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WO1998005344A1 - Therapie genique a mediation par bacteriophages - Google Patents

Therapie genique a mediation par bacteriophages Download PDF

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Publication number
WO1998005344A1
WO1998005344A1 PCT/US1997/012928 US9712928W WO9805344A1 WO 1998005344 A1 WO1998005344 A1 WO 1998005344A1 US 9712928 W US9712928 W US 9712928W WO 9805344 A1 WO9805344 A1 WO 9805344A1
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bacteriophage
phage
cell
target cell
gene
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PCT/US1997/012928
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WO1998005344A9 (fr
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Saumyendra N. Sarkar
Thomas S. Kupper
Daniel B. Dubin
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Brigham And Women's Hospital, Inc.
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Priority to AU37372/97A priority Critical patent/AU3737297A/en
Publication of WO1998005344A1 publication Critical patent/WO1998005344A1/fr
Publication of WO1998005344A9 publication Critical patent/WO1998005344A9/fr

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    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/20—Pseudochromosomes, minichrosomosomes
    • C12N2800/202—Pseudochromosomes, minichrosomosomes of bacteriophage origin
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • This invention relates to the use of a bacteriophage to express an exogenous gene in a mammalian cell.
  • the invention relates to the use of bacteriophage vectors for human gene therapy and compositions related thereto. 2 s Background Of The Invention
  • Vectors which are being studied in gene therapy trials include retroviruses, adenoviruses,
  • adeno-associated virus 3 5 adeno-associated virus, plasmids and liposomes.
  • adenovirus reportedly is a relatively efficient gene delivery vehicle for both dividing and non- dividing cells, its drawbacks include (1) limitation of inserted expression cassette to seven kilobases, (2) induction of inflammation in mammalian hosts, (3) toxicity to target cells when administered in high titers, (4) inability to target specific cell types, and (5) transient expression
  • adenoassociated virus has been proposed as an alternative to adenovirus, and does have certain advantages, including a preferential site of chromosomal integration. Although the adeno-associated virus has not been associated with inflammation, this virus' genome is only 4.7 kilobases in size and can accommodate inserts of up to 5 kb in size.
  • an "expression cassette” is a term of art that refers to an inserted exogenous DNA that optionally contains transcriptional regulatory sequences, translational regulatory sequences, coding sequences, or intervening sequences necessary for efficient expression of the included coding sequence.
  • the cost of manufacturing adeno-associated virus is high compared to that of adenovirus.
  • adeno-associated virus is difficult to grow at high titer and cannot be targeted to defined cellular targets.
  • retroviral vectors for delivering an exogenous gene into mammalian cells and are susceptible to complement-mediated destruction.
  • retroviral vectors can transduce only dividing mammalian cells.
  • the retroviruses can accommodate inserts up to only seven kilobases, thereby limiting the size of exogenous genetic material that can be delivered using this type of vector.
  • the cost of manufacturing retroviral vectors has been estimated to be approximately $100,000 per lot, reportedly due to the high costs associated with producing high titers of recombinant retroviral vectors in animal cells (D. Holzman, "Gene Therapy Depends on Finding the Right Vector", JNCI, Issue 6, vol. 87 (1995) NEWS pg 406).
  • Non-viral vectors for gene therapy that are being studied in clinical trials include liposomes and naked DNA delivery systems. Low efficiency of transfection hampers both of these methods, neither of which permit targetable delivery to specific cell types. In addition, liposomes can be toxic to cells.
  • the inadequacies of the above-identified vectors include: (1) induction of an immune response in the host; (2) possible deleterious recombination events; (3) limitations in the size of the expression cassette that can be inserted into the vector; (4) lack of specificity of the vector for delivering the expression cassette to a particular target cell; (5) inability to target a vector to both dividing and non-dividing cells;
  • bacteriophages can be modified to contain exogenous genetic material that can be transcribed and, optionally, translated in a mammalian cell. Moreover, modification of these bacteriophages to include specific ligands and gene transcription regulatory elements permits control over the cell(s) to which the expression cassette is delivered and in which the delivered genetic material will be expressed. Furthermore, these bacteriophages can be modified to include nuclear localization and endosomal lysis signals to enhance the efficiency of transfection. Despite genetic modification, these bacteriophages preferably maintain the ability to propagate to high titer in a prokaryotic host, thus greatly facilitating production.
  • the invention provides compositions and methods for using these bacteriophages to introduce an exogenous polynucleotide (e.g., a therapeutic polynucleotide) into a pre-selected target cell via receptor-mediated delivery.
  • the bacteriophages are used to deliver a therapeutic polynucleotide into a mammalian cell for human gene therapy.
  • the bacteriophages are used to deliver an exogenous polynucleotide into a mammalian cell for the production, in vitro or in vivo, of a polynucleotide transcription or translation product.
  • compositions containing the bacteriophages of the invention and methods for preparing and using the same to produce exogenous polynucleotide transcription and translation products in vitro also are provided.
  • the bacteriophage contains exogenous genetic material that can be both transcribed and translated in a mammalian cell.
  • the bacteriophage contains exogenous genetic material that is transcribed, but not translated, in a mammalian cell (e.g., ribozyme and antisense constructs).
  • Exemplary bacteriophages which can be modified to satisfy this criteria are provided in Table 1.
  • these bacteriophages can be propagated in prokaryotic cell(s), thereby permitting the large scale production of the bacteriophages of the invention at a relatively low cost in comparison with other gene therapy vectors. Furthermore, since the bacteriophage structural genes are controlled by prokaryotic promoters, no transcription of native bacteriophage genes will occur in the mammalian host cell. Therefore, cells transfected with a modified bacteriophage will not express foreign proteins on their cell surface and, thus, will not induce cell-mediated immunity.
  • the preferred bacteriophages of the invention include the lambda and pi phages. These preferred phages, after targeting modifications, have in common the ability to deliver to a mammalian cell(s) a therapeutic polynucleotide that is between about 1-100 kilobases. In particular, the preferred phages have in common the ability to deliver to a mammalian cell(s) a therapeutic polynucleotide of large size: between about 9-23 kilobases for lambda Dash II and between about 75-100 kilobases for pi .
  • the preferred modified lambda phages of the invention can accommodate between about 9-50 kilobases.
  • the invention is particularly useful for delivering to a target cell, an exogenous polynucleotide expression cassette (e.g., a therapeutic polynucleotide) containing a single gene or multi-gene complexes that are greater than 9 kilobases in length, something that cannot be accomplished by currently available viral vectors.
  • an exogenous polynucleotide expression cassette e.g., a therapeutic polynucleotide
  • an improved method for gene therapy lies in using a bacteriophage to deliver a therapeutic polynucleotide to a target cell in a mammalian recipient.
  • the invention provides, for the first time, a viral vector that delivers to a target cell, an exogenous polynucleotide containing up to 9 to 23 kilobases for lambda Dash II, up to 50 kilobases for lambda-derived cassettes containing only COS sequences and no packaging protein coding sequences (minimal lambda cassette), and up to 75 to 100 kilobases for pi.
  • the bacteriophages of the invention contain on their surfaces ligands to achieve delivery of the bacteriophages into a pre-selected target cell.
  • the pre-selected target cell contains on its surface a receptor that selectively binds to the ligand, forming a ligand- receptor complex that is internalized by the cell.
  • a receptor-mediated delivery mechanism such as that utilized by native eukaryotic viruses (e.g., adenovirus) and as that described in U.S. Patent No. 5,108,921 , issued to Low, et al. and U.S. Patent No.
  • the bacteriophage vector further contains a nuclear localization signal (e.g., retrovirus) (Naldini et al. Science 1996; 272:263) as well as an endosomal lysis signal (e.g., adenovirus)(Wagner et al. Proc Natl Acad Sci 1992;89:6009) which, alone or in combination, enhance the expression of the delivered polynucleotide.
  • a nuclear localization signal e.g., retrovirus
  • an endosomal lysis signal e.g., adenovirus
  • chloroquine or other inhibitors of lysosomal/endosomal enzymatic catabolism can be co-administered with the vector.
  • the use of nuclear localization signal(s), endosomal lysis signal(s), and/or endosomal enzyme inhibitors optimize expression of the delivered polynucleotide by minimizing the likelihood of DNA degradation that may occur as the vector traffics to the nucleus.
  • the delivered polynucleotide is designed and constructed in accordance with standard practice to integrate into the target cell chromosome.
  • the vector may be maintained episomally in the cell.
  • novel bacteriophages disclosed herein are useful for in vivo and ex vivo gene therapy, as well as for producing in culture or in animals, gene products of the therapeutic polynucleotides (e.g., transcription products such as antisense RNA or catalytic RNA (e.g., ribozyme), and translation products such as peptides and proteins).
  • the improved method for gene therapy is useful for introducing a therapeutic polynucleotide (e.g., a polynucleotide for treating or diagnosing a medical condition) into a target cell of a mammalian recipient.
  • the recipient is diagnosed as having a medical condition that is treatable by administration to the recipient of the therapeutic polynucleotide or a product thereof (e.g., a transcription product such as an antisense RNA or a translation product such a peptide or protein).
  • the improved gene therapy method involves: (1) contacting the bacteriophage with the target cell under conditions (a) to permit selective binding of a ligand on the surface of the bacteriophage to a receptor on the surface of the target cell and (b) to allow the bacteriophage to enter the target cell; and (2) allowing the target cell to live under conditions such that the therapeutic polynucleotide is transcribed therein.
  • Exemplary medical conditions and their respective therapeutic polynucleotides (or therapeutic polynucleotide products) that are useful for treating these medical conditions are provided in Table 2 (see, also, Bio World Financial Watch, Monday Sept. 19, 1994, pp4-10, American Health Consultants, Inc.; Gene Therapy A Primer for Physicians, Culver KW, Ed. 1996, Mary Ann Liebert, Inc. New York).
  • Therapeutic polynucleotides that are particularly suited for delivery using the bacteriophages of the invention are provided in Table 3.
  • Exemplary target cells to which the bacteriophages can deliver these and other therapeutic polynucleotides are provided in Table 4.
  • the preferred bacteriophage vectors are abortive to lytic growth in the natural bacterial flora of the mammalian host.
  • Such modified bacteriophage vectors are also abortive to lytic growth in Su° (natural E. Coli host without the amber suppressor gene mutation supE or supF, both of which code for tRNAs) bacterial host strains in vitro.
  • the bacteriophage vector genome is modified so as to contain an amber mutation inserted in-frame into a bacteriophage tail protein gene, e.g. J, M, or H gene, such that in an Su° bacterial host the tail protein gene is truncated and non-functional and, hence, abortive to lytic growth.
  • the bacteriophage vectors with tail protein amber mutations propagate only in E. Coli strains containing either the amber suppressor gene mutation supE or supF.
  • the bacteriophage vectors have temperature-sensitive tail protein mutations abortive to lytic growth at mammalian host physiologic temperature. At temperatures other than (greater or less than) the mammalian host physiologic temperatures, the tail proteins are expressed and function normally and can mediate prokaryotic cell infections in vitro. Accordingly, such modified bacteriophage vectors are packaged in vitro in cell free systems using purified protein packaging ⁇ tracts and engineered cosmid vectors.
  • the bacteriophage vectors have temperature-sensiti e tail protein mutations such that at temperatures other than (greater or less than) the mammalian host physiologic temperature, the tail proteins are expressed and function normally and can mediate prokaryotic cell infections; however, at mammalian physiologic temperature, the temperature sensitive mutation inactivates the wild type tail function.
  • the bacteriophage genome includes a promoter to control transcription and/or translation of the therapeutic polynucleotide in the target cell. Exemplary promoters are provided in Table 5.
  • the promoters are cell or tissue specific (i.e., they are functional only in particular types of cells or tissues), thereby providing an additional means for controlling expression (i.e., transcription and translation) of the therapeutic polynucleotide in the target cell.
  • the bacteriophage genome includes additional regulatory sequences, e.g., enhancers, for further controlling expression of the therapeutic polynucleotide in the target cell.
  • enhancers also are provided in Table 5.
  • the enhancers are target cell specific.
  • the bacteriophage genome can include eukaryotic origins of replication (e.g., from mammalian chromosomes), telomeres and centromeres to permit autonomous replication of the bacteriophage genome within the target cell and segregation of the replicated genome into the target cell progeny.
