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WO1998059065A1 - Nouveau procede de transfert d'adn dans des cellules - Google Patents

Nouveau procede de transfert d'adn dans des cellules Download PDF

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Publication number
WO1998059065A1
WO1998059065A1 PCT/IB1998/001005 IB9801005W WO9859065A1 WO 1998059065 A1 WO1998059065 A1 WO 1998059065A1 IB 9801005 W IB9801005 W IB 9801005W WO 9859065 A1 WO9859065 A1 WO 9859065A1
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Prior art keywords
toxin
dna
cells
molecule
modified
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PCT/IB1998/001005
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English (en)
Inventor
Guido Grandi
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Chiron S.P.A.
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Publication of WO1998059065A1 publication Critical patent/WO1998059065A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/235—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bordetella (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a novel method of facilitating DNA transfer into cells.
  • the present invention more specifically relates to [DNA-binding] - [cell-binding] conjugates which are capable of binding to both DNA and cell surface receptors, thereby facilitating DNA uptake by cells by receptor-mediated endocytosis.
  • the present invention relates to toxins or toxin-like molecules which have been engineered chemically or by genetic engineering to become [DNA-binding] -[ cell-binding] conjugates capable of mediating DNA transfer into cells.
  • genetic or chemical modifications can be made to cells, means have had to be devised to transfer the genetic material or chemicals necessary to accomplish the modification into the cells.
  • transferred genetic material or chemicals include nucleic acids, peptides, polypeptide ⁇ , proteins or other chemical or biochemical agents.
  • Bacterial transformation (usually with the genetic engineering workhorse, Escherichia coli ) can be carried out using bacterial cells rendered "competent” for DNA transfer (Cohen et al . , 1972) .
  • "Competent cells” are bacterial cells grown up to a particular cell density (log-phase) , chilled, pelleted, resuspended in ice-cold calcium chloride (e.g. 50 M) at a particular cell density (e.g. 10 10 cells/ml) .
  • the now "competent" calcium chloride-treated cells can be either aliquotted, frozen on dry ice in the presence of glycerol and then transferred to -80 °C for storage, or they can be used directly for transformation with DNA. Transformation protocols vary, but they usually involve incubating the competent cells on ice in the presence of the appropriate transforming DNA for about 30 min and then, depending on the protocol and cell type, subjecting the cells to a brief heat shock at 42 °C. After the heat shock, the cells are usually transferred to a non-selective nutrient medium and incubated for about 30 min to 1 hr, after which the cells are transferred to a selective medium (usually agar plates incorporating appropriate chemical selection) . Suitably transformed cells that have survived the transformation regime will be able to grow in the selective medium.
  • a selective medium usually agar plates incorporating appropriate chemical selection
  • Direct micro-injection involves the direct insertion of DNA into a cell using physical means (i.e. via injection using an ultra-thin needle). Such techniques are time-consuming (because fewer cells can be transfected) but do achieve a high transfection rate.
  • the rate of transformation or transfection not only depends on the cell type and health, the method of transfer, and the conditions of culture, but also on the DNA itself (i.e. its size, conformation, strandedness, etc.).
  • DNA transfer into cells also exist. Such methods include liposome fusion (lipofection; Deshayes et al . , 1985) and gold or tungsten microparticle bombardment of cells (biolistics; Klein et al . , 1987) in the presence of DNA.
  • Cells which are chemically modified (without actual genetic manipulation) are often those into which a selective killing agent is desired to be introduced.
  • the targeting of cytotoxic or cytolytic chemicals such as anti-cancer agents into tumour cells can often be achieved by the selective transfer (by receptor-mediated endocytosis) of the toxic or lytic agent into the desired cell type.
  • the concept of receptor-mediated uptake of selected agents into cells has been recently used to simultaneously transfer DNA and agent into cells.
  • Wagner, et al . (1990) have developed a transferrin-based receptor-mediated system for nucleic acid transfer into cells.
  • This system involves covalently linking human transferrin or its chicken homologue, conalbumin, to polycations such as the small DNA- binding protein protamine or to polylysines of various sizes via a disulphide bridge.
  • conjugated transferrin molecules maintain their ability to bind to their cognate receptors and mediate efficient iron transport into cells.
  • the transferrin-polycation conjugates can form electrophoretically stable complexes with double-stranded DNA and modified RNA molecules.
  • the transferrin-polycation- DNA conjugate can bind to the transferrin receptor of a cell and be taken up into the cell by receptor-mediated endocytosis, thereby transferring the DNA into the cell where the ingested nucleic acid is released from the endoso al vesicles and can be expressed.
  • cells can be modified not only by the uptake of genetic material, but also by the uptake of chemical agents.
  • chemical agents can either modify cells in some way or can even destroy or disable cells.
  • Toxins are chemical agents which are cytotoxic in nature and can potentially be used in modern medical treatments to selectively target and destroy improperly functioning/growing cells such as cancer cells (Pugsley, 1996) .
  • Such toxins often exert their toxic affects on cells once they have been taken up by toxin-sensitive cells (usually by receptor-mediated endocytosis) .
  • the idea of selectively targeting toxins to particular cell types is known and toxin-ligand conjugates have been developed which bind to specific cell receptors (on certain cells) . These cell-bound toxin-ligand conjugates are then endocytosed, thereby internally releasing the toxin to act upon the cell (Pugsley, 1996) .
  • the bacterial anthrax lethal toxin is a complex of protective antigen (PA) , lethal factor (LF) and edema factor (EF) .
  • LF and EF bind by means of their amino-terminal domains to PA on the surface of toxin-sensitive cells and are translocated by receptor-mediated endocytosis into the cytosol, where they act on intracellular targets (Blanke et al . , 1996).
  • Genetically fusing the amino-terminal domain of the lethal factor (LF) of anthrax toxin to certain heterologous proteins has been shown to potentiate these proteins for protective antigen (PA) -dependent delivery into mouse macrophage cells (Arora and Leppla, 1993) .
  • Blanke et al . (1996) found that short tracts of lysine, arginine, or histidine residues could be used in place of the amino-terminal sequences of LF and could also potentiate a protein (in this case, the enzymic A chain of diphtheria toxin (DTA) ) for such PA-mediated transfer into cells.
  • DTA diphtheria toxin
  • Fominaya and Wels have constructed a recombinant fusion protein which serves as a target cell-specific carrier for the transfer of DNA via receptor-mediated endocytosis.
  • the recombinant protein consists of three functional domains: (1) an ErbB-2-specific single chain antibody which confers target cell specificity, (2) the translocation domain of the bacterial Pseudomonas exotoxin A which facilitates endosome escape of the DNA, and (3) a DNA-binding domain derived from the yeast GAL4 protein which enables sequence-specific binding to DNA.
  • modified toxin or toxin-like amino acid sequences which are capable of participating in receptor-mediated endocytosis but which are non-toxic, can be used to transfer DNA into cells.
  • the toxin or toxin-like sequences are modified by the chemical or genetically-engineered addition of DNA-binding motifs thereto to produce modified toxin or toxin-like molecules capable of binding DNA molecules.
  • DNA once bound to the modified toxin or toxin-like molecule, results in a modified toxin/toxin-like- DNA conjugate which is capable of binding to toxin molecule- sensitive or toxin-like molecule-sensitive cells (i.e.
  • the DNA can then be internalised into the cells through receptor-mediated endocytosis of the modified toxin/toxin-like-DNA conjugate. Once inside the cells, the DNA is released, making it capable of transfecting the cells.
  • a modified toxin molecule or toxin-like molecule comprising:
  • toxin or toxin-like amino acid sequences are incapable of effecting toxicity or only effect low residual toxicity .
  • the toxin amino acid sequences of the modified toxin molecule are derived from one or more bacterial toxins. More preferably, the toxin sequences are derived from one or more bacterial toxins of Pseudomonas sp. , preferably Pseudomonas aeruginosa , Corynebacterium diphtheriae , Bordetella pertussis , Vibrio cholerae , Clostridium sp. , preferably Clostridium botulinum , or Escherichia coli .
