector.
FIG. 12 is a representation of a scheme for 10 constructing plasmid pU~ Star-antiCEA.
FIG. 13 is a representation of an alternative scheme for constructing plasmid LNC-antiCEA.
What follows is a procedure for the delivery of genes to target cells using targeted viral vectors. To 15 create and target a virus, the receptor recognition domain of the viral envelope protein is replaced with a ligand directed against a specific cell surface receptor.
The hybsid envelope protein is incorporated into the ~ viral envelope during the budding proce~s, producing a - ~ 20 hybrid virus ~n v~vo. Upon infection of a host, the ~ . ~
hybrid virus specifically recognizes its target cell and resultant fusion with that cell facilitates internalization (into the target cell) of viral genes, including the therapeutic gene(s) which are engineered 25 into the viral genome. Such internalization can be extremely important; for example, immunotoxins, although efficient at delivering toxin molecules to target cells, are often clinically ineffective since the cell surface molecules to which they are targQted do not ~nternalize, 30 and internalization is re~uired for entry of the toxin molecules into the cells ~Waldmann, T.A., 1991, Sc~ence 252: 1657-1662). Targeted viruses circumvent this requirement for receptor internalization since the virus itself contains the necessary cell fusion machinery (Gilbert, J.M., et al., 1990, J Virol 64: 5106-5113;

~, WO93/20221 ~ 3 3 9 1 1 PCT/US93/02957 Roizman, B. et al., 1990, in BN Fields, et al., eds.
Viroloov, Raven Press, Ltd. New York).
General Reauirements for Taraeted Viruses In general targeted viruses are constructed by 5 replacing the receptor recognition domain of the viral envelope protein with a ligand directed against a specific cell surface receptor. The ligand can be, without limitation, an immunoglobulin (e.g., FAb, dAb, Fd, or Fc), a hormone, or any other synthetic or natural 10 protein that can direct the binding of the targeted viruses to a cell surface molecule. The ligand is biologically incorporated into the viral envelope by genetic fusion with that portion of the normal viral envelope protein involved in viral assembly and budding.
15 The envelope portion of the hybrid protein consists of an envelope fragment (or analog thereof) that is sufficient to direct efficient incorporation of the envelope hybrid :`
protein into the viral envelope. Preferably, the ;~ envelope hybrid protein no longer directs an interaction 20 between the virus and ~ts normal host cell.
It has been demonstrated that changing~the receptor specificity of the envelope protein of a virus changes the virus's tropism. `For example, vesicular stomatitis virus (VSV) pseudotypes that have their virus 25 envelope replaced with that of a retrovirus acquire the ability to infect retrovirus infectable cells (Schnitzer, T.J., et al., 1977, J Gen Virol 23:449-454; Zavada. J., et al., 1~72, J Gen Virol ~:183-191), indicating that species specific protein-protein interactions between a 30 virus core protein and an envelope protein are not critical for v~rus fusion and penetration at least in these cases~ Moreover, previous experiments have also indicated that virus envelopes can tolerate changes in ~ length or conformation introduced into the envelope -~ 35 protein, e.g., by a conjugated ligand. For example, ' ;:

WO93t20221 ~ ~ 334t~ ` PCT/US93/02957 Gitman et al. have shown that Sendai virus envelo~es reconstituted with viral envelope glycoproteins, chemically cross-linked to anti-erythrocyte antibodies acquire the ability to bind to erythrocytes that had been 5 stripped of the normal virus receptor. Similarly, Sendai ; virus envelopes reconstituted with envelope proteins, chemically cross-linked to insulin molecules, were able to bind to receptor-stripped erythrocytes expressing the insulin receptor. In both cases, envelope binding but lO not fusion occurred with the receptor-stripped erythrocytes. Fusion between the conjugated envelopes and erythrocytes occurred, however, when the conjugated~
e m elopes were coreconstituted with t~e normal viral hemagglutinin/neuraminidase and fusion proteins (Gitman, 15 A.G., et al., 1985, Biochem 24:2762-2768).
, ` Preferably, the targeted virus contains l) a viral envelope derived from a host cellular membrane; 2) a transmembrane hybrid envelope protein that directs the binding and penetration of the virus to specific target 20 cells; 3) a transmembrane envelope protein that directs he fusion of the targeted virus with the cel~ular membrane of the targeted cell for viral penetration (e.g., the targeting protein itself or another envelope protein); 4) viral core proteins; 5) a foreign gene(s) of 25 interest; and 6) all necessary viral and genetic components for penetration and expression of genes contained in the viral genome. The transmembrane hybrid envelope protein consists of l) determinants that enable the hybrid protein to become processed and incorporated 30 into viral envelopes; 2) det~rminants that enable fus~on of the viral envelope with the targeted cellular membrane; these are essential for penetration of the targeted virus; 3) a ligand determinant that enables the targeted virus to recognize and bind to specific 35 receptors on target cells. The viral genome may also ~:~

W093/20221 ~ 1 3 3 4 1 1 PCT/US93/02957 include bacterial selectable markers (e.g., ampicillin resistance) andlor a mammalian cell selectable marker (e.g., neomycin resistance).
The transmembrane hybrid protein is constructed S genetically by ~plicing the cell surface receptor binding domain of a ligand gene to a portion of the viral envelope protein gene. The transmembrane hybrid protein must retain those portions of the envelope protein that direct the effioient post-translational processing, lO sorting and incorporation of the protein into the viral envelope.
The following domains must be considered in constructing the hybrid ligand-envelope protein:
l. The receptor bindina domain ;~ 15 The receptor binding domain is that portion of the envelope protein that recognizes and binds to cell ~urface receptors. In hybrid envelope proteins, this ; portion of the envelope protein is replaced with ligand sequences. The receptor binding domain of retrovirus 20 envelope proteins h~s bQen localized to the SU subunit (Coffin, J.M., l990, in BN Fields, et al., ed~. Virology, Raven Press, Ltd., New York). Since the SU protein of retroviruses is coded for 5' to the transmembrane pro~ein, replacement of the amino-terminal sequences of 25 the envelope protein with ligand sequences poses no pro~lem for the creation of a functional hybrid ligand-envelope protein.
2. Proteolytic cleavaae site The envelope protein of retroviruses is 30 synthesized as a polyprotein which is later proteolytically cleaved to form SU and TM heterodimers.
In construction of the hybrid ligand-envelope protein, the proteolytic cleavage site should b~ eliminated. Tbe proteolytic cleavage site sbould be eliminated either by 3S deletion or by site directed mutagenesis. Perez and W093/20221 ~3341~` ~ i PCT/US93/02957 Hunter have demonstrated that elimination of the proteolvtic cleavage site does not block transport or surface expression of Rous sarcoma virus envelope proteins (Perez, L.G., et al., 1987, J Virol ~1:1609-5 1614).

