Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5434—IL-12
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/555—Interferons [IFN]
  • C07K14/56—IFN-alpha
• • 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
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • 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
  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
• • 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
  • C12N2840/00—Vectors comprising a special translation-regulating system
  • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron

Definitions

• the present invention relates to gene delivery and gene therapy, and provides novel nucleic acid constructs for expression of interferon alpha in a mammal, formulations for delivery that incorporate a nucleic acid construct for expression, and methods for preparing and using such constructs and formulations.
• this invention relates to plasmid constructs for delivery of therapeutic interferon alpha encoding nucleic acids to cells in order to modulate tumor activity, methods of using those constructs (including combination therapy with other agents, such as cytokines, preferably IL-12), as well as methods for preparing such constructs .
• Plasmids are an important element in genetic engineering and gene therapy. Plasmids are usually circular DNA molecules that can be introduced into bacterial cells by transformation which replicate autonomously in the cell. Plasmids typically allow for the amplification of cloned DNA. Some plasmids are present in 20 to 50 copies during cell growth, and after the arrest of protein synthesis, as many as 1000 copies per cell of a plasmid can be generated. Suzuki et al . , Genetic Analysis, p. 404, 1989.
• Plasmid design and construction utilizes several key factors.
• plasmid replication origins determine plasmid copy number, which affects production yields. Plasmids that replicate to higher copy number can increase plasmid yield from a given volume of culture, but excessive copy number can be deleterious to the bacteria and lead to undesirable effects (Fitzwater, et al., Embo J. 7:3289-3297 (1988); Uhlin, et al., Mol . Gen . Genet . 165:167-179 (1979)). Artificially constructed plasmids are sometimes unstably maintained, leading to accumulation of plasmid-free cells and reduced production yields.
• genes that code for antibiotic resistance phenotype are included on the plasmid and antibiotics are added to kill or inhibit plasmid-free cells.
• Most general purpose cloning vectors contain ampicillin resistance ( ⁇ -lactamase, or bla ) genes. Use of ampicillin can be problematic because of the potential for residual antibiotic in the purified DNA, which can cause an allergic reaction in a treated patient.
• ⁇ -lactam antibiotics are clinically important for disease treatment. When plasmids containing antibiotic resistance genes are used, the potential exists for the transfer of antibiotic resistance genes to a potential pathogen.
• neo gene which is derived from the bacterial transposon Tnb .
• the neo gene encodes resistance to kanamycin and neomycin (Smith, Vaccine 12:1515-1519 (1994)).
• This gene has been used in a number of gene therapy studies, including several human clinical trials (Recombinant DNA Advisory Committee Data Management Report, December, 1994, Human Gene Therapy 6:535-548). Due to the mechanism by which resistance is imparted, residual antibiotic and transmission of the gene to potential pathogens may be less of a problem than for ⁇ -lactams.
• plasmid vectors have also been shown to affect transfection and expression in eukaryotic cells. Certain plasmid sequences have been shown to reduce expression of eukaryotic genes in eukaryotic cells when carried in cis (Peterson, et al., Mol . Cell . Biol . 7:1563-1567 (1987); Yoder et al., Mol . Cell . Biol . 3:956-959 (1983); Lusky et al., Nature 293:79-81 (1981); and Leite, et al., Gene 82:351-356 (1989)).
• Plasmid sequences also have been shown to fortuitously contain binding sites for transcriptional control proteins (Ghersa, et al., Gene 151:331-332 (1994); Tully et al., Biochem . Biophys . Res . Comm . 144:1-10 (1987); and Kushner, et al., Mol . Endocrinol . 8:405-407 (1994)). This can cause inappropriate levels of gene expression in treated patients.
• Interferon alpha is a gene product that has been proposed for use, either alone or in combination with other agents, in different delivery systems for the treatment of certain diseases, including particular cancers.
• International patent publication WO/97/00085 published January 3, 1997, proposes ex vivo transfection of tumor cells with interferon alpha and another immomodulatory molecule, such as IL-12. None of the previously proposed treatments have proven entirely satisfactory, due in part to the high cost and technical difficulty involved in ex vivo approaches. Thus there still remains a need in the art for improved plasmids encoding interferon alpha as well as improved treatment protocols and technologies.
• the present invention relates to gene delivery and gene therapy, and provides novel nucleic acid constructs for expression of interferon alpha in a mammal, formulations for delivery that incorporate a nucleic acid construct for expression, and methods for preparing and using such constructs and formulations.
• this invention relates to plasmid constructs for delivery of therapeutic interferon alpha encoding nucleic acids to cells in order to modulate tumor activity, methods of using those constructs (including combination therapy with other agents, such as cytokines, preferably IL-12), as well as methods for preparing such constructs.