  • the toxin-like amino acid sequences of the modified toxin-like molecule are derived from one or more ADP-ribosylating enzymes. More preferably, the toxin-like sequences are derived from ADP-ribosylating enzymes such as eukaryotic mono-ADP-ribosyltransferases (MARTs) , T-cell alloantigens, poly-ADP-ribosylpolymerases (PARPs) , the bacterial metabolism regulation factor, DraT, or enzymes encoded by T-even bacteriophages.
  • ADP-ribosylating enzymes such as eukaryotic mono-ADP-ribosyltransferases (MARTs) , T-cell alloantigens, poly-ADP-ribosylpolymerases (PARPs) , the bacterial metabolism regulation factor, DraT, or enzymes encoded by T-even bacteriophages.
  • MARTs
  • the preferred T-cell alloantigens are RT6, RT6-1, or RT6-2 and the preferred T-even bacteriophages are T2 , T4 , or T6.
  • the DNA-binding motif (s) may comprise all or part of one or more DNA-binding proteins.
  • DNA-binding proteins can be selected from, for example, those proteins with limited specificity for DNA sequence, such as histone or histone- like DNA-binding proteins or polylysine, those proteins showing DNA sequence specificity such as the yeast transcriptional activator GAL4 , and activators and repressors of transcription which selectively bind specific nucleotide motifs.
  • the DNA-binding motif (s) comprise (s) all or part of a histone or histone-like protein, optionally BpHl, or the yeast transcriptional activator GAL4.
  • the modified toxin or toxin-like molecule according to the preceding aspect can be produced chemically or by genetic engineering.
  • Such chemical or genetic engineering methods of production can include chemical alteration of the amino acid sequences of the molecule or alteration of the molecule at the DNA level using molecular biological methods well known in the art.
  • the DNA encoding the amino acid sequence (s) of the toxin or toxin-like molecule can be deleted, added to, mutated, or substituted in order to establish the desired modification of the expressed molecule.
  • modified toxin or toxin-like molecule as described above, wherein the modified toxin or toxin-like molecule is further modified to make it capable of binding to appropriate receptors other than receptors specific for all or part of the toxin or toxin-like amino acid sequences of the molecule.
  • a modified toxin or toxin-like molecule as described in any of the preceding aspects, further comprising DNA bound to the DNA-binding motif (s).
  • the modified toxin or toxin-like molecule according to the third aspect of the present invention has bound to it DNA which (i) encodes one or more bacterial, viral, fungal or parasitic proteins, cytotoxic agents, cytolytic agents, antigens, antigenic epitopes, antibodies or fragments thereof, or (ii) comprises one or more replacement genes, augmentative genes or additional genes.
  • the DNA bound to the modified toxin or toxin-like molecule can encode antigens or antigenic epitopes which are antigens or antigenic epitopes of a pathogen or, even more preferably, protective antigens or protective epitopes of a pathogen.
  • a pharmaceutical composition comprising a modified toxin or toxin-like molecule as described in any of the preceding aspects and a pharmaceutically acceptable excipient.
  • a process for the production of a modified toxin or toxin-like molecule comprising modifying the toxin or toxin-like amino acid sequences by:
  • the modification for eliminating or reducing toxicity includes addition, deletion, or substitution of amino acids.
  • the modification for incorporating the DNA-binding motif (s) is addition of all or part of one or more DNA-binding proteins.
  • DNA-binding proteins can be selected from, for example, those proteins with limited specificity for DNA sequence, such as histone or histone- like DNA-binding proteins or polylysine, those proteins showing DNA sequence specificity such as the yeast transcriptional activator GAL4 , and activators and repressors of transcription which selectively bind specific nucleotide motifs.
  • the preferred DNA-binding protein is a histone or histone-like protein, optionally BpHl, or the yeast transcriptional activator GAL4.
  • the addition of all or part of one or more DNA-binding proteins is via one or more chemical or genetically engineered linkage moieties.
  • a process for the production of a pharmaceutical composition according to the fourth aspect of the present invention comprising:
  • step (ii) combining the modified toxin or toxin-like molecule of step (i) with a pharmaceutically acceptable excipient.
  • a method of DNA transfer into cells comprising:
  • the modified toxin or toxin-like molecule according to any of the first, second or third aspects of the present invention in the in vitro or in vivo transfer of DNA into cells.
  • the DNA to be transferred into cells is bound to one or more of the DNA-binding motifs of the modified toxin or toxin-like molecule, resulting in a modified toxin/toxin-like-DNA conjugate which is then internalised into the cells by receptor-mediated endocytosis.
  • a ninth aspect of the present invention there is provided a use of the modified toxin or toxin-like molecule according to any of the first, second or third aspects of the present invention in the in vitro or in vivo transformation or transfection of cells.
  • the modified toxin or toxin-like molecule according to any of the first, second or third aspects of the present invention as a pharmaceutical.
  • the vaccination can involve activation of the cytotoxic T-cell immune response or humoral immune response.
  • the gene therapy can include the replacement, augmentation, or addition of genes.
  • the diseased cells can include cancer cells, microbially-infected cells, or abnormal cells.
  • kits for the in vitro or in vivo transfer of DNA into cells comprising the modified toxin or toxin-like molecule according to any of the first, second or third aspects of the present invention or the pharmaceutical composition of the fourth aspect of the present invention.
  • kits for the in vitro or in vivo transformation or transfection of cells comprising the modified toxin or toxin-like molecule according to any of the first, second or third aspects of the present invention
  • a significant advantage of producing heterologous proteins by recombinant DNA techniques rather than by isolating and purifying a protein from natural sources is that equivalent quantities of the protein can be produced by using less starting material than would be required for isolating the protein from a natural source.
  • Producing the protein by recombinant techniques also permits the protein to be isolated in the absence of some molecules normally present in cells. Indeed, protein compositions entirely free of any trace of human protein contaminants can readily be produced because the only human protein produced by the recombinant non-human host is the recombinant protein at issue. Potential viral agents from natural sources and viral components pathogenic to humans are also avoided.
  • Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles.
  • Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (adjuvants) .
  • Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: aluminum salts (alum) such as aluminium hydroxide, aluminium phosphate, aluminium sulphate etc. , oil emulsion formulations, with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components, such as for example (1) MF59 (Published International patent application WO-A-90/14837 , containing 5% Squalene, 0.5% Tween® 80, 0.5% Span® 85 (optionally containing various amounts of MTP-PE (see below) , although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA 02164, USA), (2) SAF, containing 10% squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a sub
  • saponin adjuvants such as StimulonTM (Cambridge Bioscience, Worcester, MA, USA) may be used or particles generated therefrom such as ISCOMS (immunostimulating complexes).
  • ISCOMS immunostain-associated complexes
  • CFA Complete Freunds Adjuvant
  • IFA Incomplete Freunds Adjuvant
  • Alum and MF59 are preferred.
  • the immunogenic compositions typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect as discussed above under pharmaceutically acceptable carriers.
  • Immunogenic compositions used as vaccines comprise an im unologically effective amount of the antigenic polypeptides, as well as any other of the above-mentioned components, as needed.
  • imunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • the immunogenic compositions are conventionally administered parenterally , e.g. by injection either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the vaccine may be administered in conjunc- tion with other immunoregulatory agents.
  • polynucleotide intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA.
  • modifications for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including, for example, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.) , those containing alkylators, those with modified linkages (e.g.,
  • a “replicon” is any genetic element, e.g., a plasmid, a chromosome, a virus, a cosmid, etc. that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control. This may include selectable markers.
  • a “vector” is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment.
  • Control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • ORF "open reading frame” is a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence.
  • a "coding sequence” is a polynucleotide sequence which is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5 ' -terminus and a translation stop codon at the 3 '-terminus.
  • a coding sequence can include, but is not limited to, cDNA, and recombinant polynucleotide sequences.
  • PCR refers to the technique of poly erase chain reaction as described in Saiki et al . , Nature, 324 : 163, 1986; Scharf et al . , Science, 233 : 1076-1078, 1986; U.S. Patent 4,683,195; and U.S. Patent 4,683,202.
  • x is "heterologous" with respect to y if x is not naturally associated with y in the identical manner; i.e., x is not associated with y in nature or x is not associated with y in the same manner as is found in nature.