3. Oligomerization domain The envelope proteins of many animal viruses associate to form trimers (Fields, B.N., et al., 1991, Virology, 2nd ed., Raven Press, Ltd., New York).
10 Trimerization of the envelope protein is thought to be essential for the proper transport and insertion of envelope proteins into the viral envelope (Singh, J. et al., l990, Embo J 9:631-639; Kreis, T.E., et al., 1986, Cell 46:929-937). Therefore it is important that this 15 domain be retained in the hybrid ligand-envelope protein.
The~tri~erization domain likely resides in the transmembrane TN protein of retroviruses (Einfeld, D., et al., 1988, Proc Natl Ac~d Sci USA 85:8688-8692); hence, creation of a functional hybrid ligand-envelope ; 2Q rètroviral protein lacking the æu subunit is possible.
Some viral envelope proteins may oligomerize to form stoichiometric combinations other than trimers.

4. Fusion domain The fusion domain is a hydrophobic stretch of 25 amino acids that is involved in fusion of the virus envelope with the cell membrane ~Wiley, D.C., et al., 1990, in Fields, B.N. et al., eds. Yiroloqy, 2nd ed., Raven Press, Ltd., New York). Viral fusion allows entry of the viral core proteins and genome into the cell. In 30 influenza virus, the fusion domain, located in the amino terminus of the envelope HA2 protein, is sequestered in the hemagglutinin trimer until a low pH-induced conformational change allows presentation of the fusion domain to the cell membrane. Trimerization of the ; 35 envelope proteins can prevent constitutive expression of , ~

:^``
wo g3/2022- ~ 1 3 3 4 1 1 rcT/us93/029s7 fusion activity by sequestering it within an internal hydrophobic pocket. A potential fusion domain has been located within the extracellular portion of the gp37 TM
protein of Rous sarcoma virus (Hunter E. et al., 1983, J
S Virol 46: 920-936). Similar hydrophobic fusion sequences have been noted in the plSE protein of Moloney murine leukemia virus (Mo-MuLV) (Chambers, P., et al.,l990, J
Gen Virol ~1: 3075-3080).
In constructing a hybrid ligand-envelope protein, 10 it may be necessary to eliminate the fusion domain to pr vent the possibility of constitutive fusion activity, ~; a state that may impair the infectivity of targeted viru-es~. Therefore two proteins may be incorporated into the viral envelope of targeting viruses. The first -15 protein i8 the hybrid ligand-envelope protein which directs targeting of the virus but lacks fusion activity.
The~s-cond protein is an envelope protein possessing fusion activity but lacking a receptor binding domain.
This type of situation is observed for paramyxoviruses 20 where one envelope protein is dedicated to targeting while ~nother carries out fusion (Xingsbury, D.W., 1990, B.N~ Fields, et al., eds. Yirology, 2nd ed., Raven Press, Ltd., New York.). Where it is not necessary to prevent constitutive fusion activity, both activities may 25 be included in one protein.

5. Transmembran~_domain The transmembrane domain is a stretch of approximately twenty or more amino acids that anchor the envelope protein to the viral envelope. It is located 30 within the pl5E protein of Moloney murine leukemia virus (Chambers, et al., supra). Retention of the transmembrane domain is thought tQ be essential since deletion of the transmembrane domain results in secretion of the synthesized envelope protein (Peres, L.G., et al., 35 1987, J Virol ~:2981-2988).

W093/20221 ~3~ 4~ PCT/US93/02957 6. Virus buddina domain Amino acid sequences within the envelope protein may be involved with the exclusive incorporation of viral envelope proteins into viral envelopes and with virus 5 budding~ The virus budding domain directs the hybrid ligand-envelope protein into the viral envelope. These ~equences are thought to reside within the portion of the envelope protein facing the inside of the virus and may involve specific protein-protein interactions between 10 envelope proteins and viral core or matrix proteins.
Although Perez Qt al. demonstrated that deletion of the carboxy-terminal sequences of the Rous sarcoma virus env ~n protein resulted in normal budding of the mutant virus (Perez, L.G., et al., 1987, J Virol 61:2981-2988), 15 evidence exi~ts that, for other viruses, interactions between envelope proteins and viral core proteins may -~ direct virus assembly and envelopment (BN Fields, et al., eds., 1990, Viro~Vgy, Raven Press, Ltd., New York).

7. Sortina 8ianal8 and other si~nals Sorting signals are determinants that direct the envelope protein to the correct intracellular location during post-translational processing. These sequences insure that the envelope protein passes through the endoplasmic reticulum, Golgi apparatus, and other 25 organelles until it eventually reaches the viral envelope. Other signals that may have to be retained in the hybrid ligand-envelope protein are glycosylation sequences and sequences involved in effective conformation of the envelope protein (e.g., disulfide 30 bonds).