  • eukaryotic origins of replication e.g., from mammalian chromosomes
  • telomeres e.g., from mammalian chromosomes
  • centromeres e.g., from mammalian chromosomes
  • the bacteriophage of the invention includes on its surface a ligand which selectively binds to a receptor on the target cell surface to form a ligand-receptor complex.
  • the complex is internalized by the target cell, presumably by receptor-mediated endocytosis.
  • Applicants do not intend to limit the invention to a particular intemalization mechanism, other than limiting the invention to exclude the natural phage transduction pathway that, as described in the literature, involves tail protein mediated injection of the bacteriophage genome into the target cell.
  • the ligand can be attached to the surface of the bacteriophage using, for example, chemical modification methods, (e.g., galactosylation), genetic engineering methods (e.g., inserting a sequence encoding the ligand into the bacteriophage genome, in frame, such that the ligand is expressed on the surface of the bacteriophage), specific adsorption (e.g., coating an antibody onto the surface of a bacteriophage) or a combination of genetic engineering and affinity binding methods (e.g., expressing avidin on the bacteriophage surface to form an "avidin-labeled bacteriophage" and binding a biotinylated ligand thereto).
  • chemical modification methods e.g., galactosylation
  • genetic engineering methods e.g., inserting a sequence encoding the ligand into the bacteriophage genome, in frame, such that the ligand is expressed on the surface of the bacteriophage
  • Such avidin-labeled bacteriophages also are useful as intermediates in attaching virtually any ligand to the surface of a bacteriophage, provided that following biotinylation, the ligand retains its functional activity (i.e., the ability to selectively bind to its receptor to form a ligand-receptor complex).
  • Potential ligands include peptide or nucleotide polymers, macromolecular aggregates, such as lipoproteins or any chemical structure, either naturally occurring, synthesized, or generated by combinatorial chemistry techniques. Exemplary receptors and preferred ligands for targeting specific cell types are provided in Table 6.
  • the preferred ligands include: low density lipoprotein (apoprotein B100), very low density lipoprotein (apoprotein E or a single chain variable immunoglobulin gene fragment that has high affinity for the VLDL receptor), HDL (apoAl), galactose, c kit ligand, transferrin, insulin, heregulin, and RGD or RGD-containing polypeptides (cyclic RGD).
  • the invention also embraces ligand-labeled bacteriophages in which the ligand is an antibody (or fragment thereof) that selectively binds to an antibody receptor (e.g., an Fc receptor) on the surface of a target cell.
  • the antibody can be attached to the surface of the bacteriophage by, for example, selectively binding an antibody to a bacteriophage surface antigen.
  • the invention is useful for targeting the delivery of an exogenous polynucleotide to virtually any Fc receptor bearing target cell in vivo or ex vivo via Fc receptor-mediated intemalization
  • the avidin labeled phage can be bound to a biotinylated antibody.
  • the ligand can be attached to the surface of a bacteriophage at a location that is not involved in phage attachment or penetration of the bacterial host.
  • Other procedures such as genetic engineering/chemical modification, can be used to interfere with the function of one or more bacteriophage encoded translation products that are essential for phage attachment and/or penetration into the bacterial host.
  • C. Merril, et al. “Long-circulating bacteriophage as antibacterial agents", PNAS USA 93:3188-3192 (1996), which describes the importance played by the amino acid glutamic acid at position 158 of the capsid E protein in the insertion, in vivo, of bacteriophage lambda into E. coli).
  • an avidin-labeled bacteriophage has a genome in which the recombinant exogenous genetic material can be transcribed and translated in a mammalian cell. Since the native bacteriophage coding sequences are regulated by prokaryotic promoters, in the ensuing discussion, the recombinant bacteriophage genome will refer only to the inserted genetic material whose expression is controlled by eukaryotic regulatory elements. Preferably, for bacteriophage vectors that are intended to produce a polypeptide, the recombinant bacteriophage genome can be both transcribed and translated in the mammalian cell.
  • the avidin-labeled bacteriophages are useful as intermediates in generating the ligand-labeled bacteriophages of the invention.
  • Alternative high affinity binding pairs can be substituted for the avidin/biotin binding pair in accordance with the methods of the invention.
  • streptavidin can be substituted for avidin to form a streptavidin-labeled bacteriophage that can be allowed to bind to a biotinylated ligand to form a streptavidin-biotin complex.
  • modified avidin or streptavidin may be employed.
  • a method for introducing an exogenous polynucleotide into a target cell preferably, a human cell.
  • the method involves: (1 ) contacting the bacteriophage with the target cell under conditions: (a) to permit selective binding of a ligand on the surface of the bacteriophage to a receptor on the surface of the target cell and
  • exogenous polynucleotides embrace the above-described therapeutic polynucleotides, as well as polynucleotides that are not intended for therapeutic applications (e.g., polynucleotides that encode a mammalian protein or protein complex for production of the protein or protein complex in cell culture, transcription regulatory elements, telomeres, centromeres, splice junctions, autonomous replicating sequences, recombination specific sequences). Exemplary bacteriophages and ligands that are useful in accordance with this method are described above in reference to the improved method for gene therapy.
  • the method further involves the step of isolating an exogenous polynucleotide product (e.g., a transcription or translation product) from the target cell.
  • an exogenous polynucleotide product e.g., a transcription or translation product
  • a bacteriophage that is useful for practicing the above-described methods for delivering an exogenous polynucleotide (e.g., a therapeutic polynucleotide) to a target cell.
  • the bacteriophage which can be propagated in prokaryotes, contains a recombinant genome that can be transcribed and, optionally, translated in a mammalian cell. More preferably, the recombinant bacteriophage genome can be both transcribed and translated in the mammalian cell.
  • the surface of the bacteriophage is modified to contain thereon a ligand that selectively binds to a receptor on the mammalian target cell.
  • the bacteriophages of the invention are useful in the preparation of a medicament for treating a medical condition that is treatable by administration to the mammalian recipient of the therapeutic polynucleotide or a product thereof.
  • the bacteriophages can be placed in a pharmaceutically acceptable carrier to form a pharmaceutical composition which can be administered to the recipient in accordance with standard clinical practice l ⁇ iown to one of ordinary skill in the art.
  • the pharmaceutical composition is contained in an implant that is suitable for implantation in the mammalian recipient.
  • the methods and compositions of the invention provide for an implantable bacteriophage gene therapy vector that is useful for delivering a therapeutic polynucleotide to the mammalian recipient over an extended period of time.
  • a kit which contains: (1) a first container containing an encapsidated bacteriophage having an appropriate surface marker and a genome (preferably, a genome having multiple cloning sites, such as the lambda DASH II genome) and instructions for inserting exogenous genetic material, which may contain coding sequence and upstream and downstream regulatory elements into the genome, preferable into the multiple cloning site, (2) a second container containing an agent for attaching a ligand to the surface of the bacteriophage, wherein the ligand is designed to bind to a receptor on the surface of a mammalian cell; and (3) instructions for attaching the agent to the surface of the bacteriophage. More preferably, the kit further includes instructions for transducing a desired target mammalian cell. Alternatively, the desired ligand coding sequence is included in the genome of the bacteriophage and the agent for attaching the ligand to the surface is unnecessary.
  • Fig. 1 is a schematic diagram describing the experimental strategy for generation of targeted bacteriophage vectors by chemical modification and use of the modified bacteriophage for gene delivery to cells of specific mammalian tissue origin.
  • Fig. 2 is a schematic diagram describing the experimental strategy for generation of targeted bacteriophage vectors by fusing the coding DNA sequences of a ligand "L" into the bacteriophage virion capsid specific "D" gene.
  • Fig. 3 is a schematic representation of a chimera of lambda DASH II /CMV promoter enhancer/beta-galactosidase gene sequences.
  • Fig. 3A shows a restriction enzyme map and schematic representation of a restriction digest of the bacteriophage vector.
  • Fig. 3B shows a schematic representation of the CMV promoter enhancer/beta-galactosidase gene sequences and ligation of this DNA to the digested bacteriophage to form the chimera, followed by (a) in vitro packaging, (b) propagation in E.
  • beta-galactosidase (beta-gal) gene under the control of the CMV promoter in mammalian cells.
  • the beta-gal gene contains a nuclear localization signal that directs localization of the translation product to the nucleus.
  • Fig. 4 is a schematic representation of a chimera of lambda DASH II/PGK promoter/- galactosidase gene sequences that are formed as described above in Fig. 3.
  • Fig. 5 is a schematic representation of a MCK/DMD/lambda bacteriophage chimeric DNA construct.
  • Fig. 6 is a schematic representation of a heregulin/lambda bacteriophage chimeric DNA constmct in which a portion of the heregulin cDNA is fused, in frame, with the 3' end of the wild type capsid D-gene.
  • Fig. 6A shows generation of the polynucleotide fragments;
  • Fig. 6B shows the joining of the polynucleotide fragments;
  • Fig. 6C shows the generation of a modified bacteriophage expressing the heregulin-protein D chimeric capsid genes.
  • Fig. 7 is a schematic representation of A) the generation of targeted bacteriophage vectors by fusing a cyclic RGD ligand onto the bacteriophage lambda virion head specific D-gene product; B) generation of fragments for gene fusion; C) joining the fragments; D) generation of targeted lambda phage vector expressing the cyclic RGD-D chimeric capsid and containing the CMV beta-gal reporter gene; E) generation of eye RGD modified lambda DASH II bacteriophage containing the murine dystophin gene expression cassette; and F) generation of eye RGD modified lambda DASH II bacteriophage containing the Factor VIII/IRES/Von Willebrand's Factor gene expression cassette.
  • primers are defined as follows: primer “a” (SEQ. ID NO. 18) contains only wt sequence of the lambda DASH II "C” gene; primer “b” (SEQ. ID NO. 19) has a 3' end that is complementary to the 3' end of the wt "D” gene and a 5' end which contains the coding sequence to cyclic RGD; primer “c” (SEQ. ID NO. 20) has a 5' end that is complementary to the 5' end of cyclic RGD and a 3' end which is complementary to the 5' end of the wt "E” gene; and primer “d” (SEQ. ID NO.
  • Fragment 1 *l-5220; Fragment 2: *5221-*6142; Fragment 3: *6143-* 15855; Fragment 4: * 15856-CMV-betagal-*41900; and Fragment 5: *41900-48000).
  • bacteriophages for delivering an exogenous polynucleotide into a target cell, preferably a mammalian cell.
  • a "bacteriophage”, for the purposes of this invention, refers to a bacteriophage that: (1) contains exogenous genetic material that can be transcribed and, optionally, translated in a mammalian cell and (2) contains on its surface a ligand that selectively binds to a receptor on the surface of a target cell, such as a mammalian cell.
  • exogenous genetic material refers to a polynucleotide (e.g., nucleic acid or oligonucleotide), either natural or synthetic, that is not naturally found in a bacteriophage, or if it is naturally found in the bacteriophage, it is not transcribed or expressed at biologically significant levels by the bacteriophage.
  • polynucleotide e.g., nucleic acid or oligonucleotide
  • Exogenous genetic material includes a non-naturally occurring polynucleotide that can be transcribed into an anti-sense RNA, as well as all or part of a "heterologous gene” (i.e., a gene encoding a protein which is not expressed or is expressed at biologically insignificant levels in a naturally-occurring bacteriophage).
  • a heterologous gene i.e., a gene encoding a protein which is not expressed or is expressed at biologically insignificant levels in a naturally-occurring bacteriophage.
  • the instant invention embraces the introduction into a mammalian cell of an expression cassette including a recombinant gene containing an inducible promoter operably coupled to a coding sequence of a therapeutic polynucleotide.
  • the exogenous genetic material of the bacteriophage can be both transcribed and translated in the mammalian target cell.
  • Exemplary bacteriophages that satisfy at least the first of these criteria are provided in Table 1 .
  • Exemplary ligands that can be attached to the bacteriophage surface, e.g., covalently coupled to the surface, expressed, or specifically adsorbed or affinity bound thereto, are provided in Table 6. (Tables 1-8 are presented at the end of the detailed description of the invention, immediately preceding the specific Examples section.)
  • the bacteriophages are useful for delivering an exogenous polynucleotide into a mammalian target cell for ex vivo and in vivo gene therapy, as well as for producing exogenous polynucleotide products (e.g., transcription products such as antisense mRNA or catalytic RNAs and translation products) in culture or in vivo.
  • the bacteriophages of the invention are particularly useful for delivering an exogenous polynucleotide containing between about one and one-hundred kilobases to a mammalian target cell, depending on the particular bacteriophage that is selected.