  • Homology refers to the degree of similarity between x and y. The correspondence between the sequence from one form to another can be determined by techniques known in the art.
  • homology can be determined by hybridization of the polynucleotides under conditions which form stable duplexes between homologous regions (for example, those which would be used prior to S digestion) , followed by digestion with single-stranded specific nuclease(s), fol- lowed by size determination of the digested fragments.
  • polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide or amino acid sequence "derived from" a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3-5 amino acids, and more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is im- munologically identifiable with a polypeptide encoded in the sequence.
  • This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.
  • the protein may be used for producing antibodies, either monoclonal or polyclonal, specific to the protein.
  • the methods for producing these antibodies are known in the art.
  • Recombinant host cells denote, for example, microorganisms, insect cells, and mammalian cells, that can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. Examples for mammalian host cells include Chinese hamster ovary (CHO) and monkey kidney (COS) cells.
  • CHO Chinese hamster ovary
  • COS monkey kidney
  • cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro . Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
  • the term "cell line” also includes immortalized cells. Preferably, cell lines include nonhybrid cell lines or hybridomas to only two cell types.
  • microorganism includes prokaryotic and eukaryotic microbial species such as bacteria and fungi, the latter including yeast and filamentous fungi.
  • Transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • genomic is meant a collection or library of DNA molecules which are derived from restriction fragments that have been cloned in vectors. This may include all or part of the genetic material of an organism.
  • cDNA is meant a complementary DNA sequence that hybridizes to a complementary strand of DNA.
  • purified and isolated is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • the term “purified” as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present).
  • the appropriate coding sequence can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, bacteria, and yeast.
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation (Sambrook et al . , "Expression of Cloned Genes in Mammalian Cells", in: Molecular Cloning: A Laboratory Manual , 2nd ed. , 1989).
  • Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumour virus LTR promoter, adenovirus major late promoter (Ad MLP) , and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible) , depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.
  • an enhancer element is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter (Maniatis et al . , Science, 236 : 1237, 1987; Alberts et al . , Molecular Biology of the Cell , 2nd ed. , 1989).
  • Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al . , EMBO J . , 4 : 761, 1985) and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al . , Proc . Natl . Acad . Sci . USA, 79 : 6777, 1982b) and from human cytomegalovirus (Boshart et al . , Cell, 41 : 521, 1985).
  • LTR long terminal repeat
  • enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion (Sassone-Corsi and Borelli, Trends Genet . , 2 : 215, 1986; Maniatis et al . , Science, 236 : 1237, 1987).
  • an inducer such as a hormone or metal ion
  • a DNA molecule may be expressed intracellularly in mammalian cells.
  • a promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N- terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
  • foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells.
  • a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells.
  • processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro .
  • the leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • the adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • the 3' terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation (Birnstiel et al . , Cell , 41 : 349, 1985; Proudfoot and Whitelaw, "Termination and 3 ' end processing of eukaryotic RNA", in: Transcription and Splicing (eds. B.D. Hames and D.M. Glover), 1988; Proudfoot, Trends Biochem . Sci . , 14 : 105, 1989).
  • transcription terminator/polyadenylation signals include those derived from SV40 (Sambrook et al . , "Expression of cloned genes in cultured mammalian cells", in: Molecular Cloning: A Laboratory Manual , 1989).
  • introns also called intervening sequences
  • splicing signals also called splice donor and acceptor sites
  • Introns are intervening noncoding sequences within a coding sequence that contain splice donor and acceptor sites. They are removed by a process called "splicing," following polyadenylation of the primary transcript (Nevins, Ann . Rev . Biochem . , 52 : 441, 1983; Green, Ann . Rev . Genet . , 20 : 671, 1986; Padgett et al . , Ann. Rev. Biochem. 55: 1119, 1986; Krainer and Maniatis, "RNA splicing", in: Transcription and Splicing (eds. B.D. Hames and D.M. Glover) , 1988) .
  • the above-described components comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs.
  • Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired.
  • Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria.
  • Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate.
  • plasmids containing the replication systems of papovaviruses such as SV40 (Gluzman, Cell , 23 : 175, 1981) or polyomaviruses, replicate to extremely high copy number in the presence of the appropriate viral T antigen.
  • mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus.
  • the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification.
  • mammalian- bacteria shuttle vectors include pMT2 (Kaufman et al . , Mol . Cell . Biol . , 9 : 946, 1989) and pHEBO (Shimizu et al . , Mol . Cell . Biol . , 6 : 1074, 1986).
  • the transformation procedure used depends upon the host to be transformed.
  • Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide (s) in liposomes, and direct microinjection of the DNA into nuclei.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS) , human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.
  • ATCC American Type Culture Collection
  • the polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector.
  • Vector construction employs techniques which are known in the art.
  • the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus- specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene into the baculovirus genome) ; and appropriate insect host cells and growth media.
  • a transfer vector usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed
  • a wild type baculovirus with a sequence homologous to the baculovirus- specific fragment in the transfer vector this allows for the homologous recombination of the heterologous gene into the baculovirus genome
  • the vector and the wild-type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine.
  • the packaged recombinant virus is expressed and recombinant plaques are identified and purified.
  • Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia , Invitrogen, San Diego, CA, USA (“MaxBac” kit) . These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No . 1555 , 1987 (hereinafter "Summers and Smith”) .
  • an intermediate transplacement construct Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above-described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector) .
  • This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements.
  • Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as a bacterium.
  • the replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.
  • pAc373 the most commonly used transfer vector for introducing foreign genes into AcNPV.
  • Many other vectors known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology, 17 : 31, 1989).
  • the plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al . , Ann . Rev . Microbiol . , 42 : 111 , 1988) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in Escherichia coli .
  • polyhedrin polyadenylation signal iller et al . , Ann . Rev . Microbiol . , 42 : 111 , 1988
  • amp prokaryotic ampicillin-resistance
  • Baculovirus transfer vectors usually contain a baculovirus promoter.
  • a baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5' to 3') transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.
  • a baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.
  • Structural genes abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein (Friesen et al . , "The Regulation of Baculovirus Gene Expression", in: The Molecular Biology of Baculoviruse ⁇ (ed. Walter Doerfler) , 1986; and EPO Publ . Nos. 127 839 and 155 476) and the gene encoding the plO protein (Vlak et al . , J . Gen . Virol . , 69 : 765, 1988) .
  • DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al . , Gene, 73 : 409, 1988).
  • the signals for mammalian cell posttranslational modifications such as signal peptide cleavage, proteolytic cleavage, and phosphorylation
  • the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells
  • leaders of non-insect origin such as those derived from genes encoding human ⁇ -interferon (Maeda et al .
  • a recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted.
  • Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.
  • recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects.
  • the leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.
  • the promoter and transcription termination sequence of the construct will usually comprise a 2-5kb section of the baculovirus genome.
  • Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art (see Summers and Smith, supra ; Smith et al . , Mol . Cell . Biol . , 3 : 2156, 1983; and Luckow and Summers, supra ) .
  • the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene (Miller et al . , Bioessays, 4 : 91, 1989).
  • the DNA sequence, when cloned in place of the polyhedrin gene in the expression vector is flanked both 5' and 3' by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.
  • the newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%) ; thus, the majority of the virus produced after co-transfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses.
  • An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished.
  • the polyhedrin protein which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles.
  • occlusion bodies up to 15 ⁇ m in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope.
  • Cells infected with recombinant viruses lack occlusion bodies.
  • the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies (Ansubel et al . (eds.), "Current Protocols in Microbiology” , Vol . 2 at 16. 8 (Supp . 10) , 1990; Summers and Smith, supra ; Miller et al . , supra) .
  • Recombinant baculovirus expression vectors have been developed for infection into several insect cells.
  • recombinant baculoviruses have been developed for, inter alia , Aedes aegypti , Autographa calif ornica , Bombyx mori , Drosophila melanogaster, Spodoptera frugiperda , and Trichoplu ⁇ ia ni (PCT Pub. No. WO 89/046699; Carbonell et al . , J . Virol . , 56 : 153, 1985; Wright, Nature, 321 : 718, 1986; Smith et al . , Mol . Cell . Biol . , 3 : 2156, 1983; and see generally, Fraser, et al . , In Vitro Cell . Dev . Biol . , 25 : 225, 1989) .
  • Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art (see, e.g., Summers and Smith, supra ) .
  • the modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid (s) present in the modified insect host.
  • the expression product gene is under inducible control, the host may be grown to high density, and expression induced.
  • the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients.
  • the product may be purified by such techniques as chromatography, e.g., HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresi ⁇ ; density gradient centrifugation; solvent extraction, or the like.
  • the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, e.g., proteins, lipids and polysaccharides.
  • host debris e.g., proteins, lipids and polysaccharides.
  • recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.
  • a bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3") transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5 ' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site.
  • a bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene.
  • Constitutive expression may occur in the absence of negative regulatory elements, such as the operator.
  • positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5 1 ) to the RNA polymerase binding sequence.
  • a gene activator protein is the catabolite activator protein (CAP) , which helps initiate transcription of the lac operon in E . coli (Raibaud et al . , Ann. Rev . Genet . , 18 : 173, 1984). Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
  • CAP catabolite activator protein
  • Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) (Chang et al . , Nature, 198 :
  • promoter sequences derived from biosynthetic enzymes such as tryptophan ( trp) (Goeddel et al . , Nuc . Acids Res . , 8 : 4057,
  • promoter systems also provide useful promoter sequences.
  • synthetic promoters which do not occur in nature also function as bacterial promoters.
  • transcription activation sequences of one bacterial or bacteriophaqe promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter (U.S. Patent No. 4,551,433).
  • the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor (Amann et al . , Gene, 25 : 167, 1983; de Boer et al . , Proc . Natl . Acad . Sci . USA, 80 : 21, 1983).
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
  • a naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.
  • the bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al . , J . Mol . Biol . , 189 : 113, 1986; Tabor et al . , Proc . Natl . Acad . Sci . USA, 82 : 1074, 1985).
  • a hybrid promoter can also be comprised of a bacteriophage promoter and an E . coli operator region (EPO Publ. No. 267 851) .
  • an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes.
  • the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon (Shine et al . , Nature, 254 : 34, 1975).
  • SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' and of E . coli 16S rRNA (Steitz et al .
  • a DNA molecule may be expressed intracellularly .
  • a promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO Publ. No. 219 237) .
  • Fusion proteins provide an alternative to direct expression.
  • a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein is fused to the 5' end of heterologous coding sequences.
  • this construct will provide a fusion of the two amino acid sequences.
  • the bacteriophage lambda cell gene can be linked at the 5' terminus of a foreign gene and expressed in bacteria.
  • the resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene (Nagai et al . , Nature, 309 : 810, 1984).
  • Fusion proteins can also be made with sequences from the lacZ (Jia et al . , Gene, 60 : 197, 1987), trpE (Allen et al . , J " . Biotechnol . , 5 : 93, 1987; Makoff et al . , J . Gen . Microbiol . , 135 : 11, 1989), and Chey (EPO Publ. No. 324 647) genes.
  • the DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site.
  • Another example is a ubiquitin fusion protein.
  • Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin specific processing- protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated (Miller et al . , Bio/Technology 7 :
  • foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria (U.S. Patent No. 4,336,336).
  • the signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria) .
  • processing sites which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.
  • DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E . coli outer membrane protein gene (o pA) (Masui et al . , in: Experimental Manipulation of Gene Expression , 1983; Ghrayeb et al . , EMBO J . , 3 : 2437, 1984) and the E . coli alkaline phosphatase signal sequence (phoA) (Oka et al . , Proc . Natl . Acad . Sci . USA, 82 : 7212, 1985).
  • o pA E . coli outer membrane protein gene
  • phoA E . coli alkaline phosphatase signal sequence
  • the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B . subtilis (Palva et al . , Proc . Natl . Acad . Sci . USA, 79 : 5582, 1982; EPO Publ. No. 244 042).
  • transcription termination sequences recognized by bacteria are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E . coli as well as other biosynthetic genes.
  • expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria.
  • a replicon such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria.
  • the replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification.
  • a replicon may be either a high or low copy number plasmid.
  • a high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150.
  • a host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.
  • the expression constructs can be integrated into the bacterial genome with an integrating vector.
  • Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome.
  • integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EPO Publ. No. 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.
  • extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.
  • Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol , erythromycin, kanamycin (neomycin) , and tetracycline (Davies et al . , Ann . Rev . Microbiol . , 32 : 469, 1978).
  • Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
  • Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
  • Expression and transformation vectors have been developed for transformation into many bacteria.
  • expression vectors have been developed for, inter alia, the following bacteria: B . subtilis (Palva et al . , Proc. Natl. Acad. Sci. USA, 79: 5582, 1982; EPO Publ. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541), E. coli (Shimatake et al . , Nature, 292: 128, 1981; Amann et al . , Gene, 40: 183, 1985; Studier et al . , J. Mol.
  • Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl 2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed (see, e.g., Masson et al . , FEMS Microbiol. Lett., 60: 273, 1989; Palva et al . , Proc. Natl. Acad. Sci. USA, 79: 5582, 1982; EPO Publ. Nos. 036 259 and 063 953; PCT Publ. No. WO 84/04541 [Bacillus], Miller et al .
  • a yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA.
  • a promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the "TATA Box") and a transcription initiation site.
  • a yeast promoter may also have a second domain called an upstream activator sequence (UAS) , which, if present, is usually distal to the structural gene.
  • the UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.
  • Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EPO Publ. No. 284 044), enolase, glucokinase, glucose-6- phosphate isomerase, glyceraldehyde-3-phosphate- dehydrogenase (GAP or GAPDH) , hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO Publ. No. 329 203).
  • ADH alcohol dehydrogenase
  • GAP or GAPDH glyceraldehyde-3-phosphate- dehydrogenase
  • hexokinase phosphofructokinase
  • 3-phosphoglycerate mutase 3-phosphoglycerate muta
  • the yeast PH05 gene encoding acid phosphatase, also provides useful promoter sequences (Myanohara et al . , Proc . Natl . Acad . Sci . USA, 80 : 1, 1983) . 59065
  • synthetic promoters which do not occur in nature also function as yeast promoters.
  • UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.
  • hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Patent Nos. 4,876,197 and 4,880,734).
  • Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2 , GAL4 , GAL10, or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164 556) .
  • a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia , Cohen et al . , Proc . Natl . Acad . Sci . USA, 77 : 1078, 1980; Henikoff et al . , Nature, 283 : 835, 1981; Hollenberg et al . , Curr . Topics Microbiol . Immunol . , 96 : 119, 1981; Hollenberg et al .
  • a DNA molecule may be expressed intracellularly in yeast.
  • a promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N- terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
  • Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems.
  • a DNA sequence encoding the N- terminal portion of an endogenous yeast protein, or other stable protein is fused to the 5' end of heterologous coding sequences.
  • this construct will provide a fusion of the two amino acid sequences.
  • the yeast or human superoxide dismutase (SOD) gene can be linked at the 5' terminus of a foreign gene and expressed in yeast.
  • the DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site (see, e.g., EPO Publ. No. 196 056).
  • a ubiquitin fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein.
  • a processing enzyme e.g. ubiquitin-specific processing protease
  • native foreign protein can be isolated (see, e.g., PCT Publ. No. WO 88/024066).
  • foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein.
  • a leader sequence fragment that provide for secretion in yeast of the foreign protein.
  • processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro .
  • the leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EPO Publ. No. 012 873; JPO Publ. No. 62,096,086) and the A-factor gene (U.S. Patent No. 4,588,684).
  • yeast invertase gene EPO Publ. No. 012 873; JPO Publ. No. 62,096,086) and the A-factor gene (U.S. Patent No. 4,588,684).
  • leaders of non-yeast origin such as an interferon leader, exist that also provide for secretion in yeast (EPO Publ. No. 060 057) .
  • a preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre” signal sequence, and a "pro” region.