8. Sional seauence The signal sequence is an amino-terminal hydrophobic stretch of amino acids that directs the envelope protein into the endoplasmic reticulum. The 35 signal sequence, which is later proteolytically cleaved, W093/20221 ~1 3 3 411 PCT/US93/02957 is essential for the hybrid ligand-envelope protein to become located in a membrane.
The diversity of signals and domains that must be considered in constructing targeted viruses requires that S precise and correct splicing of ligand and envelope genes occur. The present invention-describes a selection scheme for constructing targeted viruses whereby the ligand gene is spliced to an envelope gene fragment; this hybrid gene codes for those portions of the envelope 10 protein which are required to direct efficient incorporation of the resultant hybrid envelope-ligand protein into the mature viral particle. According to the selection scheme, cell surface receptor binding domains of ligand genes are randomly ligated to progressive 15 deletions of viral envelope genes. The correct co bination of ligand and envelope sequences is determined by a selection scheme for the production of bioiogically active targeted virus. The selection scheme -not only produces targeted virus but simplifies the 20 construction of future targeted viruses.
~; a,~peoific Exam~l~ of a Taraeted ~etrovirus There now follows an example of a recombinant retrovirus which targets and infects particular host cells for the purpose of delivering to those cells a 25 desired therapeutic gene. This example is provided for the purpose of illustrating, not limiting, the invention.
Moloney murine leukemia virus (Mo-MuLV) is a mouse ecotropic retrovirus. A recombinant Mo-NuLV based retroviral vector that is targeted to colon cancer cells 30 is constructed. The targeted retroviral vector delivers the neomycin resistance gene to colon cancer cells.
Targeting to human colon cancer cells is accompl~shed by incorporating into the viral envelope hybrid i D unoglobulin-env proteins directed against 35 carcinoembryonic antigen. Carcinoembryonic antigen (CEA) WO93/20221 2 13 3 4 ~ ~ PCT/USg3/02957 is a tumor associated antigen expressed on the surface of human colon cancer cells but not on the surface of normal adult cells. The CEA glycoprotein, possessing multiple membrane spanning alpha helices, does not internalize in 5 response to ligand (Benchimol, S. et al., 1989, Cell 57:327-334). A protein that is homologous to - carcinoembryonic antigen has recently been shown to be the receptor for mouse hepatitis virus (Dveksler, G.S., et al, 1991, J Virol 65:6881-6891).
For the purpose of this illustration, a single ~ variable region of the heavy chain of anti-CEA is fused ; to a portion of the env gene. Single variable heavy `~ chain fragments (dAb) have been shown to be as effective in antigen binding as fragmented antibodies (FAb), 15 oontaining both beavy and light chain fragments, and intact monoclonal antibodies (Ward, E.S., et al., Nature 544-546). The function of immunoglobulin-env proteins is not limited, however, to the use of dAb's and ~- can be applied with FAb's, Fv's and mAb's.
20 Modification of the retroviral vector LNCX
: ~ ~ LNCX i8 a Moloney murine leukemia virus based etroviral vector contained in the plasmid pLNCX (Miller, A.D., et al., 198~, Biotechniques 7:980-990). pLNCX
contains a unique HindIII and ClaI cloning site for 25 expression of inserted genes, a cytomegalovirus ~CMV) promoter, a polyadenylation site (pA), retroviral long terminal repeats (LTR) for retroviral RNA transcription and reverse transcription, a bacterial neomycin resistance gene (Neo) which conveys resistance to both 30 neomycin and G418, a bacterial origin of replication (Or), a bacterial ampicillin resistance gene (Amp), and a retroviral RNA packaging seguence (~). LNCX is modified to contain a unigue SalI site as shown in Figure 1.
pLNCX is Iinearized with XbaI and subcloned into the XbaI
35 site of the phagemid BluescriptII SX~ (Stratagene, La ~ " ~

WO93/20221 ~ l 3 3 4 1 1 PCT/US93/02gS7 Jolla, CA). Single stranded DNA is purified and the unique BstEII site of LNCX is converted into a SalI site by site directed mutagenesis with the oligonucleotide 5'-GCAGAAGGTCGACCCAACG-3 ' (SEQ ID NO: 1) . The BstEII
5 site is located within the extended packaging signal (~+) of Mo-NuLV RNA (Bender, M.A., et al., 1987, J V~rol Çl:1639-1646; Adam, M.A., et al., 1988, J Virol 62:3802-3806; Armentano, D. et al., 1987, J Virol 61:1647-1650).
Conversion of the BstEII site to SalI does not affect 10 packaging since this region has been determined to be dispens~ble for efficient packaging (Schwartzberg, P., et al., 1983, J V~rol 46:538-546; Mann R. et al., 1985, J
Virol 54:401-407; and Mann, R., et al., 1983, Cell 33:153-159). The BstEII site is converted into a SalI
15 site because BstEII sites, but not SalI sites, frequently occur in heavy chain genes (Chaudhary, V.K., et al, 1990, Proc Natl Acad Sci USA 87:1066-1070). The SalI
containing plasmid is recircularized with XbaI and DNA
ligase to form the plasmid pLNCX*.
20 Clonina of the Mo-Mu~V env ~rotein in p~NCX*
The Mo-NuLV env gene is cloned into pLNCX* as shown in Figure 2. The Mo-MuLV env gene is excised from plasmid p8.2 (Shoemaker, C., et al., 1980, Proc Natl Acad ' Sci USA 77: 3932-3936) as a 1.9kb ScaI-NheI fragment. The 25 l.9kb ScaI-NheI fragment contains the entire coding region for the pl5E transmembrane protein and the majority of tha coding region for the gp70 SU protein.
The 5'-protruding ends are digested with Sl-nuclease, and HindIII linkers (5-CCAAGCTTGG-3'; SEQ ID N0: 2) are 30 added. The env gene is cloned as a HindIII fragment in the HindIII site of pLNCX* to form plasmid LNCenvpA. The orient~tion of the HindIII env fragment is such th~t it can be transcribed and expressed from the cytomegalovirus ~; (CNV) promoter.