  • lambda and pi can be used to deliver exogenous polynucleotides containing up to 9 to 23 kb and up to 75 to 100 kb, respectively.
  • a minimal lambda cassette can deliver exogenous polynucleotides containing up to 50 kb.
  • conventional viral vectors for gene therapy viral vectors can accommodate, at best, a polynucleotide containing up to about seven kilobases for delivery to a mammalian cell.
  • the instant invention advantageously provides a method for delivering relatively large genes and/or multi-gene complexes to a mammalian cell for gene therapy purposes and for the in vitro or in vivo production of gene products.
  • the bacteriophages of the invention can be propagated in prokaryotic cells. Accordingly, the cost of producing the bacteriophages of the invention is relatively inexpensive compared to the cost of producing more conventional gene therapy vectors, such as retroviruses, adenovirus, or adeno-associated vims.
  • an improved method for gene therapy utilizes a bacteriophage as a vector to introduce a therapeutic polynucleotide into a target cell of a mammalian recipient.
  • the improved gene therapy method involves two steps: (1 ) contacting the bacteriophage with the target cell under conditions (a) to permit selective binding of a ligand on the surface of the bacteriophage to a receptor on the surface of the target cell and (b) to allow the bacteriophage to enter the target cell; and (2) allowing the target cell to live under conditions such that the therapeutic polynucleotide is transcribed therein.
  • the mammalian recipient is diagnosed as having a medical condition that is treatable by administration to the recipient of the therapeutic polynucleotide or a product thereof.
  • the mammalian recipient is a human.
  • Exemplary medical conditions and their respective therapeutic polynucleotides (or products thereof) that are useful for treating these conditions are provided in Table 2.
  • the medical conditions that are treatable in accordance with the methods of the invention include genetic diseases (i.e., diseases that are attributable to one or more gene defects) and acquired pathologies (i.e., pathological condition that are not attributable to an inbom genetic defect).
  • the improved method for gene therapy also embraces prophylactic processes (i.e., delaying the onset of the foregoing medical conditions).
  • the bacteriophage genome contains a therapeutic polynucleotide that encodes a therapeutic polynucleotide product which is useful for treating (i.e., delaying the onset, inhibiting or reducing the symptoms of) the medical condition.
  • a therapeutic polynucleotide refers to a polynucleotide that mediates a therapeutic benefit in a recipient of the polynucleotide or product thereof.
  • a therapeutic benefit may be an alteration of cell proliferation, a change of expression of a single or multiple genes or proteins, a cytotoxic effect against a pathogen, inhibition of viral replication, replacement of a defective gene and the like.
  • Therapeutic polynucleotides may be administered in the form of a polynucleotide operably joined to regulatory sequences, disposed in the bacteriophage vector for replication or regulated expression, or in separate non-operable pieces that can become operably joined in the target cell to yield an operable expression system.
  • Therapeutic polynucleotides include genes encoding the transcription and translation products identified in Table 2.
  • Therapeutic polynucleotides also embrace polynucleotides that encode diagnostic agents that can be detected in situ or ex vivo and that are useful in diagnosing a medical condition.
  • Therapeutic polynucleotides that encode diagnostic agents include the genes encoding, for example, an enzyme that catalyzes a reaction, in situ, to yield a detectable product.
  • a "therapeutic polynucleotide product” refers to a molecule produced as a result of transcription or translation of the therapeutic polynucleotide.
  • Therapeutic polynucleotide products include transcription products
  • RNA e.g., antisense mRNA and catalytic RNA
  • translation products e.g., proteins or peptides
  • Antisense oligonucleotides that have been approved for gene therapy protocols and/or clinical trials are provided in Table 2.
  • the phrases "antisense oligonucleotides” or “antisense” describe an oligoribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an RNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of the mRNA.
  • the antisense molecules are designed so as to hybridize with the target gene or target gene product and thereby, interfere with transcription or translation of the target mammalian cell gene.
  • the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.
  • antisense oligonucleotides Based upon the known sequence of a gene that is targeted for inhibition by antisense hybridization, or upon allelic or homologous genomic and/or cDN A sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.
  • antisense oligonucleotides should comprise at least 7 and, more preferably, at least 15 consecutive bases which are complementary to the target. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region of the gene or RNA (e.g., mRNA) transcripts
  • the antisense oligonucleotides are complementary to 5' sites, such as translation initiation, transcription initiation or promoter sites, that are upstream of the gene that is targeted for inhibition by the antisense oligonucleotides.
  • 5' sites such as translation initiation, transcription initiation or promoter sites
  • 3 '-untranslated regions or telomerase binding sites may be targeted.
  • 5' or 3' enhancers may be targeted. Targeting to mRNA splice sites has also been used in the art.
  • the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457 (1994)) and at which proteins are not expected to bind.
  • the bacteriophages of the invention by virtue of their ability to accommodate therapeutic polynucleotides which are relatively large in size, are particularly useful for delivering to the target cell a polynucleotide that encodes multiple copies of the same or different mRNAs in tandem, thereby increasing the effective concentration of antisense mRNA in the target cell.
  • the selective binding of the antisense oligonucleotide to a mammalian target cell nucleic acid effectively decreases or eliminates the transcription or translation of the mammalian target cell nucleic acid molecule. Reduction in transcription or translation of the nucleic acid molecule is desirable in those medical conditions where transcription and translation of the mammalian target cell nucleic acid leads to an adverse medical condition.
  • the antisense oligonucleotides of the invention can be used to reduce the expression of oncogenes to treat cancers whose proliferation is mediated by expression of these oncogenes.
  • the bacteriophages of the invention are also useful for delivering therapeutic polynucleotides that encode specific antigen peptides to antigen presenting cells for processing and presentation at the cell surface to enhance the immune system response of the mammalian recipient to a specific peptide antigen.
  • exemplary peptide antigens that can be expressed to induce or otherwise enhance an immune response are shown in Table 7.
  • the therapeutic polynucleotide encodes one or more peptide antigens that vaccinate the mammalian recipient against a tumor, a vims, a bacteria, or a parasite.
  • auxiliary therapeutic polynucleotides are inserted into the bacteriophage genome to enhance or otherwise improve the therapeutic efficacy of the therapeutic polynucleotide product in treating the condition.
  • auxiliary polynucleotides for delivery to the mammalian target cell include polynucleotides encoding tumor suppressor genes, polynucleotides encoding antisense mRNA or encoding catalytic RNA that inactivate oncogenes, and polynucleotides that render a target tumor cell more susceptible to an administered dmg (e.g., suicide genes encoding, for example, thymidine kinase).
  • Auxiliary polynucleotides also include polynucleotides encoding cytokines that enhance a naturally occurring anti-tumor immunity.
  • cytokines which have this function include, e.g., IL-4, TNF, IL-2, and GM-CSF.
  • the therapeutic polynucleotide is inserted into the bacteriophage genome using conventional recombinant DNA techniques. See, e.g., Methods in Enzymology, vol. 152, ed. S. L. Berger, A.R. Kimmel (1987) Academic Press, New York, NY.
  • the bacteriophage is a lambda phage and the therapeutic polynucleotide is inserted into well-defined restrictions sites in the lambda phage.
  • recombination sequences i.e., polynucleotides having a nucleic acid that allows homologous recombination
  • recombination sequences are provided at the 5' and 3' ends of the therapeutic polynucleotide to permit site-directed insertion of the therapeutic polynucleotide into a preselected location in the genomic DNA of the target cell via homologous recombination.
  • the bacteriophages of the invention can accommodate a therapeutic polynucleotide containing between about one and up to one-hundred kilobases, depending upon the particular bacteriophage selected.
  • lambda bacteriophages e.g. lambda DASH II, and pi phage can accommodate up to about 9 to 23 kb and up to about 75 to 100 kb, respectively.
  • Minimal lambda cassette such as described below, can accommodate up to about 50 kb.
  • the therapeutic polynucleotide contains between about 10 and 90 kilobases, more preferably, the therapeutic polynucleotide contains between about 15 and 85 kilobases.
  • the improved gene therapy method disclosed herein is particularly useful for gene therapy applications which require administration of a single therapeutic polynucleotide (or a product thereof) having a size within the foregoing kilobase range, as well as for delivering multiple therapeutic polynucleotides which, together, have a size within this kilobase range.
  • Exemplary therapeutic polynucleotides containing more than 7 kilobases include dystrophin, members of the globin gene complex, clotting factor VIII, von Willebrand's factor, collagen type VII, fibrillin, and any other gene(s)/gene complexes than are too large to deliver (efficiently) to mammalian cells using conventional viral vectors.
  • Additional therapeutic polynucleotides that can be delivered in accordance with the methods of the invention and that fall within the preferred kilobase size ranges can be identified by, for example, referring to the GenBank or other gene sequence data bases. See, also, Table 3 for a list of preferred therapeutic polynucleotides that can be delivered using the bacteriophages disclosed herein.
  • the delivery of a therapeutic polynucleotide containing more than 7 kilobases has not been possible using conventional gene therapy viral vectors.
  • Table 8 A summary of the insert size limitations for conventional gene therapy vectors compared to the bacteriophages disclosed herein is provided in Table 8.
  • the packaging and engineering of the lambda bacteriophage vector can be modified to permit the vector to accommodate up to approximately 50 kb of exogenous coding sequence.
  • This approach involves engineering a recombinant cosmid vector DNA constmct that contains an antibiotic resistance gene, e.g. ampicillin, a lambda origin of replication, and a DNA insert up to 50 kb in size flanked by COS (CoheSive ends of wild type bacteriophage lambda genome) sites.
  • This cosmid can be replicated to very high copy numbers in standard strains of E. Coli and then can be isolated using standard techniques for use in the packaging as described below.
  • the second component of this modified packaging system is a COS-negative lambda lysogen strain of bacteria in whose bacterial chromosome is integrated the stmctural proteins and enzymes requisite for packaging of an infective lambda vims.
  • the lysogen strain is engineered to contain modifications of certain packaging proteins such that the final modified lambda phage vector is able to effectively target the intended cell type(s), sub-cellular compartments, or sub-cellular organelles.
  • E. Coli can be infected with a modified bacteriophage that contains a fusion D gene-RGD constmct Using standard methods lysogen that contain the D gene-RGD fusion constmct can be selected .
  • bacteriophage genomes or minimal lambda cassettes can be encapsidated in vitro with a D gene-RGD fusion protein that can target the recombinant vims to cells expressing RGD's cognate receptor.
  • the D-gene is modified to include in frame a ligand, e.g. cyclic RGD, so that the vector is internalized by the targeted cell type(s).
  • This lysogen strain is incapable of producing vims because the COS sites are absent.
  • the lysogen strains are grown to large quantities using standard bacterial culture techniques.
  • the lambda packaging proteins, including any modified forms of these proteins can be obtained by standard methods, such as freeze thawing and sonication of lysogen.
  • the packaging of the high capacity bacteriophage lambda vector is accomplished by mixing the purified engineered cosmid DNA with the isolated protein extract from the above lysogen strain at approximately room temperature. This mixing results in the packaging of replication deficient modified bacteriophage lambda vims particles that contain both surface proteins as determined by the modified lysogen strain from which the packaging proteins are derived and a genome of an insert of up to about 50 kb flanked by COS sites yet lacking other bacteriophage coding sequences.
  • the bacteriophage genome further includes a regulatory sequence, e.g., a promoter region (also referred to as a "promoter”), that is operably coupled to the therapeutic polynucleotide.
  • the regulatory sequence controls the expression of the therapeutic polynucleotide in the target cell.
  • a therapeutic polynucleotide also referred to as "coding sequence” that encodes a therapeutic polynucleotide product
  • regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the transcription or the expression of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequence results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 3' or 5' non-transcribed and or non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, CAAT sequence, and the like.
  • 5' non- transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream 5' or downstream 3' transcriptional regulatory sequences as desired.
  • the bacteriophages of the invention optionally include 5' leader signal, or membrane integration sequences 5' or 3'.
  • Exemplary promoters that are useful for protecting the instant invention are provided in Table 5, which shows both constitutive promoters and regulatable promoters (e.g., cell lineage specific promoters, inducible promoters). Exemplary constitutive promoters also are included in Table 5.
  • the constitutive promoter is selected from the group consisting of a promoter of the phosphoglycerokinase gene, a long terminal repeat (LTR) of retrovimses, e.g., Rous sarcoma vims, Moloney murine leukemia vims.