  • the types of alpha-factor fragments that can be employed include the 98/59065
  • pre-pro alpha factor leader about 83 amino acid residues
  • truncated alpha-factor leaders usually about 25 to about 50 amino acid residues
  • Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alpha-factor. (See, e.g., PCT Publ. No. WO 89/02463) .
  • transcription termination sequences recognized by yeast are regulatory regions located 3 ' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes, are well known.
  • Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as yeast or bacteria.
  • the replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification.
  • yeast- bacteria shuttle vectors include YEp24 (Botstein, et al . , Gene, 8 : 17-24, 1979), pCl/1 (Brake, et al .
  • a replicon may be either a high or low copy number plasmid.
  • a high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150.
  • a host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host (see, e.g., Brake et al . , supra) .
  • the expression constructs can be integrated into the yeast genome with an integrating vector.
  • Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome (Orr-Weaver et al . , Methods in Enzymol . , 101 : 228-245, 1983) .
  • An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector (see Orr-Weaver et al . , supra ) .
  • One or more expression constructs may integrate, possibly affecting levels of recombinant protein produced (Rine et al . , Proc . Natl . Acad . Sci . USA, 80 : 6750, 1983).
  • the chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.
  • extrachro osomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.
  • Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2 , HIS4 , LEU2 , TRP1 , and ALG7 , and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively.
  • a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions (Butt et al . , Microbiol. Rev., 51: 351, 1987).
  • Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
  • Expression and transformation vectors have been developed for transformation into many yeasts.
  • expression vectors have been developed for, inter alia, the following yeasts: Candida albicans (Kurtz et al . , Mol. Cell. Biol., 6: 142, 1986), Candida maltose (Kunze et al . , J. Basic Microbiol., 25: 141, 1985), Hansenula polymorpha (Gleeson et al . , J. Gen. Microbiol., 132: 3459, 1986; Roggenkamp et al . , Mol. Gen.
  • Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed (see, e.g., Kurtz et al . , Mol. Cell. Biol., 6: 142, 1986; Kunze et al . , J. Basic Microbiol., 25: 141, 1985 [Candida], Gleeson et al., J. Gen. Microbiol., 132: 3459, 1986; Roggenkamp et al . , Mol. Gen.
  • Toxins are molecules which adhere to the surface of cells by binding to specific cell receptors. After binding, the toxins enter the cells and exert their toxic function. Numerous toxins have so far been discovered and many of them have been characterised in detail at the biochemical and genetic level (see, for example, Alouf and Freer, 1991) .
  • ADP-ribosylation is a post-translational modification performed by viral, bacterial and eukaryotic enzymes.
  • the present knowledge of these enzymes derives mainly from biochemical, genetic and three-dimensional studies on various bacterial toxins produced by Pseudomonas aeruginosa (PAETA) , Corynebacterium diphtheriae (DT) , Bordetella pertussis (PT) , Vijrio cholerae (CT) , and E. coli (LT) (Domenighini and Rappuoli, 1996). It was in these bacteria that the ADP-ribosylation enzymatic reaction was first discovered.
  • PAETA Pseudomonas aeruginosa
  • DT Corynebacterium diphtheriae
  • PT Bordetella pertussis
  • CT Vijrio cholerae
  • LT E. coli
  • amino acid sequences of more than 30 enzymes with ADP- ribosylating activity are known, including all eukaryotic mono-ADP-ribosyltransferases (MARTs) and poly-ADP- ribosylpolymerases (PARPs) , bacterial ADP-ribosylating enzymes which do not have toxic activity, and the analogous enzymes encoded by T-even bacteriophages (Domenighini and Rappuoli, 1996) .
  • toxin-like molecules are those molecules which possess similar properties to toxins.
  • Preferred examples of toxinlike molecules include ADP-ribosylating enzymes such as eukaryotic MARTs, T-cell alloantigens such as RT6, RT6-1 and RT6-2 , PARPs, the bacterial metabolism regulation factor, DraT, or enzymes encoded by bacteriophages such as T2 , T4 and T6 (Domenighini and Rappuoli, 1996) .
  • the present invention covers the novel use of toxins (and toxin-like molecules) as vehicles to deliver DNA into cells.
  • Some of the advantages of using such molecules to deliver DNA into cells over systems of DNA delivery currently in use are: (1) efficiency (a large number of toxin receptors are located on the surface of the cells) ; (2) specific delivery (the receptors for some toxins are located only on the surface of particular cells and tissues) ; and (3) built-in immunogenicity and adjuvanticity (many toxins are good immunogens and adjuvants - such properties might be particularly useful when the methods described herein are used for DNA immunization) .
  • E . coli enterotoxin LT
  • LF lethal factor
  • DT diphtheria toxin
  • other toxins or toxin-like molecules could also be used.
  • Figure 1 shows the construction of plasmid pT7/LTbpHl through the cloning of the LT/bpHl hybrid gene into the vector pT7-7.
  • the gene coding for the A and B subunits of LT of E . coli , where the region coding for the Al domain carrying the toxic activity of LT was replaced with a 165 bp fragment of the bpHl sequence of B . pertussis was cloned into pT7-7 to express the LT/BpHl fusion protein.
  • Figure 2 shows a South-Western analysis of radiolabelled DNA probe binding to LT/BpHl fusion protein.
  • Bacterial total protein extracts were fractionated on a 15% SDS-acrylamide gel and blotted onto a nitrocellulose filter. The filter was hybridised with pGEM®-3 radiolabelled DNA.
  • Lane 1 B . pertussis total protein extract
  • Lane 2 pT7/LTbpHl-transfor ed E . coli total protein extract after induction of expression of the hybrid gene;
  • Lane 3 pT7/LTbpHl-transformed E . coli total protein extract without induction of expression of the hybrid gene.
  • Figure 3 shows plasmid pGST/LF N bpHl , which was constructed through the cloning of LF N and bpHl into the pGEX-KG vector.
  • LF N sequence coding for amino acid residues 1-254 and the bpHl sequence coding for amino acid residues 1-55 were cloned into the pGEX-KG vector to express a GST/LF N BpHl fusion protein; the GST protein is then cleaved by thrombin digestion.
  • Figure 4 shows a South-Western analysis of radiolabelled DNA probe binding to GST/LF N BpHl fusion protein.
  • Bacterial total protein extracts were fractionated on a 12% SDS- acrylamide gel and blotted onto a nitrocellulose filter. The filter was hybridised with pGEM®-3 radiolabelled DNA.
  • Lane 1 pGST/LF N bpHl-transformed E . coli total protein extract without induction of expression of the hybrid gene
  • Lane 2 pGST/LF N bpHl-transformed E . coli total protein extract after induction of expression of the hybrid gene
  • Lane 3 B . pertussis total protein extract.
  • Figure 5 shows the gal/dtb hybrid gene as cloned in plasmid pTrcHis-GAL/DTB.
  • Plasmid pTrcHis-GAL/DTB was constructed through the cloning of the gal /dtb hybrid gene into the pTrcHis A vector.
  • GAL4 sequence, coding for amino acid residues 2-147, and toxl97 sequence, coding for amino acid residues 195-560, were cloned into the pTrcHis A vector to express the GAL/DTB fusion protein. Nucleotides between restriction sites SacI and Kpnl belong to pGEM®-3 sequence.
  • Figure 6 shows a South-Western analysis of radiolabelled DNA probe binding to GAL/DTB fusion protein.
  • Lane 1 2 ⁇ g of DT protein
  • Lane 2 9 ⁇ g of GAL/DTB fusion protein before dialysis
  • Lane 3 2 ⁇ g of GAL/DTB fusion protein after dialysis in
  • Figure 7 shows a Fluorescence Activated Cell Sorter (FACS) analysis of the binding of the GAL/DTB fusion protein to Vero cells.
  • FACS Fluorescence Activated Cell Sorter
  • Figure 8 shows a FACS analysis of the intemalisation of the GAL/DTB fusion protein into Vero cells.
  • the strategy used to modify LT is described in Figure 1.
  • the gene encoding the A and B subunits of LT of E . coli was isolated from the plasmid pBS/LT (Pizza et al . , 1994) and the region coding for the Al domain, carrying the toxic activity, was replaced with a 165 bp DNA fragment isolated by PCR from Bordetella pertussis chromosomal DNA.