:; :

WO93/20221 ~33 ~ PCT/US93/02957 Modification of LNCenvpA to LNCenv LNCenvpA is cloned as an XbaI fraament in phagemid pBluescript II SK~ for additional site directed mutagenesis (Figure 3). The env encoding HindIII
5 fragment contains a polyadenylation signal that may interfere with the polyadenylation signal provided by the viral vector. The AAUAAA polyadenylation signal is therefore changed to AAGAAA by site directed mutagenesis with the oligonucleotide 5'-GTTTTCTTTTATC-3' (SEQ ID NO:
10 3). The HindIII site located at the 3' end of the env gene is eliminated by site directed mutagenesis with the oliqonucleotide 5-CAAGCATGGCTTGCC-3' (SEQ ID NO: 4). The env containing retroviral vector is recircularized by XbaI restriction and ligation to form plasmid LNCenv.
15 Molecular clonina of anti-CEA immunoalobulin aenes cDNA encoding the mature variable region domain of ~ ~ anti-CEA heavy chain genes is cloned as an XhoI-SpeI
;~ fragment using the polymerase chain reaction (PCR) and RNA template. RNA is derived from the spleen of mice 20 immunized against purified carcinoembryonic antigen.
~::
Alternat~vely, RNA can be derived from hybridoma cell lines that ~ecrete monoclonal antibodies against CEA, e.g., 1116NS-3d (American Type Culture Collection CRL80~9) or CEA 66-E3 (Wagener, C., et al., 1983, J
2S Immunol 130:2308-2315).
The following PCR primers hybridize to cDNA
encoding the aminoterminal end of mature heavy chain genes (Stratacyte, Inc.). The degenerate primers introduce an XhoI site which is underlined.

W~93/20221 ~ ~ 3 ~ PCT/US93/02~7 5' AGGTGCAGCTGCTCGAGTCGGG 3' (SEQ ID NO: 5) 5' AGGTGCAACTGCTCGAGTCGGG 3' (SEQ ID NO: 6) S~ AGGTGCAGCTGCTCGAGTCTGG 3' (SEQ ID NO: 7) 5' AGGTGCAACTGCTCGAGTCTGG 3' (SEQ ID NO: 8~
5' AGGTCCAGCTGCTCGAGTCTGG 3' ~SEQ ID NO: 9) XhoI

The f~llowing PCR primer hybridizes to immunoglobulin heavy chain mRNA within the region coding for the J-region and introduces SpeI and BstEII sites.
5' CTATTAACTAGTGAC~GTTACCGTGGTCCCTTGGCCCCA 3' (SEQ
ID NO: 10) SpeI BstEII
The amplified anti CEA variable hea~y chain DNA is cloned as an XhoI-SpeI fragment in an ImmunoZ~P H vector (Stratacyte, Inc.) (Mullinax, R.I. et al., 1990, Proc Natl Acad Sci USA 87: 8095-8099). ImmunoZAP H is a modified lambdaZAP vector that has been modified to express in E.coli immunoglobulin variable heavy chain fragments behind a pelB signal sequence. The procadure 20 could similarly be performed by expressing immunoglobulin variable light chain fragments in a packaging cell line.
Identi~ication of hiah affinitY anti-CEA clones Clones expressing high affinity anti-CEA
antibodies axe identified by a f ilter binding assay . The 25 anti-CEA phage library i6 screened by nitrocellulose plaque lifts with ~l25I]bovine serum albumin conjugated to CEA, as previously described (Huse, W.l)., et al., 1989, Science 246:12~5-1281). High and intermediate affinity anti-CEA clones are chosen for further manipulation.
30 Construction of plasmid LNC-immuno~lobulin Two strategies are presented for creating plasmid LNC-immunoglobUlin ( in this example, LNC-an~iCEA, which codes for an anti-CEA immunoglobulin gene). LNC-immunogl o~ul in vectors encode an immunoglobulin peptide W O 93/20221 ~1 ~3~ P~r/US93/02957 fused to an amino-terminal signal se~uence. Some amino acidæ at the amino-terminal end of the mature immunoglobulin peptide have been modified by the PCR
primers used to generate the immunoZAP library.
5 Moreover, the design of the LNC-antiCEA plasmid results in the insertion of an extra amino acid at the amino terminal end. These amino acid changes do not affect antigen binding because 1) ~he amino acid changes are conservative; 2) the affected amino acids are normally 10 variable at those sites; and 3) the affected amino acids occur within the framework region of immunoglobulins`
which has been shown not to participate in antigen binding or conformation of the antibody (Relchman et al.
1988 Nature 332:323-327). It is for these same reasons 15 that cleavage o* the signal seguence from mature peptide will not be affected.
~ Both strategies for creating LNC-immunoglobulin ; ~ rely on the use of plasmid pUC Star-Sig, the construction of which is presented below.
An immunoglobulin signal sequence is cloned into a modified pUCll9 vector to create pUC Star-s~g.as follows (Figure 4). pUCll9 is a phagemid containing a polylinker cloning site. The multiple cloning sites of pUCll9 are replaced with new restriction sites by insertion of the 25 following polylinker into the HindIII and XbaI sites of pUCll9.