  • LTR long terminal repeat
  • tissue or cell specific transcriptional regulatory sequences are derived from the genes encoding the following proteins: tyrosinase, lipoprotein lipase, albumin, muscle creatine kinase, keratin (K14/K10), globin gene cluster, immunoglobulin heavy chain gene cluster, and involucrin.
  • liver-specific promoters such as the albumin promoter/enhancer
  • PCT application number PCT/US95/1 1456 having international publication number WO96/09074, entitled "Use of a Non-mammalian DNA Vims to Express an Exogenous Gene in a Mammalian Cell," hereinafter WO 96/09074, and the references cited therein.
  • the alpha-feto protein promoter can be used to effect expression of a therapeutic polynucleotide(s) in liver tumor cells (but not normal liver cells) for treating liver cancer.
  • Exemplary inducible promoters are identified in Table 5 and are described in the following references: Science 268: 1786 (1995); TIBS 18:471 (1993); PNAS 91 :3180 (1994); PNAS
  • the preferred inducible promoter system is the tetracycline inducible system.
  • An exemplary repressible promoter, the tetracycline repressible system, is identified in Table 5 and is described in PNAS 89:5547 (1992).
  • the IRES allows initiation of translation of the trans- activating polypeptide or the coding sequence of interest, independently, from a single polycistronic message.
  • the transactivator is not activated and the tetO driven transcription of the coding sequence of interest is substantially reduced to negligible levels.
  • the bacteriophage genome further includes an enhancer region ("enhancer").
  • enhancer region that are useful for practicing the instant invention are provided in Table 5.
  • the preferred enhances are selected from the group consisting of the following: a locus control
  • the bacteriophage genome can be engineered to contain an origin of replication to effect autonomous replication and facilitate persistence of the therapeutic polynucleotide in the mammalian cell. Origins of replication derived from mammalian target cells have been identified (see, e.g., ⁇ o Burhans, et al., 1994, Science 263 : 639-640).
  • the bacteriophages optionally contain one or more sequences that are suitable for use in the identification of cells that have or have not been transfected.
  • Transfection refers to the introduction of the bacteriophage genome into the target cell. Markers to identify cells that have been transfected include, for example, genes encoding proteins that increase or i5 decrease resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes having activities that are detectable by standard assays known in the art and genes which detectably (e.g. visibly) effect the phenotype of the transfected target cells, hosts, or plaques. Exemplary genes that are suitable as markers include the lacZ genes, chloramphenicol acetyltransferase genes, alkaline phosphatase genes, luciferase genes, and green fluorescent
  • the bacteriophages of the invention contain on their surface a ligand that selectively binds to a receptor expressed on the surface of the target cell.
  • the bacteriophage ligand binds to the target cell receptor to form a ligand-receptor complex that is internalized by the target cell. In this manner, the bacteriophage can be targeted for delivery to a pre-selected cell or tissue type,
  • ligand/receptor pair wherein the receptor is selectively expressed on a specific population of cells.
  • Exemplary pairs of ligands/receptors which are useful in accordance with the methods of the invention are provided in Table 6.
  • the preferred ligand/receptor pairs for use in accordance with the methods of the invention include the following: insulin/insulin receptor, heregulin/heregulin receptor, 0 keratinocyte growth factor/keratinocyte growth factor receptor, hepatic growth factor/hepatic growth factor receptor, RGD peptides/integrin alpha-5:beta-l, interleukin-2/interleukin-2 receptor, galactose/asialoglycoprotein, low density lipoprotein (LDL) or apoBlOO/LDL receptor, very low density lipoprotein (VLDL). apoE/VLDL receptor, or HDL or apoAl/HDL receptor.
  • insulin/insulin receptor heregulin/heregulin receptor
  • 0 keratinocyte growth factor/keratinocyte growth factor receptor hepatic growth factor/hepatic growth factor receptor
  • RGD peptides/integrin alpha-5:beta-l interleukin-2/interleukin-2 receptor
  • antibodies can be attached to the bacteriophage via interaction of a bacteriophage coat protein specific antibody with its cognate antigen or via interaction of a recombinant bacteriophage coat protein that contains avidin and a biotinylated antibody. Once the antibody is tightly bound to the bacteriophage as described above, the antibody can direct the bacteriophage either to cells that express the Fc receptor in the case of a coat protein specific IgG antibody or to cells that express the cognate antigen of the attached biotinylated antibody.
  • Selective ligand-receptor interaction also is useful for mediating intemalization of the bacteriophage into subcellular locations e.g., the nucleus, mitochondria, and other membranes-bound organelles or cytoplasmic molecular aggregates of protein and/or nucleic acid.
  • novel ligands can be identified using phage display procedures such as those described in (S. Hart, et al., J. Biol. Chem. 269(17): 12468 (1994)). While such filamentous phages could, of course, never be used to deliver genetic material to a cell (because they are single stranded), this methodology is potentially very useful in the discovery of novel receptor ligand interactions.
  • phage display libraries using, e.g., Ml 3 or fd phage are prepared using conventional procedures such as those described in the foregoing reference.
  • the libraries display inserts containing from 4 to 80 amino acid residues.
  • the inserts optionally represent a completely degenerate or a biased array of peptides.
  • Ligands that bind selectively to a particularly type of target cell are obtained by selecting those phages which express on their surface a ligand that binds to the target cell of interest. These phages then are subjected to several cycles of reselection to identify the peptide ligand-expressing phages that have the most useful binding characteristics.
  • phages that exhibit the best binding characteristics are further characterized by nucleic acid analysis to identify the particular amino acid sequences of the peptides expressed on the phage surface and the optimum length of the expressed peptide to achieve optimum binding to the target mammalian cell.
  • peptide ligands can be selected from combinatorial libraries of peptides containing one or more amino acids. Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. These novel ligands can be attached to the bacteriophage surface to deliver the phage to the particular target cell of interest.
  • the ligands may be selected from polynucleotide libraries, as polynucleotides can also demonstrate specific affinity for polypeptide cell surface receptors.
  • Exemplary screening methods for selecting bacteriophage vectors that transduce mammalian target cells via receptor-mediated endocytosis and target cells that internalize the bacteriophage vectors by this mechanism are described in the Examples.
  • At least four general procedures are available for attaching a ligand to the surface of a bacteriophage. These include (1) chemical modification of the bacteriophage surface (e.g., galactosylation, cross linking reactions); (2) modification of the bacteriophage genome to express a ligand on the bacteriophage surface (e.g., a fusion protein formed between the ligand and a functional viral packaging protein); (3) selective binding of a ligand (e.g., a monoclonal antibody, a polyclonal antibody, or functionally active fragments thereof containing an Fc domain) to a bacteriophage surface antigen to mediate targeting of the bacteriophage to cells that express an Fc receptor on their surface; and (4) modification of the bacteriophage genome to express a surface avidin-bacteriophage coat protein fusion product to which a biotinylated ligand (e-g-, antibody) can be attached.
  • chemical modification of the bacteriophage surface
  • the simplest method for attaching a ligand to the surface of a bacteriophage is a chemical modification reaction in which the surface of the bacteriophage is subjected to galactosylation or lactosylation via N-linked glycosidic covalent linkages so as to attach galactose or lactose, respectively, to the bacteriophage surface.
  • galactosylation or lactosylation via N-linked glycosidic covalent linkages so as to attach galactose or lactose, respectively, to the bacteriophage surface.
  • Galactose- or lactose-labeled bacteriophages selectively bind to asialoglycoprotein receptors on the surface of hepatocytes to form a ligand-bacteriophage complex that is internalized by the target cell.
  • Chemical modification also can be used to attach a peptide ligand to the bacteriophage surface.
  • peptide ligands containing a free amine group, carboxyl group, or sulfhydryl group can be attached to the bacteriophage surface using conventional procedures known to those of ordinary skill in the art for cross linking proteins. See, e.g., U.S. Patent No. 5,108,921, issued to
  • peptide ligand does not have a free amine or carboxyl group
  • such a group can be introduced by, for example, introducing a cysteine (containing a reactive thiol group) into the peptide ligand by site directed mutagenesis.
  • Disulfide linkages can be formed between thiol groups in, for example, the peptide ligand and a protein expressed on the surface of the bacteriophage.
  • covalent linkages can be formed using bifunctional crosslinking agents that are known by those of ordinary skill in the art to have utility with respect to crosslinking peptides and proteins.
  • Exemplary crosslinking agents include bismaleimidohexane (which contains thiol-reactive maleimide groups and which forms covalent bonds with free thiols). See, also, the Pierce Co. Immunotechnology Catalogue and Handbook Vol. 1 for a list of exemplary homo- and hetero-bifunctional crosslinking agents, thiol -containing amines and other molecules with reactive groups for a comprehensive list of commercially available agents and corresponding peptide coupling chemistries that can be used to attach a peptide ligand to, for example, an amino acid functional group (e.g., amine) on the surface of a bacteriophage.
  • an amino acid functional group e.g., amine
  • exemplary coupling chemistries that are suitable for this purpose include methods which utilize the following crosslinking agents: glutaraldehyde (M. Riechlin, Methods in Enzymology 70:159- 165 (1980); N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (T.L. Goodfriend, et al., Science 144: 1344-1346 (1964); and N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (M.H. Klapper and I.M. Klotz, Methods in Enzymology 25:531-536 (1972)).
  • glutaraldehyde M. Riechlin, Methods in Enzymology 70:159- 165 (1980
  • N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide T.L. Goodfriend, et al., Science 144: 1344
  • the ligand-labeled bacteriophages can be prepared by using well-known methods for forming amide, ester or imino bonds between acid, aldehyde, hydroxy, amino, or hydrazo groups on the respective bacteriophage and ligand.
  • reactive functional groups that are present in the amino acid side chains of the bacteriophage extracellular proteins or peptide ligands preferably are protected with a protecting group prior to coupling the ligand to the bacteriophage.
  • protecting group refers to a molecule which is bound to a functional group on a bacteriophage or peptide ligand and which may be selectively removed therefrom to expose the functional group in a reactive form.
  • the protecting groups are reversibly attached to the functional groups and can be removed therefrom using, for example, chemical or other cleavage methods.
  • side-chain-blocked amino acids e.g., FMOC- derived amino acids from Advanced Chemtech. Inc., Louisville, KY
  • side-chain-blocked amino acids can be used to synthesize peptide ligands in accordance with standard peptide synthesis methods to obtain peptide ligands having side-chain-blocked amino acids.
  • the peptide ligand side chains can be reacted with protecting groups after peptide ligand synthesis, but prior to the covalent coupling reaction.
  • the ligand-labeled bacteriophages of the invention can be prepared in which the amino acid side chains of the peptide ligand do not participate to any significant extent in the coupling of the peptide ligand to functional groups on the surface of the bacteriophage.
  • ligand coding sequence can be inserted, in frame, into the bacteriophage genome adjacent to or within a bacteriophage protein that is expressed on the bacteriophage surface to provide a fusion protein that contains both functional ligand and a functional viral packaging protein.
  • the ligand-labeled bacteriophage is formed by selectively binding an antibody or functionally active fragment thereof (i.e., an antibody fragment containing at least one antigen-binding site) to an antigen that is contained on the surface of the bacteriophage (e.g., a bacterial extracellular protein).
  • the selectively bound antibodies mediate targeting of the bacteriophage to a target cell that contains on its surface an Fc receptor (e.g., a phagocyte or antigen presenting cell).
  • Antibodies that are useful in accordance with this aspect of the invention are antibodies that exhibit a sufficiently high binding affinity for a bacteriophage antigen to result in little or no dissociation of the antibody-antigen complex under physiological conditions.
  • such antibody binding to the surface of the bacteriophage is performed by contacting the bacteriophage with an antibody that selectively binds to an antigen expressed on the bacteriophage surface under the same conditions that are used for performing an immunoassay, e.g., an ELISA, RIA.
  • an immunoassay e.g., an ELISA, RIA.
  • Exemplary conditions are described in Current Protocols in Immunology, ed. Coligan, J.E., et al., National Institutes of Health, John Wiley and Sons, Inc. ( 1994).
  • the ligand is attached to the surface of the bacteriophage by means of an avidin/biotin complex.
  • avidin or “avidin peptide” refers to an avidin molecule, a streptavidin molecule, or a fragment or variant thereof that binds to biotin with an affinity that is approximately the same (i.e., within 10%) or greater than the affinity with which streptavidin binds to biotin.
  • the bacteriophage is modified to express on its surface avidin or a portion thereof that selectively binds to biotin with the requisite binding affinity.