  • the protein encoded by the amplified fragment has been described as a double-stranded DNA-binding protein homologous to a histone HI (Scarlato et al . , 1995) and named BpHl.
  • the hybrid gene was inserted into the expression vector pT7-7 (Tabor and Richardson, 1985) and the plasmid (pT7/LTbpHl) was used to transform E . coli cells.
  • the recombinant clones obtained were able to express a fused product (detectable by Western blot) which was able to bind DNA as judged by Southwestern analysis ( Figure 2).
  • a 580 bp gene fragment encoding the first 28 amino acids of the Al subunit of LT was derived by Smal /Xbal digestion of plasmid pBS/LT.
  • PCR polymerase chain reaction
  • PCR was performed on 200 ng of plasmid pG23.1EB as template containing the 901 bp EcoRI-BamHI fragment of bpHl (Scarlato et al . , 1995). The reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl pH 8.3 , 50 mM KCl, 1.5 mM MgCl 2 , 0.001% w/v gelatin, 2% DMSO, 50 pmole of each primer, 200 ⁇ M of each dNTP, 2.5 U Taq DNA polymerase (Perkin Elmer, USA) .
  • DNA was denaturated for 5 min at 97 °C and PCR performed for 30 cycles: 30 sec at 97°C, 20 sec at 64°C, 20 sec at 72 °C.
  • the amplified fragment was digested with Xbal and Hindlll restriction enzymes and gel-purified.
  • the 210 bp sequence coding for the A2 domain of LT was amplified by PCR using the pBS/LT plasmid DNA as template. The PCR reaction was performed using the following synthetic oligodeoxynucleotides:
  • PCR was performed on 200 ng of plasmid pBS/LT as template containing the 1840 bp Smal-Hindlll fragment of LT.
  • the reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl pH 8.3, 50 mM KC1, 1.5 mM MgCl 2 , 0.001% w/v gelatin, 50 pmole of each primer, 200 ⁇ M of each dNTP, 2.5 U Taq DNA polymerase (Perkin Elmer, USA) .
  • DNA was denaturated for 5 min at 94 °C and PCR performed for 30 cycles: 30 sec at 94°C, 30 sec at 60°C, 30 sec at 72°C.
  • the amplified fragment was digested with Hindlll restriction enzyme and gel-purified.
  • the 210 bp LT-A2 amplified region was then cloned 3' of the LT-Al/bpHl hybrid gene fragment into the Hindlll restriction site for the construction of the LT-A/bpHl hybrid sequence.
  • Several positive clones were obtained, as determined by restriction enzyme analysis, and the plasmid isolated from one positive clone was named pEM/LTAbpHl.
  • the sequence of the plasmid was analyzed by automatic sequencing.
  • plasmid pT7-7 (Tabor and Richardson, 1985) that contains a T7 promoter and is used to express genes using T7 RNA polymerase.
  • a 460 bp DNA fragment encoding the LT-A/BpHl fused protein was amplified by PCR using the DNA of plasmid pEM/LTAbpHl as template. The fragment obtained lacked the LT constitutive promoter region.
  • the PCR reaction was performed using the following synthetic oligodeoxynucleotides :
  • Ndel restriction site (underlined lower case letters) was introduced at the 5' end of the DNA fragment.
  • PCR was performed on 20 ng of plasmid pEM/LTAbpHl as template. The reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl pH 8.8 , 10 mM KC1, 0.002% v/v Tween 20, 1 mM MgCl 2 , 50 pmole of each primer, 40 ⁇ M of each dNTP, 3 U UlTma DNA polymerase (Perkin Elmer, USA) . DNA was denaturated for 2 min at 94 °C and PCR performed for 30 cycles: 30 sec at 94°C, 30 sec at 60°C, 30 sec at 72°C. The amplified fragment was digested with Ndel/EcoRI restriction enzymes and gel-purified.
  • the DNA fragment coding for the B domain of the LT toxin was isolated from plasmid pBS/LT by EcoRI /HindiII restriction enzyme digest.
  • the two DNA fragments coding for the LT-A/BpHl and the LT-B domains of the fused protein were then cloned into the Ndel /Hindlll sites of the pT7-7 vector thereby obtaining pT7/LTbpHl clones.
  • the plasmid DNA was transformed into the E . coli BL21 (DE3) strain carrying the T7 RNA polymerase of bacteriophage ⁇ DE3 integrated into the chromosome (Studier and Moffatt, 1986) .
  • E . coli BL21 cells carrying plasmid pT7/LTbpHl were grown at 37 °C in Luria Bertani (LB) medium containing 50 ⁇ g/ml of ampicillin to A 600nm of 0.6. Protein expression was induced by addition of 0.5 mM isopr ⁇ pyl- -D-thiogalactoside (IPTG) and incubation was continued for an additional two hours. Cells were harvested by centrif gation, the cell pellet was resuspended in 50 mM Tris-HCl, pH 8.0, 25% saccharose.
  • LB Luria Bertani
  • IPTG isopr ⁇ pyl- -D-thiogalactoside
  • fusion protein derived from pT7/LTbpHl containing the BpHl binding protein domain was determined in a South- estern assay. 20 ⁇ l of the bacterial suspension was subjected to electrophoresis in a 15% gel of acrylamide-containing SDS and blotted onto a nitrocellulose filter. The filter was saturated in 3% dried milk in PBS for 30 min at room temperature and rinsed with TE Buffer. The filter was hybridized using as a probe pGEM®-3 (Promega Corporation, Madison, WI, USA) DNA, linearised at the EcoRI site.
  • PA 63 is the carboxyl terminal 63 kDa domain of the PA protein that binds to a specific cell surface receptor (Escuyer and Collier, 1991) .
  • Cell-bound PA 63 has been found to expose a site that can bind LF N , thereby forming an LF N /PA complex.
  • the LF N /PA complex is then able to be internalised by receptor-mediated endocytosis and delivered to the endosome, where the low pH environment triggers translocation of the LF N to the cytoplasm.
  • the gene sequence encoding the amino terminal domain of LF (LF N : residues 1-254) of the anthrax toxin of Bacillus anthracis was isolated from plasmid pLF7 (Robertson and Leppla, 1986) .
  • the hybrid gene was inserted into the expression vector pGEX (Smith and Johnson, 1988) and the plasmid was used to transform E. coli cells.
  • the recombinant clones obtained were able to express a fused product which was able to bind DNA as judged by South-western analysis ( Figure 4) .
  • the LF N sequence was amplified from plasmid pLF7 (Robertson and Leppla, 1986) using the same primers as described by Arora et al . (1994), where the Xjal restriction site was inserted at the 5 ' end of the LF N fragment and the Ncol site was added at the 3' end of the fragment.
  • the LF N PCR product was digested sequentially with Xbal and Ncol restriction enzymes.
  • the digested PCR product was then cloned into the pGEX-KG vector (Smith and Johnson, 1988) that had been previously digested with the same enzymes.
  • the vector pGEX-KG is designed for inducible high level intracellular expression of a gene or gene fragment of interest as a fusion protein with Schistosoma japonicum Glutathione S-transferase (GST) .
  • a thrombin cleavage site is downstream from the multiple cloning site.
  • the vector is derived from pGEX-2T (Pharmacia Biotech, Inc., Uppsala, Sweden) and has the coding sequence for five glycines inserted between the thrombin cleavage site and the multiple cloning site (Guan and Dixon, 1991) .
  • the gene expression is controlled by the tac promoter, which is induced using the lactose analogue isopropyl-AD-thiogalactoside (IPTG) .
  • PCR polymerase chain reaction
  • Ncol and Xhol restriction sites (underlined lower case letters) were introduced at the 5* and 3' ends, respectively.
  • PCR was performed on 100 ng of plasmid pG23.1EB as template containing the 901 bp jE.coRI-_3a;nHI fragment of bpHl.
  • the reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl pH 8.3 , 50 mM KCl, 1.5 mM MgCl 2 , 0.001% w/v gelatin, 2% DMSO, 50 pmole of each primer, 200 ⁇ M of each dNTP, 2.5 U Taq DNA polymerase (Perkin Elmer, USA) .