Hind~I P~tI XhoI BclI SpeI NotI ClaI Xbal 5'-AGCTTCTGCAGGCTCGAGTGATCAACTAGTGCGGCCGCATCGATT-3' (SEQ

ID N0: 11) 30 3'-AGACGTCCGAGCTCACTAGTTGATCACGCCGGCGTAGCTAAGATC-5'(SEQ
ID N0:12) The modified pUCll9 is called pUC Star-l (Figure 4). The restriction sites may be further separated by small W093/20221 ~ 1 3 3 g 11 rCT/US93/02957 linkers, if adjacent restriction siteæ interfere with one another during digestion.
The signal sequence from an anti-NP immunoglobulin heavy chain gene is isolated from plasmid pcDFL.l (Ucker, S D.S., et al., 1985, J Immnol 135:4204-4214) as a -330bp PstI fragment. The 330bp PstI fragment is subcloned into pUC Star-l to yield plasmid pUC Star-Sig (Figure 4). The PstI fragment is oriented so that the signal sequence can be expressed.
o a. Cons~uç ~ on of L;NC-immunoalobulin through plasmid LNC-Sig. an immunoalobulin expression vector.
LNCX* is converted into a eukaryotic immunoglobu}in expression vector (Figures 4 and 5). An immunoglobulin heavy chain signal sequence and XhoI-SpeI
15 cloning sites are inserted behind the CMV promoter of plasmid LNCX* to allow expression of the PCR amplified im~unoglobulin genes. Conversion of LNCX* is as follows.
The immunoglobulin heavy chain signal sequence is recovered from pUC Star-Sig a8 a HindIII-ClaI restriction 20 fragment ~nd cloned into the HindIII-ClaI sites of LNCX*.
The resulting plasmid, LNC-Sig contains a retroviral vector with the immunoglobulin heavy chain signal sequence under control of the CMV promoter (Figure 5).
An anti-CEA gene from the immunoZAP library is 25 then subcloned into LNC-Sig to form plasmid LNCanti-CEA.
This generates an anti-CEA variable heavy chain gene containing a signal sequence (Figure 6). The anti-CEA
gene is first excised from immunoZAP phage DN~ as a Bluescript SK- phagemid (see lambdaZAP protocols, 30 Stratagene, Inc. La Jolla, CA). The anti-CEA gene is purified as an XhoI-SpeI fragment and l~gated to XhoI-SpeI restricted LNC-S~g. LNC-Stg contains three SpeI
sites. Therefore, to generate plasmid LNCanti-CEA, the ligation mix is transformed into neomycin-sensitive, 35 ampicillin-sensitive E. coli and neomycin-resistant, WO93/20221 ~ 3 3 4 l i PCT/US93/02957 ampieillin-resistant transformants are selected for.
Plasmid LNCanti-CEA is sereened from neomycin-resistant, ampieillin-resistant trans~ormants by using the SpeI-XhoI
anti-CEA restrietion fragment from Bluescript SK-anti-CEA
5 as a probe. The SpeI site is used because of dependenee upon the available sites in the ImmunoZap expression veetor. To simplify eonstruetion of LNCanti-CEA, a unique NotI site ean be introdueed into the ImmunoZap H
expression veetor so that NotI sites ean be used instead lO of SpeI sites.
. Construetion of LNC-immunoalobulin throuah Plasmid pUC Star-Sig An anti-CEA gene from the immunoZAP library is subeloned into plasmid pUC Star-Si~ to form plasmid pUC
15 Star-anti-CEA. This generates an anti-CEA variable heavy ehain gene eontaining a signal sequenee (Figure 12). The anti-CEA gene is exeised from immunoZAP phage DNA as a Blueseript SK-phagemid (~ee lambdaZAP protoeols, Stratagene, Ine., La Jolla, CA). Blueseript SX-anti-CEA
; 20 double ~tranded DNA ~8 prQpared and restrieted with XhoI
and SpeI. The anti-CEA eontaining XhoI-SpeI f~agment is purified by eleetroelution and ligated to Xho-SpeI
restrieted pUC Star-Sig to ereate plasmid pUC Star-antiCEA ~Figure 12).
The antiCEA gene is transferred from pUC Star-anti CEA to LNCX* as a HindIII-ClaI fragment to ereate plasmid LNC-antiCEA (figure 13). The antiCEA-eontaining HindIII-ClaI fragment is purified from pUC Star-antiCEA by eleetroelution. Phosphatase treated, HindIII-ClaI
30 restrieted LNCX* is ligated with the purified HindIII-ClaI antiCEA fragment to generate LNC-antiCEA ~figure 13).
Strateov for generatina targeted viruses The starting materials for generation of targeted 35 viruses are the LNC~nv and LNC-immunoglobulin lin this ~ .

.~'`~, .
W O 93/20221 . X 1 ~ 3 4 1 1 P{~r/US93/02957 example, LNC-antiCEA) plasmids shown in Figure 7.
Figures 8-11 diagram the general principle for the primary generation of targeted viruses. Hybrid i Dunoglobulin-env proteins are generated that target 5 viruses to cells expressing carcinoembryonic antigen.
Since the location of important determinants for envelope protein sorting (S), trimeriza~ion (T), and fusion (F) iæ
; not known with certainty, the i D unoglobulin gene is ligated to progreæsive deletions of the env gene and 10 functional i D unoglobulin-env hybrids are selected for.
Useful Envelo~e Fraoments or Analogs The envelope portion of the fusion protein may consist of any portion of the envelope protein (or any analog thereof) which is sufficient to direct efficient 15 incorporation of the envelope fusion protein into the viral coat ~upon budding of the recombinant virus from a producer cell line). Suc~ fragments or analogs may be determined using the following general selection scheme which generally involves ligation of cell surface 2Q receptor binding domains of ligand genes to progessive deletions of viral envelope genes. The correct combination of ligand and envelope sequences is determined by a selection scheme for the production of biologically active targeted virus. The selection scheme 25 not only produces targeted virus but simplifies the construction of future targeted viruses.
Construction of hvbrid immunoalobulin-env genes in v~txo Plasmid LNCenv contains the coding region for the Mo-MuLV env polyprotein (Figure 8). LNCenv is first 30 linearized by HindIII restriction. A rangQ of deletions extending into the env gene is created by colleoting aliquots of Exonuclease III treatqd DNA over time and removing 5'-processive ends with Sl-nuclease (Guo, I.H., et al., 1983, Methods Enzymol 100:60; and Sambrook, J., 3S et al., 1989, Molecular Clonina, Cold Spring Harbor ` ~