  • Modification of the bacteriophage to express avidin is most easily accomplished by inserting the nucleic acid encoding avidin or a functionally active portion thereof into the bacteriophage genome such that the avidin or avidin portion is expressed on the bacteriophage surface.
  • the avidin can be inserted, in frame, into the D gene of the lambda coat protein, using well-defined restriction sites in the lambda phage. (See, e.g., the Examples and figures.) In this manner, an avidin-expressing bacteriophage is produced which serves as an intermediate for attachment of a biotinylated ligand to the bacteriophage surface.
  • avidin or a functionally active portion thereof can be chemically coupled to the bacteriophage surface using standard cross-linking chemistries, such as those described above.
  • the avidin-labeled bacteriophage permits non-covalent, yet high affinity, attachment of pre-selected biotinylated ligands to the bacteriophage surface for receptor-mediated targeted delivery to the mammalian target cell.
  • Exemplary ligands which can be biotinylated in accordance with standard procedures are provided in Table 6.
  • the bacteriophage can be biotinylated and an avidin-labeled ligand can be used to form the ligand-labeled bacteriophages described herein.
  • the bacteriophages of the invention are contacted with the target cell under conditions to permit selective binding of the ligand on the surface of the bacteriophage to the receptor on the surface of the target cell and to allow the bacteriophage to enter the target cell.
  • Conditions which permit the binding of a receptor to its cognate ligand are the physiological conditions (e.g., the particular pH, ionic strength, viscosity) at which the ligands and receptors are known to bind to one another in vivo and the conditions at which the ligands and receptors are known to bind to one another in vitro, such as in receptor assays for determining the presence of a ligand in, for example, a biological fluid.
  • Such conditions are known to those of ordinary skill in the art of receptor-mediated processes, such as receptor-based binding assays and receptor-mediated delivery of therapeutic agents to preselected tissues in situ.
  • the conditions that allow the target cell to live and transcribe the therapeutic polynucleotide are the same conditions that permit selective binding of the ligand to the receptor and that allow the bacteriophage to enter the target cell.
  • the conditions that allow the cell to transcribe the therapeutic polynucleotide further include the addition of an inducer (see, e.g., Table 5) that activates an inducible promoter to induce transcription and translation of the therapeutic polynucleotide.
  • an inducer see, e.g., Table 5
  • the optimum conditions for inducing the transcription and translation of a therapeutic polynucleotide that is under the control of a particular inducible promoter can be determined by one of ordinary skill in the art using no more than routine experimentation.
  • the mammalian cell can be allowed to live on a substrate containing collagen, e.g., type I collagen, or a matrix containing laminin, such as described in PCT application number PCT/US95/1 1456, having international publication number WO96/09074, entitled "Use of a Non-mammalian DNA Vims to Express an Exogenous Gene in a Mammalian Cell," and the references cited therein.
  • collagen e.g., type I collagen
  • laminin such as described in PCT application number PCT/US95/1 1456, having international publication number WO96/09074, entitled "Use of a Non-mammalian DNA Vims to Express an Exogenous Gene in a Mammalian Cell," and the references cited therein.
  • contacting in reference to the bacteriophage and the target cell, refers to bringing the bacteriophage into sufficiently close proximity to the target cell to permit the receptor on the target cell to selectively bind to the ligand on the bacteriophage.
  • Such conditions are well known to those of ordinary skill in the art and are exemplified by the procedure provided in the Examples. See also, e.g., U.S. patent No. 5,108,921 , issued to Low et al. which reports the conditions for receptor-mediated delivery of "exogenous molecules” such as peptides, proteins and nucleic acids to animal cells and U.S. patent No.
  • the bacteriophage can be contacted with the targeted mammalian cell in vitro, for example, for ex vivo gene therapy or production of a catalytic RNA or recombinant protein in cell culture, or in vivo for in vivo gene therapy or in vivo production of a polynucleotide transcription or translation product.
  • a "mammalian target cell” refers to a mammalian cell (preferably, a human cell) which contains on its surface a receptor for the ligand that is contained (e.g., expressed) on the surface of the bacteriophage.
  • any mammalian cell can be targeted in accordance with the methods of the invention.
  • the cell may be a primary cell or may be a cell of an established cell line.
  • Exemplary cell types that can be targeted in accordance with the methods of the invention are provided in Table 4.
  • the mammalian cell is a hepatocyte (liver cell), a breast epithelial cell, a keratinocyte, a melanocyte, or a hematopoietic cell, e.g., erythrocyte, leukocyte, monocyte, or a lymphocyte.
  • Screening methods can be used to confirm that these and other target cells internalize the bacteriophage vectors of the invention via receptor-mediated endocytosis and, further, that these target cells express detectable levels of the exogenous polynucleotide insert.
  • Such high-throughput screening methods can be used to select target cells that satisfy the above-noted criteria using no more than routine experimentation.
  • screening assays are predictive of receptor-mediated endocytosis of target cells in vivo.
  • the target cell subsequently can be introduced into the mammal (e.g., into the portal vein or into the spleen) if desired.
  • expression of the therapeutic polynucleotide is accomplished by allowing the cell to live or propagate in vitro, in vivo, or in vitro and in vivo, sequentially.
  • the invention is used to express a therapeutic polynucleotide in more than one cell, a combination of in vitro and in vivo methods are used to introduce the therapeutic polynucleotide into more than one mammalian cell.
  • the cells are removed from a subject and a therapeutic polynucleotide is introduced (i.e., transfected) into the cells in vitro.
  • a therapeutic polynucleotide is introduced (i.e., transfected) into the cells in vitro.
  • the transfected cells are expanded in culture before being reimplanted into the mammalian recipient.
  • the procedure for performing ex vivo gene therapy is outlined in U.S. Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents.
  • ex vivo gene therapy involves the introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject.
  • the functional copy of the gene is under the operable control of regulatory elements which permit expression of the gene in the genetically engineered cell(s).
  • the target cells are not removed from the patient.
  • the therapeutic polynucleotide is introduced into the cells of the mammalian recipient in situ, i.e., within the recipient.
  • the improved gene therapy method disclosed herein is practiced by using the ligand-labeled bacteriophage of the invention in place of the gene therapy vectors disclosed in the prior art (e.g., adenoviral vectors, modified adenoviral vectors, retroviral vectors, plasmids, liposomes) in the procedures currently used for administering these vectors (or cells containing these vectors) to the subjects.
  • adenoviral vectors e.g., adenoviral vectors, modified adenoviral vectors, retroviral vectors, plasmids, liposomes
  • Such procedures are known to those of skill in the art of human gene therapy. (See, also, the references identified below with respect to in vivo gene therapy.)
  • a particularly preferred embodiment of the invention is illustrated in the Examples, which describes the constmction of a lambda bacteriophage for use as a gene therapy vector.
  • a bacteriophage containing an exogenous polynucleotide (encoding beta-galactosidase) was chemically modified (galactosylated) to contain galactose residues on the bacteriophage surface.
  • the galactose-labeled bacteriophage was internalized by HepG2 cells (a hepatoma cell line) in culture, presumably, by binding of the labeled bacteriophage to the asialoglycoprotein receptor present on the surface of the hepatocytes.
  • Insertion of the therapeutic polynucleotide into the target cell genome may be either transient or permanent.
  • transient it is meant that the bacteriophage genome lacks the capacity to replicate and/or to segregate to progeny cells.
  • the therapeutic polynucleotide may be epigenetic and without the capacity to replicate and segregate to progeny cells (e.g., lacks an origin of replication, appropriate telomere and centromere stmctures).
  • Transient insertion into the target cell also occurs, for example, when the bacteriophage is used to infect cells of limited replicative capacity, i.e., non-stem cells.
  • Permanent insertion of the therapeutic polynucleotide into the target cell is accomplished by, for example, (1 ) infecting stem cells that produce bacteriophage-bearing progeny; or (2) including recombination sequences in the bacteriophage genome on either side of the therapeutic polynucleotide so as to promote reasonably efficient homologous recombination of the therapeutic polynucleotide into a defined sequence of the target cell genome or (3) random integration into the host cell chromosomal
  • Permanent insertion also can be achieved by including in the bacteriophage an origin of replication, telomeres and centromeres to obtain a bacteriophage that autonomously replicates (i.e., an "artificial chromosome") and is capable of segregating into progeny cells. If the bacteriophage genome is autonomously replicating, it is preferred to further include in the bacteriophage genome appropriate enhancer-promoter sequences, such as those described in the aforementioned tables.
  • the bacteriophage is administered to the mammalian recipient, for example, intravascularly, intraluminally (introduction of the bacteriophage into body cavities and lumens, such as the genital urinary tract, gastrointestinal tract, trachea-bronchopulmonary tree or other internal tubular structures), direct injection into a tissue (e.g., muscle, liver), topical application (e.g., eye drops or aerosol application to mucosal surfaces), or intracavitary (e.g., intraperitoneally or intrathecally (introduction into the cerebrospinal fluid).
  • a tissue e.g., muscle, liver
  • topical application e.g., eye drops or aerosol application to mucosal surfaces
  • intracavitary e.g., intraperitoneally or intrathecally (introduction into the cerebrospinal fluid).
  • an implantable pump or other device or implant to effect the sustained release of the bacteriophage can be used to facilitate delivery of the bacteriophage to the mammalian targeted cell over a pre-selected period of time (e.g., sustained release over a period of days to sustained release over a period of weeks to months).
  • a pre-selected period of time e.g., sustained release over a period of days to sustained release over a period of weeks to months.
  • the ligand receptor-mediated delivery of the bacteriophage is the predominant mechanism for targeting delivery of the bacteriophage to a particular cell type, delivery to the target cell can further be modulated by regulating the amount of bacteriophage administered to the mammalian recipient and or by controlling the method of delivery.
  • intravascular administration of the bacteriophage to the portal vein or to the hepatic artery can be used to facilitate targeting the bacteriophage to a liver cell.
  • the bacteriophage can be administered to the mammalian recipient using the same modes of administration that currently are used for adenovims-mediated gene therapy in humans.
  • Such conditions are adequate for contacting the bacteriophage and the target cell under conditions to permit selective binding of a ligand on the surface of the bacteriophage to a receptor on the surface of the target cell and to allow the bacteriophage to enter the target cell.
  • immunosuppressive dmgs such as glucocorticosteroids or cyclophosphamide are co- administered with the bacteriophage to suppress a primary immune response that may be triggered by an initial exposure to a foreign antigen.
  • Mammalian cells which have been transfected with the bacteriophage ex vivo can be introduced into the mammalian recipient using the known methods for implanting transfected cells into a human for gene therapy. See, e.g., U.S. Patent No. 5,399,346 ("Gene Therapy") issued to Anderson et al.; PCT International application no. PCT/US92/01890 (Publication No.
  • the invention is not limited in utility to human gene therapy, but also can be used in the manufacture of a wide variety of proteins and nucleic acids that are useful in the fields of biology and medicine.
  • the bacteriophages of the invention advantageously provide a method for synthesizing gene products from genes which range in size from about one to one-hundred kilobases. Further, the invention provides a method for providing the bacteriophage vectors at a low cost, namely, by propagating the bacteriophages in a prokaryotic host. Moreover, the invention provides a simple method for preparing mammalian proteins, including proper post- translational modifications, in vitro.
  • the invention provides an improved method for introducing an exogenous polynucleotide into a mammalian cell.
  • the improved method involves contacting the bacteriophages of the invention (which contain the exogenous polynucleotide) with the mammalian cell and allowing the bacteriophage to enter the cell, gain access to the nucleus, and replicate the exogenous polynucleotide therein.
  • the bacteriophage contains on its surface the ligand that selectively binds to a receptor for the ligand that is contained on the surface of the mammalian cell.
  • exogenous polynucleotide refers to a nucleic acid that is not normally present in the bacteriophage genome and that is inserted into the bacteriophage using recombinant engineering methodology.
  • exogenous polynucleotides include the above described therapeutic polynucleotides, as well as regulatory polynucleotides which are not intended for therapeutic applications (e.g., polynucleotides that are introduced into the mammalian cell in vitro or in vivo for the purpose of producing a mammalian protein/protein complex in vitro or in vivo).
  • the invention provides a generic mechanism for forming a bacteriophage which contains on its surface virtually any type of ligand.
  • the avidin-labeled bacteriophage can be provided as a component of a kit for labeling a bacteriophage with a ligand of choice.