• the pharmaceutical acceptable, cost effective and highly efficient delivery system presented herein represents an unanticipated improvement over the art.
• the invention features a plasmid that contains a CMV promoter and optionally a synthetic 5' intron transcriptionally linked with an interferon alpha coding sequence, and a 3 ' -untranslated region (UTR) .
• the 3' UTR is a 3 ' growth hormone UTR.
• plasmid refers to a construct made up of genetic material (i.e., nucleic acids). It includes genetic elements arranged such that an inserted coding sequence can be transcribed in eukaryotic cells. Also, while the plasmid may include a sequence from a viral nucleic acid, such viral sequence does not cause the incorporation of the plasmid into a viral particle, and the plasmid is therefore a non-viral vector. Preferably a plasmid is a closed circular DNA molecule.
• Cytomegalovirus promoter refers to one or more sequences from a cytomegalovirus which are functional in eukaryotic cells as a transcriptional promoter and an upstream enhancer sequence.
• the enhancer sequence allows transcription to occur at a higher frequency from the associated promoter.
• transcriptionally linked means that in a system suitable for transcription, transcription will initiate under the direction of the control sequence (s) and proceed through sequences which are transcriptionally linked with that control sequence (s). Preferably no mutation is created in the resulting transcript, which would alter the resulting translation product.
• coding region refers to a nucleic acid sequence which encodes a particular gene product for which expression is desired, according to the normal base pairing and codon usage relationships.
• the coding sequence must be placed in such relationship to transcriptional control sequences (possibly including control elements and translational initiation and termination codons) that a proper length transcript will be produced and will result in translation in the appropriate reading frame to produce a functional desired product.
• the interferon alpha coding sequence is for human interferon alpha and preferably is a synthetic sequence having optimal codon usage, such as the nucleotide sequence of SEQ ID NO: 11 or semi-optimal codon usage, such as the nucleotide sequence of SEQ ID NO: 12.
• coding regions suitable for use in the plasmids of this invention are the natural sequences coding for human interferon alpha.
• coding region has a nucleotide sequence which is the same as SEQ ID NO: 10, which is the natural nucleotide sequence encoding human interferon alpha.
• an interferon alpha coding sequence which is a synthetic coding sequence.
• the interferon alpha coding sequence is a synthetic sequence utilizing optimal or semi-optimal codon usage, preferably the sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12.
• a “sequence coding for the human interferon alpha” or "a human interferon alpha coding sequence” is a nucleic acid sequence which encodes the amino acid sequence of human interferon alpha, based on the normal base pairing and translational codon usage relationships. It is preferable that the coding sequence encode the exact, full amino acid sequence of natural human interferon, but this is not essential.
• the encoded polypeptide may differ from natural human interferon alpha, so long as the polypeptide retains interferon alpha activity, preferably the polypeptide is at least 50%, 75%, 90%, or 97% as active as natural human interferon alpha, and more preferably fully as active as natural human interferon alpha.
• the polypeptide encoded by the interferon alpha coding sequence may differ from a natural human interferon alpha polypeptide by a small amount, preferably less than a 15%, 10%, 5%, or 1% change.
• a change may be of one of more different types, such as deletion, addition, or substitution of one or more amino acids.
• transcriptional control sequence refers to sequences which control the rate of transcription of a transcriptionally linked coding region.
• the term can include elements such as promoters, operators, and enhancers.
• the transcriptional control sequences will include at least a promoter sequence.
• a "growth hormone 3' untranslated region” is a sequence located downstream (i.e., 3') of the region encoding material polypeptide and including at least part of the sequence of the natural 3' UTR/poly(a) signal from a growth hormone gene, preferably the human growth hormone gene. This region is generally transcribed but not translated.
• sequence which signals the addition of a poly-A tail For expression in eukaryotic cells it is generally preferable to include sequence which signals the addition of a poly-A tail.
• a synthetic 3' UTR/poly(A) signal has a sequence which differs from naturally-occurring UTR elements.
• the sequence may be modified, for example by the deletion of ALU repeat sequences. Deletion of such ALU repeat sequences acts to reduce the possibility of homologous recombination between the expression cassette and genomic material in a transfected cell.
• the plasmid preferably includes a promoter, a TATA box, a Cap site and a first intron and intron/exon boundary in appropriate relationship for expression of the coding sequence.
• the plasmid may also include a 5' mRNA leader sequence inserted between the promoter and the coding sequence and/or an intron/5' UTR from a chicken skeletal ⁇ - actin gene.