Laboratory Press, Cold Spring Harbor). NotI linkers (5' AGCGGCCGCT 3 ' SEQ ID N0: 13) are ligated onto the blunt end termini and restricted with NotI. This results in a NotI restriction overhang at the 5'-border of every 5 deletion within the env gene. The NotI overhangs at the other end of the molecules are removed by SalI
restriction of the reaction mixture. The reaction mixture is then treated with phosphatase to prevent circularization of the re~ction products.
The reaction mixture is ligated to a SalI-NotI
restriction fragment from LNC-antiCEA that contains the ~` anti-CEA variable heavy chain gene. This creates a pool of functional retroviral vectors encoding an anti-CEA
peptide fused to a series of env deletions.
15 Generation of Dooled virus constructions The total reaction mixture from above is transformed into ampicillin-sensitive E. coli and ampicillin resistance is selected for tFigure 9).
Reoombinants containing functional retroviral vectors are 20 selected for since only they contain the ampicillin resistance gene. Plasmid DNA is prepared from~
transformants grown in liquid culture to create a pool of retroviral vectors containing different immunoglobulin-env fusion genes.
The DNA is tranæfected into the crip2 retroviral packaging cell line (Danos, 0., et al., 1988 , Proc Natl Acad Sci USA 85: 6460-6464). Alternatively, DNA is transfected into a packaging cell line that does not encode wild-type env protein. The transfected packaging 30 cell line synthesizes each of the different hybrid immunoglobulin-env protein~ as well as the wild type env protein (encoded by an env gene contained in the cell line). The transfected packaging cell line secretes a pool of enveloped retroviruses containing the different 35 `retroviral genomes encoding hybrid immunoglobulin-env `` WO93/20221 ~ 1 3 3 ~ PCT/USg3/02957 genes. If the hybrid immunoglobulin-env protein retained all of the neeessary determinants for efficient incorporation into viral envelopes then the hy~rid-env protein ean be incorporated into viral envelopes. Wild 5 type env proteins eneoded for by the packaging eell line are also ineorporated into the viral envelopes. This ereates a virus eontaining both wild type and hybrid env proteins in the viral envelope. This system therefore seleets for immunoglobulin-env hybrids that ean 10 ineorporate their gene produets into the viral envelope.
Virus pools are harvested from media filtered at 0.45~ to remove contaminating G418-resistant paekaging eells.
Seleetion and eharaeterization of targeted virus G418-sensitive target eells are exposed to virus pools by standard proeedures, and G418-resistant eells are seleeted for. The target eells ean be any non-mouse eell line (uninfeetable by wild type No-MuLV) that expresses eareinoembryonie antigen. Examples inelude 20 ATCC COLO 205, a human eell line isolated from the aseites of a patient with eareinoma of the eo~on (A.T.C.C.#CCL 222); LR-73 CEA, a ehinese hamster ovary eell line transfeeted with a mouse eareinoembryonie antigen gene (Benehimol, S. et al., supr~); and HCT48, a 25 human eolon adenoeareinoma eell line (Shi, 2.R., et al., 1883, Can~er Res 43:4045-4049).
G418-resistant eells ean only have arisen from transduetion of the neomyein resistanee gene by targeted virus. This system therefore seleets for reeombinant 30 viruses that have hybrid immunoglobulin-~nv proteins that have retained all the neeessary determinants for viral targeting and fusion.
Reseue of inteorated i~munoalobulin-~nv gene Infeetion by targeted virus results in integration 35 of the hybrid envelope gene that ereated the targeting :

WO93/20221 PCT/US93/02~7 ~1 3 ~ 411 _ 30 ~
protein. The integrated hybrid immunoglobulin-env gene is rescued from the host DNA by polymerase chain reaction (PCR) with the following primers:

PCR 5' Rescue primer:
5 5'-CCAGCCTCCGCGGCCCCAAGCTTCTGCA-3' (SEQ ID NO: 14) HindIII
PCR 3' Rescue primer:
5'-GGTTC~SIaÇaAACTGCTGAGGGC-3' (SEQ ID NO: 15) XbaI

PCR amplification with these primers generates the immnoglobulin-env gene bordered by HindIII and XbaI
sites. The amplified DNA is restricted with HindIII and XbaI to create sticky ends and the DNA is ligated into HindIII-XbaI cut LNCX*. When transfected into crip2 15 packaging cells, this generates a retroviral vector targeted to cells expressing cell surface carcinoembryonic antigen (e.g., colon cancer cells).
The retroviral vector produced in the above selection scheme is targeted to both CEA-expressing human 20 cells (directed by the hybrid envelope protein) and normal mouse cells (directed by the wild type envelope protein) when produced in crip2 packaging cells. To create viruses that infect target cells only, the retroviral vector will first be tested to determine if 25 incorporation of the hybrid envelope protein alone is sufficient to direct virus fusion. This is accomplished by transfecting DNA into a modified packaging cell line that does not encode wild type env.
If fusion functions are found to have been 30 supplied from the wild type envelope protein, targeted viruse~ will be created as follows. A packaging cell line will be created that encodes an env gene containing mutations in the receptor binding domain. When W093~20221 PCT/US93/02g57 `~13~