  • the kit includes instmctions for forming a ligand-labeled bacteriophage by allowing the avidin-bacteriophage to react with a biotinylated ligand under conditions to permit selective binding of the avidin-labeled bacteriophage to the biotinylated ligand.
  • the kit further includes reagents, and appropriate instmctions, for biotinylating a ligand of choice.
  • the invention provides other compositions and kits which are useful for practicing the above-described methods. According to a particularly preferred aspect of the invention, a bacteriophage of the invention is provided.
  • the bacteriophage contains (a) a bacteriophage genome containing an exogenous polynucleotide that can be transcribed in a mammalian cell; and (b) a ligand contained on the surface of the bacteriophage that selectively binds to a receptor expressed on the surface of a mammalian cell.
  • the bacteriophages of the invention optionally are contained in a pharmaceutically acceptable carrier to form a pharmaceutical composition.
  • the pharmaceutical compositions should be sterile and contain a therapeutically effective amount of the bacteriophages (or target cells containing the bacteriophages) in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • the preferred pharmaceutical composition is contained in an implant that is suitable for implantation into the mammalian recipient.
  • implant that is suitable for implantation into the mammalian recipient.
  • Exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System", claiming priority to U.S. patent application serial no. 213,668, filed March 15, 1994).
  • PCT/US/0307 describes a biocompatible, preferably biodegradable polymeric matrix for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrix is used to achieve sustained release of the exogenous gene in the patient.
  • the bacteriophage particles described herein are encapsulated or dispersed within the biocompatible, preferably biodegradable polymeric matrix disclosed in PCT/US/03307.
  • the polymeric matrix preferably is in the form of a micro particle such as a micro sphere (wherein the bacteriophage particle is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein the bacteriophage particle is stored in the core of a polymeric shell).
  • Other forms of the polymeric matrix for containing the bacteriophage particle include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix device is implanted.
  • the size of the polymeric matrix further is selected according to the method of delivery which is to be used, typically injection into a tissue or administration of a suspension by aerosol into the nasal ⁇ ' or pulmonary areas.
  • the polymeric matrix composition can be selected to have both favorable degradation rates and also to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when the polymeric matrix is administered to a mucosal surface.
  • the matrix composition also can be selected not to degrade, but rather, to release by diffusion over an extended period of time. Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the bacteriophage particles of the invention to the subject. Biodegradable matrices are preferred.
  • Such polymers may be natural or synthetic polymers.
  • Synthetic polymers are preferred.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross- linked with multi-valent ions or other polymers.
  • the bacteriophage particles of the invention are delivered using the bioerodible implant by way of diffusion, or more preferably, by degradation of the polymeric matrix.
  • exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terphthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose a
  • biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly( valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules. 1993, 26, 581 -587, the teachings of which are incorporated herein, polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • the invention provides a composition of the above-described bacteriophages for use as a medicament, methods for preparing the medicament and methods for the sustained release of the medicament in vivo.
  • the bacteriophage is selected from the group of bacteriophages shown in Table 1 and is labeled with an appropriate ligand as described above.
  • the bacteriophage is selected from the group consisting of a lambda phage, a PI phage, a P22 phage, and an SPPl phage; more preferably, the bacteriophage is a lambda phage or a PI phage; most preferably, the bacteriophage is a lambda phage.
  • a bacteriophage particle serves as a vector for gene delivery to a mammalian cell.
  • the bacteriophage capsid and/or tail are modified to contain a ligand (or an adapter, e.g., avidin, for a ligand) that can bind to a receptor contained on the surface of a mammalian cell and facilitate entry therein.
  • a ligand or an adapter, e.g., avidin, for a ligand
  • the recombinant bacteriophage can be internalized via receptor mediated endocytosis or other receptor-mediated mechanisms of intracellular transport.
  • the bacteriophage genome is engineered to include transcriptional control signals, which allow expression of an exogenous polynucleotide in the mammalian cell.
  • the recombinant bacteriophage further contains an endosomal lysis signal, such as that contained within the capsid-stabilizing protein VI and L3/p23 of adenovims (Greber UF et al. EMBO J, 1996; 15 : 1766) to promote export of the bacteriophage genome into the cytoplasm prior to fusion of the endocytosed vesicles with mammalian cell lysosomes and enzymatic degradation of the bacteriophage genome.
  • an endosomal lysis signal such as that contained within the capsid-stabilizing protein VI and L3/p23 of adenovims (Greber UF et al. EMBO J, 1996; 15 : 1766) to promote export of the bacteriophage genome into the cytoplasm prior to fusion of the endocytosed vesicles with mammalian cell lysosomes and enzymatic degradation of the bacteriophage genome.
  • adenovims endosomal lysis signal can be inserted in-frame into or appended to the bacteriophage tail fiber J gene.
  • the bacteriophage genome is delivered to the nucleus.
  • the genome of the bacteriophage used in the invention is efficiently delivered to the nucleus during mitosis when the nuclear membrane dissipates and reforms.
  • the phage particle is modified to include nuclear localization signals (NLS) that mediate transport of molecules or macromolecular aggregates, such as viral DNA across the nuclear membrane and into the nucleoplasm.
  • NLS nuclear localization signals
  • Such nuclear localization signals are known to those of ordinary skill in the art and include portions of the human immunodeficiency vims (HIV) proteins matrix (MA) and Vpr (Naldini L et al. Science 1996; 272: 263; Bukrinsky MI et al, Nature 1993; 365: 666), large T antigen of SV40 (Kalderon D et al. Cell 1984; 39: 499; Drosophila Antennapedia (Derossi D et al. J Biol Chem 1996; 271: 18188), VP22 of Herpes vims (Elliott G. et al., Cell 1997; 88: 223), X. laevis nucleoplasmin, X.
  • the HIV or SV40 nuclear localization signal can be inserted in frame into or appended to the tail fiber J protein or the H protein which is thought to bind tightly to the bacteriophage genome.
  • Exemplary nuclear localization signals as disclosed in the above-identified references are provided in SEQ. ID NOS. 7-14.
  • the bacteriophage genome further includes adeno-associated viral sequences that mediate integration into specific chromosomal regions of the mammalian cell to effect delivery of the transduced bacteriophage genome to a specific mammalian cell chromosomal location.
  • adeno-associated viral sequences are known to those of ordinary skill in the art.
  • the bacteriophage has a genome of sufficient size (e.g., PI phage) to permit the further inclusion of nucleic acid sequences located 5' and 3' of the exogenous polynucleotide to promote site directed homologous recombination.
  • the bacteriophage has a genome of sufficient size (e.g., PI phage) to permit insertion of a large genetic unit containing not only one or more coding sequences of interest and their associated control regions, but in addition, mammalian autonomous replicating sequences, as well as centromere and telomere sequences in a self replicating format to form, in effect, an artificial chromosome.
  • PI phage a genome of sufficient size
  • mammalian autonomous replicating sequences as well as centromere and telomere sequences in a self replicating format to form, in effect, an artificial chromosome.
  • exogenous genetic material of large size e.g., a complete gene, including regulatory sequences
  • Example I illustrates the construction of three bacteriophage ⁇ vectors containing exogenous genetic material
  • Example II illustrates the post- packaging modification of bacteriophage ⁇ capsid and tail proteins to form a galactose-labeled bacteriophage ⁇
  • Example III illustrates the targeted delivery of a modified bacteriophage of the invention to human hepatoma G2 (HEP G2) cells grown in vitro
  • Example IV illustrates the targeted delivery of a modified bacteriophage of the invention to hepatocytes in the liver of live mice in vivo
  • Example V illustrates the constmction of bacteriophage vectors containing modified tail proteins
  • Example VI illustrates screening methods for selecting bacteriophages and target cells that transduce mammalian cells via receptor-mediated endocytosis.
  • each of the procedures described in reference to the bacteriophage ⁇ can be performed using any of the bacteriophages identified in Table 1 by: (1) obtaining the published restriction maps for these bacteriophages; (2) selecting an exogenous polynucleotide (e.g., a gene having a published sequence that encodes a therapeutic polynucleotide product, such as identified in Tables 2 or 3); (3) inserting the gene into the well-defined restriction sites of the bacteriophage genome using substantially the same procedures described herein (e.g., blunt- ended ligation into bacteriophage lambda) or altemative procedures known to one skilled in the art for the insertion of a polynucleotide into a restriction site of a bacteriophage; (4) modifying the surface of the bacteriophage to include a ligand that targets delivery of the modified bacteriophage to a mamm
  • substantially the same conditions it is meant that the conditions are modified to substitute a different bacteriophage for bacteriophage lambda in the procedure and to use the published restriction enzymes, buffers, incubation times, size inserts and so forth that are known to be appropriate for modifying the different bacteriophage to include an exogenous polynucleotide and contain a surface ligand.
  • the CMV ⁇ -gal 1 chimeric DNA sequences used in these experiments was constmcted as follows.
  • a 10.7 kilobase pair (kb) Eco Rl fragment excised from plasmid adCMV/ NLS ⁇ - gal (obtained from Dr. Ronald Crystal, Columbia University; see Figure 3) that contained the reporter gene expression cassette was ligated to Eco Rl/ BamHI double-digested left and right arm of ⁇ DASH II (Stratagene, La Jolla, CA), a derivative of ⁇ 2001, which contains an extended range of cloning sites.
  • the genetic and physical map information used in this invention was obtained from published wild type ⁇ DNA sequences and from the GenBank data base (accession number J02459).
  • the 10.7 kb reporter gene expression cassette included DNA sequences corresponding to the CMV promoter/ enhancer linked to the E. coli ⁇ -gal gene.
  • the expression cassette also included sequences encoding simian vims 40 (SV40) RNA splicing donor / acceptor sites and polyadenylation signals.
  • SV40 simian vims 40
  • CMV ⁇ -gal containing recombinant ⁇ phage particles were generated by packaging the ligated CMV ⁇ -gal ⁇ DASH II chimeric DNA with an in vitro packaging extract, Giga pak Gold II (Stratagene, La Jolla, CA), according to the manufacturer instmctions.
  • a chimeric PGK (phosphoglycerokinase) promoter/enhancer/ ⁇ -gal/ ⁇ DASH II (Fig. 4) was constructed as follows. Eco Rl and Bal I digested 0.6 kb fragment released from the PGK promoter containing plasmid (Gene, 80:65, 1987) was subcloned into Eco Rl Xho 1/ blunt site of adaCMV/ NLS/ beta-gal plasmid.
  • the resulting plasmid (designated PGK-NLS- ⁇ -gal) was Eco Rl digested and ligated to ⁇ DASH II sequences. After ligation, the chimeric PGK-NLS- ⁇ -gal ⁇ DASH II DNA was packaged in vitro and subsequently propagated in E. coli for large scale production of phage particles.
  • bacteriophage ⁇ used in this invention is that it can be readily engineered to permit large gene-containing expression cassettes that could never be achieved by viral vectors currently used for delivery to mammalian cells.
  • An example is a phage vector containing the MCK-DMD gene, which at 20.3 kb (Fig 5) greatly exceeds the capacity of currently used vectors. This vector was constmcted as follows. The 20.3 kb expression cassette containing muscle creatine kinase (MCK) promoter/enhancer 5' to the full length DMD cDNA
  • ⁇ phage particles displaying the ligand "L" heregulin for targeted gene delivery can be used for delivering the gene to a specific cell or tissue expressing the corresponding receptor via receptor/ligand mediated endocytosis.
  • DNA sequences that encode ligand(s) known to be endocytosed after interaction with cognate receptor(s) were inserted, in frame, into the D-gene locus of the ⁇ phage genome to produce a transcription template for a chimeric D gene-ligand bifunctional protein that expresses the fusion protein (including the ligand(s) of choice) on the bacteriophage surface.
  • ligand directed ⁇ phage targeting of specific mammalian cells is achieved by expressing chimeric ⁇ D-gene and immunoglobulin single chain variable fragments (SCVF) directed against receptors such as LDL and IL-2 , both of which are known to be internalized after the antibody-receptor interaction.
  • SCVF immunoglobulin single chain variable fragments
  • Bacteriophage ⁇ particles having a capsid that displays a chimeric ⁇ D-gene-avidin fusion protein also can be used to effect targeted delivery of the phage particles to a specific mammalian cell. Since the avidin molecule has very high affinity for biotin, any peptide or polypeptide ligand that can be biotinylated without adversely affecting the ability of the receptor to mediate endocytosis can be used as described herein.