• the plasmid may have a nucleotide sequence which is the same as the nucleotide sequence of plasmid pIF0921, as shown in Figure 5.
• the plasmid may also include: (a) a first transcription unit containing a first transcriptional control sequence transcriptionally linked with a first 5 ' -untranslated region, a first intron, a first coding sequence, and a first 3 ' -untranslated region/poly (A) signal, wherein the first intron is between the control sequence and the first coding sequence; and (b) a second transcription unit containing a second transcriptional control sequence transcriptionally linked with a second 5 ' -untranslated region, a second intron, a second coding sequence, and a second 3'- untranslated region/poly (A) signal, wherein the second intron is between the control sequence and the second coding sequence; wherein the first and second coding sequences contain a sequence having the sequence of SEQ ID NO: 2, 3, 4 or 25 coding for a human IL-12 p40 subunit, and a sequence having the sequence of SEQ ID NO: 6, 7, 8 or 24 coding for a human IL-12 p35 sub
• transcription unit or "expression cassette” refers to a nucleotide sequence which contains at least one coding sequence along with sequence elements which direct the initiation and termination of transcription.
• a transcription unit may however include additional sequences, which may include sequences involved in post-transcriptional or post-translational processes.
• the first transcriptional control sequence or the second transcriptional control sequence contain one or more cytomegalovirus promoter sequences.
• the first and second transcriptional control sequences can be the same or different.
• a “5' untranslated region” or “5' UTR” refers to a sequence located 3' to promoter region and 5' of the downstream coding region. Thus, such a sequence, while transcribed, is upstream of the translation initiation codon and therefore is not translated into a portion of the polypeptide product.
• one or more of a promoter, 5' untranslated region (5' UTR), the 3' UTR/poly(A) signal, and introns may be a synthetic sequence.
• synthetic means that the sequence is not provided directly by the sequence of a naturally occurring genetic element of that type but rather is an artificially created sequence (i.e., created by a person by molecular biological methods) . While one or more portions of such a synthetic sequence may be the same as portions of naturally occurring sequences, the full sequence over the specified genetic element is different from a naturally occurring genetic element of that type. The use of such synthetic genetic elements allows the functional characteristics of that element to be appropriately designed for the desired function.
• a “synthetic intron” refers to a sequence which is not a naturally occurring intron sequence but which will be removed from an RNA transcript during normal post transcriptional processing. Such introns can be designed to have a variety of different characteristics, in particular such introns can be designed to have a desired strength of splice site.
• a "subunit" of a therapeutic molecule is a polypeptide or RNA molecule which combines with one or more other molecules to form a complex having the relevant pharmacologic activity. Examples of such complexes include homodimers and heterodimers as well as complexes having greater numbers of subunits. A specific example of a heterodimer is IL-12, having the p40 and p35 subunits.
• the "p40 subunit” is the larger of the two subunits of the IL-12 heterodimer. Thus, it is capable of association with p35 subunit to form a molecule having activities characteristic of IL-12.
• Human p40 has the amino acid sequence of SEQ ID NO:l. Those skilled in the art will recognize that the molecule may have a number of changes from that sequence, such as deletions, insertions or changes of one or a few amino acids, while still retaining IL-12 activity when associated with p35. Such active altered molecules are also regarded as p40.
• p35 subunit is the smaller of the two heterodimeric subunits of IL-12.
• p35 has the amino acid sequence of SEQ ID NO: 5.
• p35 may have a low level of alterations from that sequence while still being regarded as p35.
• a particular example of coding regions suitable for use in the plasmids of this invention are the natural sequences coding for the p40 and p35 subunits of human IL-12.
• the first and second coding regions are coding regions for those sequences and are preferably in the order p40 then p35 in the 5' to 3' direction.
• sequence coding for the p40 subunit of human IL-12 is a nucleic acid sequence which encodes the human p40 subunit as described above, based on the normal base pairing and translational codon usage relationships.
• sequence coding for p35 subunit of human IL-12 is similarly defined.
• sequence coding for the p40 subunit of human IL-12 is 5' to the sequence coding for the p35 subunit of human IL-12.
• interferon alpha, p35 subunit and p40 subunit may all be on a single transcription unit, that all three may be on separate transcription units, or that any two coding sequences may be on one transcription unit and the other coding sequence on another transcription unit.
• the plasmid may also contain an intron having variable splicing, a first coding sequence, and a second coding sequence, wherein the first and second coding sequences include a sequence having the sequence of SEQ ID NO: 2, 3, 4 or 25 coding for a human IL-12 p40 subunit, and a sequence having the sequence of SEQ ID NO: 6, 7, 8 or 24 coding for a human IL-12 p35 subunit.