  • DNA was denaturated for 5 min at 97 °C and PCR performed for 30 cycles: 30 sec at 97°C, 30 sec at 64°C, 30 sec at 72°C.
  • the amplified fragment was digested with Ncol and Xhol restriction enzymes and gel-purified.
  • the 165 bp bpHl amplified region was cloned 3' of the GST-LF N gene into the Ncol /Xhol sites of the pGEX expression vector for the construction of the GST-LF/bpHl hybrid sequence.
  • Several positive clones were obtained, as determined by restriction enzyme analysis, and named pGST/LF N bpHl.
  • the sequences of 3 positive clones were analyzed by automatic sequencing.
  • the plasmid DNA of the 3 positive clones (clones 4, 10 and 17) was transformed into the E . coli expression host TGI (Gibson, 1984).
  • E . coli TGI cells carrying plasmid pGST/LF N bpHl were grown at 37 °C in LB medium containing 50 ⁇ g/ml of ampicillin to ⁇ soo nm °f 1-0. Protein expression was induced by addition of 0.2 mM isopropyl-/?-D-thiogalactoside and incubation was continued for an additional three hours. Cells were harvested by centrifugation, the cell pellet was resuspended in 50 mM Tris-HCl, pH 8.0, 25% saccharose. Cell lysates were then prepared by mild sonication on ice and cleared by centrifugation. Samples were analyzed for the expression of the fusion protein by SDS-PAGE analysis.
  • DNA-binding activity of the fusion protein containing the bpHl-binding protein domain was determined in a Southwestern assay. 20 ⁇ l of the bacterial suspension was subjected to electrophoresis in a 12% SDS-polyacrylamide gel and blotted onto a nitrocellulose filter. The filter was saturated in 3% dried milk in PBS for 30 min at room temperature and rinsed with TE buffer. The filter was hybridized using as a probe pGEM®-3 (Promega Corporation, Madison, WI , USA) DNA, linearised at the EcoRI site. 100 ng of pGEM®-3 DNA was uniformly labelled with 32 P dATP and dCTP using a random priming kit (Amersha , UK) . Protein-DNA complexes were allowed to form for 20 min at room temperature in TE buffer, rinsed twice in TE buffer, and subjected to autoradiography (Figure 4).
  • the fusion proteins LT/BpHl (Example I) and LF N /BpHl (Example II) , containing detoxified toxin residues, can be incubated with an appropriate amount of transfecting DNA in order to bind the DNA to the BpHl residues of the fusion proteins.
  • the transfecting DNA can essentially be any double-stranded DNA capable of transfecting (through current methods of transfection) the toxin-sensitive cells.
  • a suitable DNA would be a plasmid containing a selection marker for eukaryotic cells that would allow selection in culture of positively transfected cells.
  • a plasmid containing the gene sequence of the Green Fluorescence Protein (GFP) (Clontech, Palo Alto, CA, USA) would allow selection of positive cells by monitoring the expression of GFP in cells by flow cytometry.
  • GFP Green Fluorescence Protein
  • a plasmid containing the Luciferase reporter gene (Prcmega Corporation, Madison, WI , USA) would allow the visualisation of positive cells by using a chemiluminescence assay.
  • the gene encoding the CRM197 diphtheria toxin (DT) mutant depleted of its toxic moiety (A domain) was amplified from a recombinant M13 mp8 plasmid carrying a 1.88 kb fragment encoding the tox operon (Giannini et al . , 1984).
  • the amplification product was fused to a 439 bp fragment isolated by PCR from plasmid pGBT9 (Clontech, Palo Alto, CA, USA) .
  • the 439 bp amplified fragment encodes amino acids 2-147 of the yeast protein GAL4 , a transcriptional activator which is able to bind to a specific double-strand DNA sequence (Carey et al .
  • the hybrid gene was inserted into the expression vector pTrcHis A (Invitrogen Corporation, San Diego, CA, USA) and the resulting plasmid pTrcHis-GAL/DTB was used to transform E . coli cells.
  • the recombinant clones obtained were able to express a fused protein which specifically recognized the GAL4-specific DNA sequence.
  • the fusion protein termed GAL/DTB, was also able to bind to DT receptors, as judged by binding analysis on African green monkey Vero cells.
  • a 1120 bp DNA fragment encoding amino acids 195-560 of CRM197 was isolated by PCR from plasmid M13 p ⁇ using the following synthetic oligodeoxynucleotides:
  • PCR was performed on 70 ng of plasmid M13 mp8 as template, containing the 1683 bp EcoRI/HindiII fragment of toxl97.
  • the reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl pH 8.3 , 1.5 M MgCl 2 , 50 M KC1, 2% DMSO, 50 picomoles of each primer, 200 ⁇ M of each dNTP and 5 U of Taq DNA polymerase (Boehringer Mannheim, Germany) .
  • DNA was denatured for 5 min at 94 °C and PCR was performed for 30 cycles: 30 sec at 94°C, 30 sec at 42°C, 1 min at 72°C.
  • the amplified fragment was digested with Sail and Hindlll restriction enzymes and gel-purified.
  • the GAL4 439 bp DNA fragment encoding amino acids 2-147 was amplified by PCR from plasmid pGBT9 (Clontech, Palo Alto, CA, USA) using the following synthetic oligodeoxynucleotides :
  • PCR was performed on 50 ng of plasmid pGBT9 as template, containing the 460 bp HindlH/EcoRI fragment of the GAL4 gene.
  • the reaction was performed in a volume of 100 ⁇ l containing 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl 2 , 50 mM KCl, 2% DMSO, 50 picomoles of each primer, 200 ⁇ M of each dNTP and 5 U of Taq DNA polymerase (Boehringer Mannheim, Germany) .
  • DNA was denatured for 5 min at 94 °C and PCR was performed for 30 cycles: 30 sec at 94°C, 30 sec at 42°C, 1 min at 72 °C.
  • the amplified fragment was digested with Kpnl and Sail restriction enzymes and gel-purified.
  • the 439 bp KpnI /Sal l fragment of the GAL4 gene obtained by PCR was cloned into KpnI /Sal l sites of pGEM®-3 cloning vector (Promega Corporation, Madison, WI , USA). Subsequently, the 1120 bp toxl97 amplified region was cloned 3' of the GAL4 gene fragment into the Sall/Hindlll sites for the construction of the GAL/DTB-encoding hybrid sequence, giving rise to plasmid pGEM-GAL/DTB.
  • the plasmid pTrcHis A (Invitrogen Corporation, San Diego, CA, USA) was used. This plasmid contains a Trc promoter and a histidine tag to easily purify the resulting protein.
  • the DNA fragment coding for the GAL/DTB fusion protein was isolated from plasmid pGEM-GAL/DTB by Sacl/Hindlll restriction enzyme digestion (note that nucleotides between Sad and Kpnl restriction sites of the Sacl/Hindlll fragment belong to the pGEM®-3 sequence) .
  • Sacl/Hindlll fragment derived from plasmid pGEM-GAL/DTB was cloned into the Sacl/Hindlll sites of pTrcHis A ( Figure 5) , giving rise to the expression plasmid pTrcHis-GAL/DTB.
  • E. coli DH5 ⁇ cells (Hanahan, 1983) carrying plasmid pTrcHis- GAL/DTB were grown at 37 °C in 1.7 1 of Luria-Bertani (LB) medium containing 100 ⁇ g/ml ampicillin to A 590nm of 0.7. Protein expression was induced by addition of 1 mM IPTG and incubation was continued for an additional 4 hrs . Cells were harvested by centrifugation and resuspended by stirring for 1 hr at room temperature in 15 ml of 6 M guanidine-HCl, 100 mM NaH 2 P0 4 , 10 mM Tris-HCl pH 8 (buffer A) .
  • LB Luria-Bertani
  • the mix was added to 4 ml of nickel-conjugated agarose (Qiagen GmbH, Germany) equilibrated in buffer A and, after 30 min stirring at room temperature, it was loaded on a 1.6 cm diameter column.
  • the column was washed with 10 volumes of buffer A and 5 volumes of 8 M urea, 100 mM NaH 2 P0 4 , 10 mM Tris-HCl pH 8. Additional washing with 8 M urea, 100 mM NaH 2 P0 4 , 10 mM Tris-HCl pH 6 were performed until O.D.,,,- of the eluate was ⁇ 0.01.