transfected with the targeted viral vector DNA, targeted viruses expressing both hybrid ligand-env proteins and env proteins with mutated binding sites will be produced.
The viruses will exclusively infect target cells.
5 The taraeted viral vector is a universal vector The viral vector that is constructed by the above procedure is a universal targeted vector (Figure 11).
Targeting to other cells is accomplished by replacing the XhoI-SpeI anti-CEA fragment with any XhoI-SpeI fragment 10 encoding an in-frame immunoglobulin or ligand directed against specific cell surface proteins. For example, an XhoI-SpeI i D unoglobulin-containing fragment from an immunoZAP library can be fused in frame behind a signal sequence and subcloned into LNCX* through the pUC Star-15 Sig plasmid, as outlined above. Substituting a SalI-NotI
fragment from another LNC-immunoglobulin plasmid into the universal vector would create another targeted virus vector.
Other Viral Vectors Any enveloped virus may be used as a vector for the targeted delivery of a therapeutic gene. Particular examples include both DNA and RNA viruses, such as Herpesviridae, e.g., herpes simplex type 1 or 2, Paramyxoviridae, Retroviridae, Hepadnaviridae, 25 Poxviridae, Iridoviridae, Togaviridae, Flaviviridae, Coronaviridae, Rhabodoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, or Arenaviridae, or any other, yet unclassified, enveloped virus.
An extensive selection of these viruses is 30 available, e.g., from the American Type Culture Collection.
Taraetina Liaands Any molecule that is capable of directing specific interaction with a target host cell (e.g., by specific 35 recognition of and binding to a host cell surface ~ if ~
W093/20221 2133 ~i 1 i PCT/USg3/02g57 protein) may be used as the targeting ligand portion of the envelope fusion protein. Preferably, such a protein is derived from one member of a ligand:receptor pair.
The targeting ligands are not limited to proteins.
5 Carbohydrate and lipid moieties can be attached to the envelope protein via protein fragments containing consensus ~equences for glycosylation and lipidation.
Immunoglobulin genes can be used as ligands, as shown in the example above. Genes for high affinity lO immunoglobulins are screened from a lambda or bacterial expression library by a filter binding assay with sI] bovive serum albumin conjugated to antigen, as previously described (Huse, et al. Sup~a ) .
Cell surface molecules such as integrins, adhesion l5 molecules or homing receptors can be used as cell-speaific ligands since they are involved in cell-cell interactions via receptors on other cells. Genes encoding these molecules can be identified by the panning method of Seed and Aruffo (Seed. B., et al., 1987, Proc 20 Natl Acad Sci USA 84:3365-3369).
Hormones that bind to specific receptors can be used as targeting ligands as well as viral proteins, such as ~IV envelope protein gpl20, and modifications of naturally occuring ligands.
25 Thera~eutic Genes Therapeutic genes useful in the invention include the following. l) Genes that are therapeutic to cancer cells may include a) antisense oncogenes; b) tumor suppressor genes, such as p53 or the retinoblastoma gene 30 product Rb, c) destructive toxin genes such as a diphtheria toxin gene; d) cytokines æuch as tumor necrosis factor or interferons; or e) any other therapeutic gene. 2) Therapeutic genes targeted to cells that are infected with HIV. Specific examples 35 include antisense DNA complementary to essential genes ` wo g3/2022l ~ 1 3 3 9 1 1 PCT/US93/02~7 for HIV, e.g., polymerase; destructive toxin genes, e.g., diphtheria toxin; and genes that will invoke intracellular immunity, e.g., HIV enhancer sequences that titrate and remove HIV regulatory proteins (Baltimore, 5 D., 1988, Nature 335:395-396). 3) Genes to correct inherited deficiencies. Examples include, but are not limited to, insulin genes delivered specifically to pancreatic beta cells, or the cystic fibrosis transmembrane regulator (CFTR) gene delivered to the 10 appropriate lung cells of cystic fibrosis patients. The expression of targeted genes can be further accomplished through the use of tissue specific enhancers that regulate the transgene.
Therav For any gene therapy described herein, the appropriate recombinant virus, as described above, is administered to a patient in a pharmaceutically-acceptable buffer (e.g., physiological saline). The therapeutic preparation is administered in accordance 20 with the condition to be treated. For example, to treat an HIV-infected individual, the virus is administered by direct injection, e.g., by intravenous, intramuscular, or intraperitoneal injection, at a dosage that provides suitable targeting and lysis of HIV-infected host cells.
25 Alternatively, it may be necessary to administer the targated virus surgically to the appropriate target tissue, or via a catheter, or a videoscope. It may be convenient to administer the therapeutic orally, nasally, or topically, e.g., as a liquid or spray. Again, an 30 appropriate dosage is an amount of therapeutic virus which effects a reduction in the disease.
Targeted virus can also be administered by implanting viral packaging cells into a patient. The cells can be enclosed in a semi-permeable container, 35 e.g., permeable to a virus but not permeable to a ~3~4~

WO93/20221 PCT/USg3/02957 packaging cell. The implanted container may be removable. Alternatively, the container may be hooked up to a patient intravenously, so that virus enters the patient through a needle or through a catheter. In this 5 way the patient receives a continuous dose of viral gene therapy.
Other Embodiments Other embodiments are within the following claims.
For example, replication competent viruses may be lO used in certain cases. In other cases, where replication-deficient viruses are necessary, it may be efficacious to administer modified packaging cells, rather than the targeted virus, to patients. By this method a non-proliferating dose of recombinant virus is 15 delivered to a local area, and then the virus locates the specific target cell. For example, tumor infiltrating lymphocytes (TIL), which surround cancer cells, can be modified to secrete locally high concentrations of cancer cell-targeted virus. Treatment may be repeated as 20 necessary. Immune response against targeted viruses can be overcome with immunosuppressive drugs.
In addition to colon cancer celLs, the ~irus of the invention may be used to target other cancer cells, e.g., ovarian, breast, or lung cancer cells, or cells 25 affected with hereditary diseases such a~ muscular dystrophy, Huntington's disease, or cells with a defect in adenosine deaminase. Herpesviridae viruses may include Herpes simplex type 1 or type 2, Epstein-Barr virus, or Cytomegalovirus. Sendai virus and Vaccinia 30 virus may also be adapted to this method.