• the plasmid also has: (a) a transcriptional control sequence transcriptionally linked with a first coding sequence and a second coding sequence; (b) a 5 ' -untranslated region; (c) an intron 5' to the first coding sequence; (d) an alternative splice site 3' to the first coding sequence and 5' to the second coding sequence; and (e) a 3 ' -untranslated region/poly (A) signal.
• the transcriptional control sequence preferably includes a cytomegalovirus promoter sequence.
• the plasmid also has: (a) a transcriptional control sequence transcriptionally linked with a first coding sequence, an IRES sequence, a second coding sequence, and a 3 ' -untranslated region/poly (A) signal, wherein the IRES sequence is between the first coding sequence and the second coding sequence; and (b) an intron between the promoter and the first coding sequence; wherein the first and second coding sequences include a sequence having the sequence of SEQ ID NO: 2, 3, 4 or 25 coding for a human IL-12 p40 subunit, and a sequence having the sequence of SEQ ID NO: 6, 7, 8 or 24 coding for a human IL-12 p35 subunit.
• the transcriptional control sequence preferably includes a cytomegalovirus promoter sequence and the IRES sequence preferably is from an encephalomyocarditis virus.
• the invention features a composition containing a plasmid as described above and a protective, interactive non-condensing compound (PINC) .
• PINC protective, interactive non-condensing compound
• the PINC enhances the delivery of the nucleic acid molecule to mammalian cells in vivo, and preferably the nucleic acid molecule includes a coding sequence for a gene product to be expressed in the cell.
• the relevant gene product is a polypeptide or protein.
• the PINC is used under conditions so that the PINC does not form a gel, or so that no gel form is present at the time of administration at about 30-40DC.
• the PINC is present at a concentration of 30% (w/v) or less. In certain preferred embodiments, the PINC concentration is still less, for example, 20% or less, 10% or less, 5% or less, or 1% or less.
• compositions differ in compound concentration and functional effect from uses of these or similar compounds in which the compounds are used at higher concentrations, for example in the ethylene glycol mediated transfection of plant protoplasts, or the formation of gels for drug or nucleic acid delivery.
• the PINCs are not in gel form in the conditions in which they are used as PINCs, though certain of the compounds may form gels under some conditions.
• the term "protects" or “protective” refers to an effect of the interaction between such a compound and a nucleic acid such that the rate of degradation of the nucleic acid is decreased in a particular environment.
• Such degradation may be due to a variety of different factors, which specifically include the enzymatic action of a nuclease.
• the protective action may be provided in different ways, for example, by exclusion of the nuclease molecules or by exclusion of water.
• Some compounds which protect a nucleic acid and/or prolong the bioavailability of a nucleic acid may also interact or associate with the nucleic acid by intermolecular forces and/or valence bonds such as: Van der Waals forces, ion-dipole interactions, ion-induced dipole interactions, hydrogen bonds, or ionic bonds.
• Piggyback endocytosis is the cellular uptake of a drug or other molecule complexed to a carrier that may be taken up by endocytosis.
• CV Uglea and C Dumitriu-Medvichi Medical Applications of Synthetic Oligomers, In: Polymeric Biomaterials, Severian Dumitriu ed., Marcel Dekker, Inc., 1993, incorporated herein by reference.
• the compounds which protect the nucleic acid and/or prolong the bioavailability of a nucleic acid have amphiphilic properties; that is, have both hydrophilic and hydrophobic regions.
• the hydrophilic region of the compounds may associate with the largely ionic and hydrophilic regions of the nucleic acid, while the hydrophobic region of the compounds may act to retard diffusion of nucleic acid and to protect nucleic acid from nucleases. Additionally, the hydrophobic region may specifically interact with cell membranes, possibly facilitating endocytosis of the compound and thereby also of nucleic acid associated with the compound. This process may increase the pericellular concentration of nucleic acid.
• Agents which may have amphiphilic properties and are generally regarded as being pharmaceutically acceptable are the following: polyvinylpyrrolidones; polyvinylalcohols; polyvinylacetates; propylene glycol; polyethylene glycols; poloxamers (Pluronics) ; poloxamines (Tetronics) ; ethylene vinyl acetates; methylcelluloses, hydroxypropylcelluloses, hydroxypropylmethylcelluloses; heteropolysaccharides (pectins) ; chitosans; phosphatidylcholines (lecithins) ; miglyols; polylactic acid; polyhydroxybutyric acid; xanthan gum.