  • Elution of the GAL/DTB fused protein was achieved by a linear gradient of pH (from 6.5 to 4) in 60 ml of 8 M urea, 100 mM NaH 2 P0 , 10 mM Tris-HCl; the fused protein eluted at a pH between 4 and 4.5.
  • fractions containing GAL/DTB protein were pooled and dialysed overnight at room temperature against 4 M urea, 100 mM NaH 2 P0 , 10 mM Tris-HCl pH 7 , 5% glycerol.
  • the resulting GAL/DTB solution was again dialysed overnight at 4°C against 2 M urea, 100 mM NaH 2 P0 4 , 10 mM Tris-HCl pH 7, 10% glycerol.
  • the 2 M urea solution of GAL/DTB protein was diluted to 100 ⁇ g/ml in 500 mM L-arginine, 50 mM phosphate buffer pH 7, 0.5 mM glutathione (oxidated), 5 mM glutathione (reduced), 10% glycerol and dialysed overnight at 4°C against the same buffer. (6) Analysis of DNA-binding capacity of GAL/DTB protein
  • African green monkey kidney Vero cells were maintained in D-MEM medium supplemented with 5% heat-inactivated foetal calf serum (FCS) at 37 °C in a humidified atmosphere of air and 5% C0 2 .
  • FCS foetal calf serum
  • cells were recovered, resuspended in PBS containing 5% FCS (PBS-5% FCS) and seeded on a 96-well tissue culture plate at a density of 10 5 cells/well in a volume of 10 ⁇ l.
  • PBS-5% FCS 5% calf serum
  • Increasing amounts of GAL/DTB protein final concentration ranging from 0.006 ⁇ g/ml to 54 ⁇ g/ml
  • GAL/DTB-cells complexes were incubated overnight at 4°C, rinsed twice in PBS-5% FCS, resuspended in 10 ⁇ l of PBS-5% FCS and stained for 45 min on ice with a polyclonal anti-GAL4 (1-147) antibody raised in rabbit (Santa Cruz Biotechnology, CA, USA) at a final concentration of 1 ⁇ g/ml in each well.
  • Cells were rinsed twice with PBS-5% FCS, resuspended in 10 ⁇ l of PBS-5% FCS and stained for 30 min on ice with anti-rabbit FITC- conjugated antibody (Boehringer Mannheim, Germany) at a final concentration of 12.5 ⁇ g/ml in each well. After a final washing with PBS-5% FCS, the cells were resuspended in 200 ⁇ l of PBS-5% FCS and subjected to FACS analysis (Figure 7).
  • GAL/DTB binds Vero cells in a suitable manner as determined by fluorescence intensity measurement.
  • the minimal saturating concentration was in the region of 2 ⁇ g/ml.
  • African green monkey kidney Vero cells were maintained in D-MEM medium supplemented with 5% heat-inactivated foetal calf serum (FCS) at 37 °C in a humidified atmosphere of air and 5% C0 2 .
  • FCS foetal calf serum
  • cells were recovered, resuspended in PBS-5% FCS and seeded on a 96-well tissue culture plate at a density of 10 5 cells/well in a volume of 10 ⁇ l.
  • GAL/DTB protein was added to each well at a concentration of 1.5 ⁇ g/ml in a final reaction volume of 20 ⁇ l.
  • GAL/DTB-cells complexes were incubated overnight at 4°C and then incubated at 37°C for 0, 5, 10, 30 or 60 min. After two rinsing steps in PBS-5% FCS, the cells were 8/59065
  • the mean fluorescence intensity value decreases with time, indicating that the hybrid GAL/DTB protein is internalised by Vero cells.
  • the intemalisation process appears to be quite rapid, being 60% accomplished within 5 min of the temperature shift.

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Abstract

La présente invention concerne un nouveau système de transfert d'ADN dans des cellules, lequel fait appel à une endocytose dont le médiateur est un récepteur pour l'introduction de l'ADN dans la cellule. L'invention se rapporte à des molécules de toxine ou de type toxine modifiées comprenant des motifs structuraux fixant l'ADN. Ces molécules de toxine ou de type toxine, qui sont capables d'entrer dans la cellules par une endocytose dont le médiateur est un récepteur, sont rendues non toxiques et peuvent se lier à l'ADN pour former des conjugués toxine/ADN de type toxine modifiés. Ces conjugués toxine/ADN de type toxine modifiés, une fois entrés dans la cellule, libèrent leur 'ADN passager' et, ce faisant, transforment ou transfectent la cellule. L'invention se rapporte également à des procédés de transfert d'ADN faisant appel aux molécules de toxine ou de type toxine modifiées et à l'utilisation desdits procédés.
PCT/IB1998/001005 1997-06-20 1998-06-18 Nouveau procede de transfert d'adn dans des cellules WO1998059065A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9713122.1A GB9713122D0 (en) 1997-06-20 1997-06-20 Novel method of DNA transfer into cells
GB9713122.1 1997-06-20

Publications (1)

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WO1998059065A1 true WO1998059065A1 (fr) 1998-12-30

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PCT/IB1998/001005 WO1998059065A1 (fr) 1997-06-20 1998-06-18 Nouveau procede de transfert d'adn dans des cellules

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WO (1) WO1998059065A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1314808C (zh) * 2000-12-28 2007-05-09 中国医学科学院基础医学研究所 绿脓毒素基因及其包装与导向蛋白复合物对癌的靶向基因治疗
US9902960B2 (en) 2013-06-21 2018-02-27 University Of Greenwich Antisense oligonucleotide compositions

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004696A1 (fr) * 1992-08-25 1994-03-03 Miles Inc. Apport nucleaire de macromolecules facilite par un signal de translocation
WO1995022618A1 (fr) * 1994-02-22 1995-08-24 Dana-Farber Cancer Institute Systeme de liberation d'acide nucleique, son procede de synthese et ses utilisations
WO1995028494A1 (fr) * 1994-04-15 1995-10-26 Targeted Genetics Corporation Proteine de fusion d'apport de gene
WO1996013599A1 (fr) * 1994-11-01 1996-05-09 Winfried Wels Systeme de transfert de l'acide nucleique
WO1997005267A2 (fr) * 1995-07-26 1997-02-13 Maxim Pharmaceuticals Apport de polynucleotides dans les muqueuses
WO1997023236A1 (fr) * 1995-12-13 1997-07-03 President And Fellows Of Harvard College Utilisation de peptides de toxine et/ou de marqueurs d'affinite pour l'introduction de composes dans des cellules

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004696A1 (fr) * 1992-08-25 1994-03-03 Miles Inc. Apport nucleaire de macromolecules facilite par un signal de translocation
WO1995022618A1 (fr) * 1994-02-22 1995-08-24 Dana-Farber Cancer Institute Systeme de liberation d'acide nucleique, son procede de synthese et ses utilisations
WO1995028494A1 (fr) * 1994-04-15 1995-10-26 Targeted Genetics Corporation Proteine de fusion d'apport de gene
WO1996013599A1 (fr) * 1994-11-01 1996-05-09 Winfried Wels Systeme de transfert de l'acide nucleique
WO1997005267A2 (fr) * 1995-07-26 1997-02-13 Maxim Pharmaceuticals Apport de polynucleotides dans les muqueuses
WO1997023236A1 (fr) * 1995-12-13 1997-07-03 President And Fellows Of Harvard College Utilisation de peptides de toxine et/ou de marqueurs d'affinite pour l'introduction de composes dans des cellules

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1314808C (zh) * 2000-12-28 2007-05-09 中国医学科学院基础医学研究所 绿脓毒素基因及其包装与导向蛋白复合物对癌的靶向基因治疗
US9902960B2 (en) 2013-06-21 2018-02-27 University Of Greenwich Antisense oligonucleotide compositions
US10400241B2 (en) 2013-06-21 2019-09-03 University Of Greenwich Antisense oligonucleotide compositions

Also Published As

Publication number Publication date
GB9713122D0 (en) 1997-08-27

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