  • the avidin gene or portion thereof encoding the polypeptide that selectively binds to biotin, is inserted, in frame, into the bacteriophage genome using the procedures described herein for forming a chimeric ⁇ D-gene and the published cDNA nucleic acid sequence for avidin (Gope, L. Mohan, et al., Nucleic Acid
  • Fig. 6 illustrates the constmction of a ⁇ phage particle expressing the heregulin-D-gene chimeric DNA sequences on its capsid.
  • the D gene heregulin sequences were first fused by generating 3 fragments (AB, CD, and EF) by polymerase chain reaction (PCR) in accordance with standard procedures using Taq polymerase and 6 sets of primers
  • primer a SEQ. ID NO. 1 ATACCGAGGGCTGCAGTGTACA primer b (SEQ. ID NO. 2) CTCTTTCAATTGGGGAGGCAAAACGATGCTGATTGCCGTTC primer c (SEQ. ID NO. 3) TTGCCTCCCCAATTGAAAGAG primer d (SEQ. ID NO. 4)
  • CAATC primer e SEQ. ID NO. 5
  • Gel purified fragments AB, CD and EF were treated with T4 polymerase to remove the overhanging nucleotide "A" in the fragment generated by terminal transferase activity present in the Taq polymerase used for PCR amplification. This step ensures the joining "in frame" of the coding sequences of both the ⁇ D-gene and heregulin.
  • fragments AB and CD were joined by first denaturing and then annealing the partial overlapping sequences, followed by extension with Taq polymerase, and then amplification after the addition of primers a and d.
  • the fragment AD was gel purified, treated with T4 polymerase, and subsequently used for joining fragment EF (as described above).
  • the fused ⁇ D-gene/heregulin gene containing fragment AF was subcloned into the TA cloning vector (Invitrogen, San Diego, CA).
  • the clones containing the AF fragment were identified by determining the sequences with a double-stranded sequencing method using the Sequenase 2.0 kit (USB, Cleveland, OH).
  • the AF fragment was generated after digestion of the TA plasmid clone with the restriction enzyme BsrGI and ligated into the necessary fragments of ⁇ (as shown in Fig. 6).
  • This chimeric DNA constmct was used to generate ⁇ phage particles having a genome that includes the recombinant D-gene/heregulin constmct and a capsid that displays this chimeric protein.
  • the particular procedure for modifying the bacteriophage ⁇ capsid and tail proteins to form a galactose-labeled bacteriophage ⁇ is described herein.
  • the galactose- labeled bacteriophage particles can be used to target liver cells which express a unique asialo-glycoprotein receptor. This procedure is based upon the published procedures for forming an artificial asialo-glycoprotein containing lactose (Neda, H., et al., JBC 296: 14143-14146 (1991)) or galactose (Human Gene Therapy 5:429-435 (1994)).
  • PFU/ml were galactosylated in 2 ml reaction volume containing 60mg of galactose and 100 mg of 1 ethyl-3-(3-diethylaminopropyl) carbodiaminide (EDC, Sigma Chemical. Corp., Saint Louis, MO) in sodium chloride solution.
  • EDC ethyl-3-(3-diethylaminopropyl) carbodiaminide
  • the pH of the unbuffered solution was adjusted to 7.5 with NaOH and the reaction mixture was incubated at room temperature for various lengths of time ranging from 24 to 48hrs.
  • the galactose associated with phage particles was determined using ,4 [C]-labeled galactose.
  • small peptide ligands such as insulin, epidermal growth factor (EGF), keratinocyte growth factor (KGF), Fab fragments for anti-polymeric immunoglobulin receptors can be covalently linked to ⁇ phage using the hetero-bifunctional crosslinking reagent N-succinimidyl 3-(2-pyridyl dithio) propionate (SPDP) or other bifunctional crosslinking agents in accordance with manufacturer's instmctions. Additional crosslinking agents are provided in the description and are known to those of ordinary skill in the art.
  • the modified bacteriophage is used to selectively deliver the gene of interest to target cells that express a cognate receptor for the ligand (Example III).
  • HEP G2 cells Conventional tissue culture methods were used to grow HEP G2 cells. HEP G2 cells were cultured in minimal essential medium as modified by Eagle (EMEM) containing 10% FBS. Cells were seeded one day prior to the addition of ⁇ phage particles for gene transfer experiments. In vitro targeted delivery of the ⁇ -gal gene to HEP G2 cells was accomplished by allowing the phage particles to interact with the cells in tissue culture growth medium for about 6-10 hrs; more preferably, for 8-10hrs. In general, after galactosylation, 10 -10 total phage particles/ml for 6- 10 hrs, preferably 10 particles/ml for 10 hours, are needed for efficient transduction of targeted cells. After exposing the cells to phage particles for the appropriate time, the phage-containing medium was removed and replaced with fresh media.
  • EMEM minimal essential medium as modified by Eagle
  • Detection of intemalization of bacteriophage vectors and gene expression After ligand-receptor mediated endocytosis of a bacteriophage vector into a mammalian cell, the expression of the exogenous genetic material in the mammalian cell can be monitored using standard methodologies. For example, delivery of a bacteriophage modified with a CMV ⁇ -gal recombinant constmct and a galactose ligand to a HEP G2 cell receptor can be measured by detecting bacteriophage DNA or RNA by Southern or northern blotting or in situ hybridization with or without amplification by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Radioactively-labeled DNA or RNA probes that selectively hybridize to unique portions of the phage genome e.g., portions corresponding to a regulatory sequence, such as a promoter, or portions corresponding to the exogenous genetic material, such as the reporter gene ⁇ -galactosidase, can be designed and constmcted using standard molecular biology techniques.
  • the modified bacteriophage is to be used for delivering exogenous genetic material to a mammalian cell in vivo (e.g., to hepatocytes in murine liver)
  • delivery of the phage to the specific cell can be detected by obtaining the targeted cells in a biopsy and assaying the biopsied cells using, for example, the above- mentioned conventional methods (e.g., Southern or northern blotting).
  • RNA or protein analysis for detecting specific transcription or translation products can be performed in accordance with standard practice.
  • Exemplary detection techniques to measure gene expression include one or more of the following techniques, alone or in combination: northern or western blotting, in situ hybridization, reverse transcription, PCR amplification, immunostaining, RIA and ELISA.
  • Such routine techniques also can be used to measure the stability of expression and maintenance of the delivered exogenous genetic material in the mammalian target cell by, for example, measuring the expression of one or more reporter (e.g., marker) genes as a function of time following transduction.
  • a reporter gene #1 initially is delivered to the target mammalian cells.
  • reporter gene #1 expression is assayed.
  • a reporter gene #2 is transduced into the same population of target cells.
  • the ratio of reporter gene #1 to reporter gene #2 is determined by, for example, immunostaining (e.g., using a different dye for each immunohistologic assay) the target tissue to assess the stability and maintenance of expression of the exogenous genetic material in the target cell population.
  • a diminution of reporter gene #1 expression compared to reporter gene # 2 expression can indicate either immune attack against transduced cells or transduction of short-lived, differentiated cells.
  • An exemplary procedure for processing a target tissue to which a ⁇ -galactosidase reporter gene expression has been delivered to determine whether the reported gene is expressed therein includes: a) snap freezing the tissue in isopentane chilled with liquid nitrogen, b) mounting the tissue on cryomold (Tissue -Teck, Miles, Elkhart, IN) using OCT and freezing, c) cutting the frozen tissue with a microtome at -20 °C into lOum sections , d) staining the tissue for ⁇ -galactosidase reporter gene expression with X-gal (lmg/ml) (a reporter gene substrate, Boehringer Mannheim, Indianapolis, IN), potassium ferro- and ferricyanide (35mM each) in phosphate buffered saline solution.
  • X-gal lmg/ml
  • a reporter gene substrate Boehringer Mannheim, Indianapolis, IN
  • potassium ferro- and ferricyanide 35mM each
  • mice were hepatocvtes in the liver of live mice in vivo.
  • 10 phage particles either modified with an average of 50 galactose residues/phage particle or unmodified (control)
  • mice were injected into mice intraperitoneally.
  • liver and kidney tissue biopsies were prepared.
  • Frozen sections were cut and fixed with 1% glutaraldehyde and subsequently stained with X-gal. After staining, tissue sections were analyzed by light microscopy. Unmodified phage (control) injection did not result in ⁇ -gal expression in either liver or kidney tissue sections that were stained with X-gal as detailed above.
  • bacteriophage with wild type packaging proteins and the beta-galactosidase gene are not effective genetic vectors for transducing Hep G2 cells.
  • endocytosis inhibition agents e.g., colchicine, cytochalasin B and D, and monodansylcadaverine
  • endocytosis inhibition agents e.g., colchicine, cytochalasin B and D, and monodansylcadaverine
  • endocytosis inhibition agents are used to arrest cell-mediated endocytosis in a mammalian cell and, thereby, select for bacteriophages whose mechanism of transfer does not depend on injection of genetic material into the mammalian call as is observed in the naturally occurring bacteriophage transduction of prokaryotic hosts.
  • antibodies that bind to bacteriophage tail proteins and block their function can be used to demonstrate that wild type tail function is not required for the transfer of the bacteriophage genome to the host cell nucleus.
  • the methods also are useful for identifying target cells that contain an appropriate receptor in sufficient quantity to internalize the preferred bacteriophage vectors via receptor-mediated endocytosis.
  • the screening assays disclosed herein demonstrate that bacteriophage vectors which include one or more non-functional tail protein(s) and a standard genomic expression marker, e.g. beta-galactosidase or other detectable protein, are incapable of transferring genetic material to the target cells in the presence of the endocytosis inhibition agents but are capable of transduction when contacted with the target cells in the absence of such inhibition agents.
  • the assay is useful for identifying novel bacteriophages which are structurally and functionally distinct from naturally-occurring bacteriophages and modified bacteriophage such as those described in PCT publication no. WO 96/21007, entitled, "Bacteriophage-mediated Gene Transfer Systems Capable of
  • Standard recombinant methods and screening technology are used to prepare a preferred bacteriophage vector with genetically modified tail protein genes that either contain amber mutations or sequences that enhance binding of the bacteriophage vector to the mammalian cell surface and subsequent intemalization via receptor-mediated endocytosis.
  • exemplary essential tail proteins in the lambda phage that can be modified to prepare preferred embodiments of the invention include: H, J, M proteins.
  • essential tail proteins refers to those proteins that are essential for facilitating the injection of the bacteriophage genetic material into its natural prokaryotic host.
  • bacteriophage can be selected or designed to have tail proteins that are capable of facilitating the injection of the bacteriophage genetic material into a eukaryotic host
  • the phrase "essential tail proteins" is also meant to embrace the tail proteins of such hypothetical eukaryotic cell-injecting bacteriophage.
  • One or more of these or other tail proteins can be rendered non-functional (i.e., incapable of facilitating the injection of the genetic material into the host cell) using recombinant, mutagenesis, and/or chemical methods in accordance with procedures known to one of ordinary skill in the art.
  • Such procedures can, of course, be applied to the preparation of other types of modified bacteriophages by, e.g., identifying the essential tail proteins of one or more bacteriophages in Table 1 and modifying the essential tail proteins as described herein.
  • the modifications to the essential tail protein(s) renders the bacteriophage incapable of mediating the transfer of genetic material into a mammalian host via an injection mechanism analogous to that responsible for the naturally occurring bacteriophage transduction of prokaryotic hosts.
  • modification s) of the tail proteins further render the bacteriophage incapable of infecting and/or propagating within its natural prokaryotic host (Su° E.Coli bacteria).
  • tail mutant bacteriophage vectors are packaged in vitro using specific purified protein packaging extracts and recombinant bacteriophage genomes or in Su + E. Coli bacteria (for amber mutant containing bacteriophage vectors).
  • the benefit of such preferred bacteriophage vectors is that these vectors are incapable of propagating in the host organism's natural flora.
  • the tail proteins are modified so that they function to properly package the vims' genome but have lost their ability to mediate injection across bacterial cell membranes. Phages containing these modified tail-proteins require packaging in a cell-free system as described above.
  • the mutations in the tail proteins are temperature sensitive such that at temperatures other than mammalian physiologic temperature, the tail protein functions normally and can mediate prokaryotic cell infection; however, at mammalian physiologic temperature, the temperature sensitive mutation inactivates the wild type tail function.
  • Such mutant bacteriophage vectors can be propagated in a prokaryotic host and packaged using standard procedures.