~ 1 3 ~ 4 1 1 WO93/20221 . j!. PCT/US93/02~7 BBOUBNCE ~I8TING
BNERAL INFORMATION:
(i) APPLICANT: Alexander T. Young (ii) TITLB OF IN~ENTTON: GENE THERAPY USING
TARGETED VIRAL VECTORS
(~ii) NnMBBR OF 8BQ~ENCE8: 15 (iv) CORRE8POND~NCB ADDRB88:
(A) ADDRE88~B: Fish & Richardson (B) 8TR~T: 225 Franklin Street ~C) CITYs Boston (D) 8TATE: Massachusetts ~B) CO~NTRY: U.S.A.
(F) 8IP: 02110-2804 ~v) COMP~TBR R~ADABLB FORM:
(A) NEDI~ T~PB: 3.5" Diskette, 1.44 Nb (B) CO~P~TDR: IBM PS/2 Model 50Z or 55SX
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~A) APPLICATION N~NB~R:
(B) FILING DATE:
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~A) APPLI~ATIO~ N~NBER: 07/862,~95 ~B) FILING DATE: April 3, 1992 (vi~) A~TO~N~Y/AG~NT INFORMATION:
~A) NAME: Paul T. Clark (B) REGI8TRATION N~MBER: 30,162 ~C) REFERBNCE/DOC~ET NUMBER:05140/002002 ;~8) TELBCOMM~NICATION INFORMATION:
~A) TELBP~ON~: (617) 542-5070 ~B) TE$EFAS: (617) 542-8906 (C) TELE~: 200154 t WO 93/20221 ~13 ~ 4 ~ r/us93/o2957 ~2) INFORMATION FOR 8BQ~ENCB IDENTIFICATION N~NBER: 1:
(i) 8BQVBNCB C~ARACTBRI8TIC8:
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Claims (39)

1. A method for expressing a nucleic acid of interest in a heterologous host cell, said method comprising (a) providing a virus whose genome comprises (i) said nucleic acid of interest and (ii) a hybrid envelope gene, said hybrid gene encoding an envelope fragment joined to a targeting ligand, whereby said envelope fragment does not facilitate recognition or binding of its normal host cell but which does facilitate efficient incorporation of said virus into a mature viral particle and whereby said targeting ligand facilitates targeting and binding of said mature viral particle to the surface of said heterologous host cell, and (b) administering said virus so as to permit viral infection of said heterologous host cell.
2. The method of claim 1, wherein said virus is an envelope virus.
3. The method of claim 2, wherein said envelope virus is a Herpesviridae.
4. The method of claim 2, wherein said envelope virus is a Retroviridae.
5. The method of claim 4, wherein said Retroviridae is a Moloney murine leukemia virus.
6. The method of claim 1, wherein said nucleic acid of interest is DNA.
7. The method of claim 1, wherein said nucleic acid of interest is RNA.
8. The method of claim 1, wherein said heterologous host cell is infectious.
9. The method of claim 1, wherein a portion of said hybrid envelope fragment consists of a receptor binding domain, an oligomerization domain, a transmembrane domain, a virus budding domain, sorting signals, and a signal sequence.
10. The method of claim 9, wherein said envelope fragment further consists of a fusion domain.
11. The method of claim 1, wherein the fusion activity of said envelope fragment is performed by a second protein.
12. The method of claim 1, wherein said administration is by implanting a container enclosing said virus into a patient.
13. The method of claim 12, wherein said virus is inside a packaging cell.
14. A virus, the genome of which encodes a hybrid envelope protein, said hybrid protein comprising an envelope fragment joined in frame to a targeting ligand, whereby said envelope fragment does not facilitate recognition or binding of its normal host cell but which does facilitate efficient incorporation of said hybrid envelope protein into a mature viral particle and whereby said non-viral protein facilitates targeting and binding of said mature viral particle to the surface of a cell not normally infected by said virus.
15. The virus of claim 14, wherein said virus is an envelope virus.
16. The virus of claim 15, wherein said envelope virus is a Herpesviridae.
17. The virus of claim 15, wherein said envelope virus is a Retroviridae.
18. The virus of claim 17, wherein said Retroviridae is a Moloney murine leukemia virus.
19. The virus of claim 14, wherein said nucleic acid of interest is DNA.
20. The virus of claim 14, wherein said nucleic acid of interest is RNA.
21. The virus of claim 14, wherein said heterologous host cell is infectious.
22. The virus of claim 14, wherein a portion of said hybrid envelope protein consists of a receptor binding domain, an oligomerization domain, a transmembrane domain, a virus budding domain, sorting signals, and a signal sequence.
23. The virus of claim 22, wherein said envelope fragment further consists of a fusion domain.
24. The virus of claim 14, wherein the fusion activity of said envelope fragment is performed by a second protein.
25. The virus of claim 14, wherein said administration is by implanting a container enclosing said virus into a patient.
26. The virus of claim 25, wherein said virus is inside a packaging cell.
27. A method for delivering a nucleic acid of interest to a heterologous host cell, said method comprising a) providing a virus whose genome comprises (i) said nucleic acid of interest and (ii) a hybrid envelope gene, said hybrid gene encoding an envelope fragment joined to a targeting ligand, whereby said envelope fragment does not facilitate recognition or binding to its normal host cell but does facilitate efficient incorporation of said virus into a mature viral particle and whereby said targeting ligand facilitates targeting and binding of said mature viral particle to the surface of said heterologous host cell, and b) administering said virus so as to permit viral infection of said cell.
28. The method of claim 27, wherein said virus is an envelope virus.
29. The method of claim 28, wherein said envelope virus is a Herpesviridae.
30. The method of claim 28, wherein said envelope virus is a Retroviridae.
31. The method of claim 30, wherein said Retroviridae is a Moloney murine leukemia virus.
32. The method of claim 27, wherein said nucleic acid of interest is DNA.
33. The method of claim 27, wherein said nucleic acid of interest is an RNA.
34. The method of claim 27, wherein said heterologous host cell is infectious.
35. The method of claim 27, wherein a portion of said hybrid envelope fragment consists of a receptor binding domain, an oligomerization domain, a transmembrane domain, a virus budding domain, sorting signals, and a signal sequence.
36. The method of claim 35, wherein said envelope fragment further consists of a fusion domain.
37. The method of claim 27, wherein said the fusion activity of said envelope fragment is performed by a second protein.
38. The method of claim 27, wherein said administration is by implanting a container enclosing said virus into a patient.
39. The method of claim 38, wherein said virus is inside a packaging cell.
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