  • the tail protein is modified to include signals that target and/or facilitate entry of the vector's genetic material into subcellular organelles, including the nucleus.
  • signals for targeting the nucleus include polypeptides derived from the matrix or Vpr proteins of HIV or the large T-antigen of SV40.
  • signals can be inserted into the lambda or other bacteriophage genomes using standard procedures.
  • signals are integrated into the lambda phage genome in frame either within or appended to the J or H genes.
  • the genome of the modified bacteriophage described in Example 3 could be further modified in the following way.
  • Such signals can be inserted anywhere in the bacteriophage packaging protein coding sequence provided that such insertion does not interfere with receptor-mediated endocytosis and or expression of the exogenous polynucleotide within the target cell.
  • insertion of these signals into the bacteriophage also inactivates wild type function and, optionally, further serves to enhance bacteriophage penetration of the outer membrane of the mammalian cell.
  • the following assay is useful for selecting target cells and bacteriophage vectors that depend on receptor mediated endocytosis for transfer of the bacteriophage genome to the target cell nucleus.
  • the modified bacteriophages are grown to high titer 10 12 to 10 13 by standard methods employing either endogenous packaging in a prokaryotic host or in vitro packaging with proteins extracted from appropriately engineered lysogen strains.
  • these modified bacteriophages may be modified chemically after packaging using standard methods.
  • the coding sequence for beta-galactosidase or other easily detectable gene expression indicators is incorporated into the genome of the test bacteriophages using standard procedures.
  • purified bacteriophage is then added to sub-confluent cultures on cover slips of mammalian test cells that bear the receptor to which a modified bacteriophage has been targeted.
  • a test cell line is HepG2 cells in which the asialoglycoprotein mediated endocytosis of galactose bearing oligosaccharides and macromolecules is well studied (described above).
  • the screening assay is performed in the presence and absence of endocytosis inhibitors.
  • inhibitors of endocytosis are added to the cell medium containing the target cells either singly or in various combinations at concentrations such that receptor mediated endocytosis is optimally inhibited without irreversibly injuring the test target cells.
  • Exemplary endocytosis inhibitors include colchicine, taxol, monodansylcadaverine, cytochalasin B, or cytochalasin D.
  • cells not treated with endocytosis inhibitors are infected with test bacteriophages in parallel.
  • test cells are incubated with bacteriophage for about 48 hours or other suitable time to allow transduction and expression.
  • the plated cells are fixed for x-gal staining, immunostaining, in situ hybridization, electron microscopy or other standard methods that are known to one skilled in the art for detecting the inserted exogenous polynucleotide or its expression product.
  • X-gal staining is used to indicate whether or not the transduced genetic material (including a beta- galactosidase marker) is expressed in the test cells.
  • immunostaining with specific antibodies or anti-sense in situ hybridization probes employed to detect either the translation or transcription of a gene inserted into the vector's genome.
  • antibodies with specific affinity for the modified bacteriophage surface protein(s) are used to detect the localization of viral coat proteins within test cells, including organelles within the test cells, using confocal immunofluorescence microscopy.
  • transmission electron microscopy is employed to determine the sub cellular localization of modified bacteriophages at various time points after introduction of the modified bacteriophage into the test cell media.
  • bacteriophage genome which appends, in frame, the coding sequence cyclic RGD ligand 3' to the coding sequence of the native D-gene.
  • This bacteriophage genome was packaged in vitro and propagated in E. Coli so as to produce productive phage which contains the D-gene-RGD fusion protein product on its surface.
  • the effect of displaying this cyclic RGD ligand on the surface is to promote uptake of this bacteriophage by mammalian cells bearing the alpha- 5/beta-l integrin receptor via cell- mediated endocytosis.
  • This modified bacteriophage is referred to as lambda DASH II- RGD.
  • Example VIII r Delivery and expression of human factor VIH/von Willebrand factor containing vector
  • CMV promoter-human factor VIII cDNA (Seq ID No. 15) - internal ribosomal entry site ⁇ o (IRES)-human von Willebrand factor cDNA (Seq. ID No. 17).
  • IRES cassette was obtained from Clontech (Palo Alto, CA). The total size of this linear DNA constmct is 20.6 kilobases.
  • Example IX Deliverv and expression of a murine dystrophin containing vector
  • this dystrophin bacteriophage vector To test the expression capacity of this dystrophin bacteriophage vector, we injected 50 microliters of 10'° phage particles/mL into the gastrocnemius muscle of a dystrophin-deficient mouse (mdx). The phage vector was co-injected with a sublethal dose of India ink. This permitted accurate localization of the myocytes in proximity to the needle tract. Employing a polyclonal antibody against dystrophin (Cox et al. Nature, 264,
  • Lambda phage, pi phage, T even and T odd phages e.g., Tl, T2, T3, T4, T5, T6 and TT;
  • CFRT cystic fibrosis transmembrane conductance regulator
  • Cystic fibrosis Use of replication deficient recombinant 3/02/93 adenovims vector to deliver human CFTR cDNA to the lungs
  • MDR Advanced cancer Human multiple-drug resistance
  • Dystrophin-Duchenne muscular dystrophy Globin gene complex-Hemoglobinopathies e.g. sickle cell anemia, thalassemias
  • Clotting factor VIII-Hemophilia A von Willebrand's factor-von Willebrand's disease Collagen type VII-Epidermolysis bullosa dystrophica
  • cytokines or co-stimulatory immune modulators e.g., IL-1 , IL-2, I 12, GM-CSF, TNF ⁇ , IL4, B7-Neo ⁇ lastic processes Thymidine kinase-Suicide gene for neoplastic, hyperplastic or hypertrophic processes
  • Polypeptide antigens in conjunction with tolerance inducing sequences e.g. ribozyme against B7-1 -Treatment for autoimmune disease, e.g. rheumatoid arthritis, psoriasis, multiple sclerosis, alopecia areata Combinations of ribozyme(s), antisense RNA(s), or polypeptide coding sequences(s) for biopolymers that interfere with human viral infections, e.g. HIV, CMV, Hepatitis
  • Hematopoietic cells e.g. lymphocytes, erythrocytes, leukocytes, monocytes, progenitor and stem cells
  • Antigen presenting cells e.g. macrophages, B-cells, Langerhan's cells
  • LTR Long terminal repeat
  • Hematopoietic cells Promoters c fins (monocytes, trophoblasts)
  • Myosin light chain-2 ⁇ -myosin heavy chain (cardiac and slow twitch skeletal) ⁇ -cardiac myosin heavy chain Cardiac alpha actin
  • Elastin fibroblasts and smooth muscle cells
  • Aromatase cytochrome P450 (adipocytes, brain, ovary)
  • Non specific enhancer elements SV40 CMV LTR
  • Growth Factor/Cytokine receptors hepatocyte growth factor epidermal growth factor insulin-like growth factor I, II interleukin-la/b interleukin-2, IL-7, IL-4 ⁇ -interferon ⁇ -interferon keratinocyte growth factor
  • Hormone receptors prolactin thyroglobulin growth hormone insulin glucagon leutinizing hormone human choriogonadotrophic hormone
  • Antigen presenting cell receptors immunoglobulin G-Fc receptor -62-
  • Kidney cells angiotensin II vasopressin
  • Keratinocvte and skin fibroblast receptors very low density lipoprotein low density lipoprotein integrins that bind to RGD bearing polypeptides collagen laminin
  • ligands low density lipoprotein apoprotein B 100
  • very low density lipoprotein apoprotein E
  • galactose c kit ligand transferrin insulin heregulin RGD or RGD-containing polypeptides
  • Melanoma or other tumor specific antigens include leishmaniasis antigens; helicobacter pylori specific antigens (e.g., urease B); hepatitis B antigens; hepatitis C antigens;
  • Tuberculosis antigens cytomegalovirus antigens; lyme disease antigens; malaria antigens; respiratory syncytial vims antigens; leprosy antigens; toxoplasmosis antigens; pneumocytis carinii antigens; schistosomiasis antigens; chlamydial antigens;
  • HTLV-1 antigens enterococcal antigens (e.g., VRE); gonococcal antigens; treponemal antigens; clostridium difficile antigens;
  • Staphylococcus aureus antigens e.g., MRSA
  • trypanosomal antigens filarial antigens
  • salmonella antigens salmonella antigens
  • shigella antigens e.g., pneumococcal antigens (e.g., penicillin resistant strains); pseudomonal antigens -64 -
  • Phage bacteriophage yes none controlled high Vectors lambda persistent expression
  • NAME BRIGHAM AND WOMEN'S HOSPITAL, INC. 03
  • STREET 75 FRANCIS STREET
  • NAME KUPPER, THOMAS S.
  • ADDRESSEE WOLF, GREENFIELD & SACKS, P.C.
  • B STREET: 600 ATLANTIC AVENUE
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Bacteriophage lambda
  • Val Arg Thr Thr Lys Gly Lys Arg Lys Arg lie Asp Val 1 5 10
  • Lys lie Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys His 1 5 10 15
  • CTGTGCCTTA CCTACTCATA TCTTTCTCAT GTGGACCTGG TAAAAGACTT GAATTCAGGC 720
  • AAGACTCACA TTGATGGCCC ATCATTATTA ATTGAGAATA GTCCATCAGT C ⁇ GGCAAAAT 3300 ATATTAGAAA GTGACACTGA GTTTAAAAAA GTGACACCTT TGATTCATGA CAGAATGCTT 3360
  • GTCCTACTTA CATAGTTGAA ATATCAAGGA GGTCAGAAGA AAATTGGACT GGTGAAAACA 8340 GAAAAAACAC TCCAGTCTGC CATATCACCA CACAATAGGA TCCCCCTTCT TGCCCTCCAC 8400
  • CTACACAGAA CTCTCCTGAT AGTAAAGGGG GCTGGAGGCA AGGATAAGTT ATAGAGCAGT 8520
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 16:
  • GGCAGGTCAT CCACGGCCCG ATGCAGCCTT TTCGGAAGTG ACTTCGTCAA CACCTTTGAT 240
  • CTGAAGAGCA CCTCGGTGTT TGCCCGCTGC CACCCTCTGG TGGACCCCGA GCCTTTTGTG 840
  • AAAATTGGTG AAGCCGACTT CAACAGGAGC AAGGAGTTCA TGGAGGAGGT GATTCAGCGG 4680 ATGGATGTGG GCCAGGACAG CATCCACGTC ACGGTGCTGC AGTACTCCTA C ⁇ TGGTGACC 4740
  • TGCGATGTGT GCACCTGCAC CX ⁇ CATGGAG GATGCCGTGA TGGGCCTCCG OSTGGCCCAG 7500 TGCTCCCAGA AGCCCTGTGA GGAICAGCTGT CGGTCGGGCT TCACTTACGT TCTGCATGAA 7560
  • AGAACAACTG AACAGCCGGT GGACAGAATT CTGCCAATTG CTGAGTGAGA GAGTTAACTG 2700
  • GCTAGAGTAT CAAACCAACA TCATTACCTT TTATAATCAG CTACAACAAT TGGAACAGAT 2760 GACAACTACT GCCGAAAACT TGTTGAAAAC CCAGTCTACC ACCCTATCAG AGCCAACAGC 2820
  • AAAACAGCTC AAACAATGCA GACTTTTAGT TGGTGATATT CAAACAATTC AGCCCAGTTT 3540
  • GTTTCGAAGA CTC ⁇ ACTTTG CACAAATTCA CACTCTCCAT GAAGAAACTA TGGTAGTGAC 6180 GACTGAAGAT ATGCCTTTGG ATOTTTCTTA TGTGCCTTCT ACTTATTTGA CCGAGATCAG 6240

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Abstract

Procédé perfectionné permettant d'apporter un gène exogène, un polynucléotide thérapeutique, par exemple, jusqu'à une cellule de mammifère. Ce procédé consiste à utiliser un bactériophage comme vecteur pour apporter le gène exogène jusqu'à une cellule cible présélectionnée. Ce bactériophage contient un matériau génétique exogène qui peut être transcrit, et, éventuellement, traduit dans une cellule de mammifère, et comprend sur sa surface un ligand qui se lie à une récepteur sur la cellule cible. Le bactériophage est incapable d'injecter le matériau génétique exogène dans la cellule. Les bactériophages peuvent être utilisés dans des applications de thérapie génique et pour produire des produits géniques exogènes in vitro.
PCT/US1997/012928 1996-08-05 1997-07-03 Therapie genique a mediation par bacteriophages WO1998005344A1 (fr)

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