• copolymer systems such as polyethylene glycol- polylactic acid (PEG-PLA), polyethylene glycol- polyhydroxybutyric acid (PEG-PHB) , polyvinylpyrrolidone- polyvinylalcohol (PVP-PVA) , and derivatized copolymers such as copolymers of N-vinyl purine (or pyrimidine) derivatives and N-vinylpyrrolidone.
• PEG-PLA polyethylene glycol- polylactic acid
• PEG-PHB polyethylene glycol- polyhydroxybutyric acid
• PVP-PVA polyvinylpyrrolidone- polyvinylalcohol
• derivatized copolymers such as copolymers of N-vinyl purine (or pyrimidine) derivatives and N-vinylpyrrolidone.
• non- condensing means that an associated nucleic acid is not condensed or collapsed by the interaction with the PINC at the concentrations used in the compositions.
• the PINCs differ in type and/or use concentration from such condensing polymers.
• Examples of commonly used condensing polymers include polylysine, and cascade polymers (spherical polycations) .
• the term “enhances the delivery” means that at least in conditions such that the amounts of PINC and nucleic acid is optimized, a greater biological effect is obtained than with the delivery of nucleic acid in saline.
• the level of expression obtained with the PINC: nucleic acid composition is greater than the expression obtained with the same quantity of nucleic acid in saline for delivery by a method appropriate for the particular PINC/coding sequence combination.
• the PINC is polyvinyl pyrrolidone (PVP) , polyvinyl alcohol (PVA), a PVP-PVA co-polymer, N-methyl-2-pyrrolidone (NM2P) , ethylene glycol, or propylene glycol.
• PVP polyvinyl pyrrolidone
• PVA polyvinyl alcohol
• NM2P N-methyl-2-pyrrolidone
• ethylene glycol or propylene glycol.
• the nucleic acid is preferably not a viral vector, i.e., the nucleic acid is a non-viral vector.
• the PINC is bound with a targeting ligand.
• targeting ligands can be of a variety of different types, including but not limited to galactosyl, residues, fucosal residues, mannosyl residues, carntitine derivatives, monoclonal antibodies, polyclonal antibodies, peptide ligands, and DNA-binding proteins.
• the targeting ligands may bind with receptors on cells such as antigen-presenting cells, hepatocytes, myocytes, epithelial cells, endothelial cells, and cancer cells.
• the term "bound with” means that the parts have an interaction with each other such that the physical association is thermodynamically favored, representing at least a local minimum in the free energy function for that association.
• Such interaction may involve covalent binding, or non-covalent interactions such as ionic, hydrogen bonding, van der Waals interactions, hydrophobic interactions, and combinations of such interactions.
• the targeting ligand may be of various types, in one embodiment the ligand is an antibody. Both monoclonal antibodies and polyclonal antibodies may be utilized.
• the nucleic acid may also be present in various forms. Preferably the nucleic acid is not associated with a compounds (s) which alter the physical form, however, in other embodiments the nucleic acid is condensed (such as with a condensing polymer) , formulated with cationic lipids, formulated with peptides, or formulated with cationic polymers.
• the protective, interactive non-condensing compound is polyvinyl pyrrolidone, and/or the plasmid is in a solution having between 0.5% and 50% PVP, more preferably about 5% PVP.
• the DNA preferably is at least about 80% supercoiled, more preferably at least about 90% supercoiled, and most preferably at least about 95% supercoiled.
• the invention features a composition containing a protective, interactive non-condensing compound and a plasmid containing an interferon alpha coding sequence.
• the invention provides a composition containing a plasmid of the invention (or a plasmid containing an interferon alpha coding sequence) and a cationic lipid with a neutral co-lipid.
• the cationic lipid is DOTMA and the neutral co-lipid is cholesterol (chol) .
• DOTMA is 1,2-di-O- octadecenyl-3-trimethylammonium propane, which is described and discussed in Eppstein et al., U.S. Patent 4,897,355, issued January 30, 1990, which is incorporated herein by reference.
• other lipids and lipid combinations may be used in other embodiments. A variety of such lipids are described in Gao & Huang, 1995, Gene Therapy 2:710-722, which is hereby incorporated by reference.
• the charge ratio of the cationic lipid and the DNA is also a significant factor, in preferred embodiments the DNA and the cationic lipid are present is such amounts that the negative to positive charge ratio is about 1:3. While preferable, it is not necessary that the ratio be 1:3. Thus, preferably the charge ratio for the compositions is between about 1:1 and 1:10, more preferably between about 1:2 and 1:5.
• cationic lipid refers to a lipid which has a net positive charge at physiological pH, and preferably carries no negative charges at such pH.
• An example of such a lipid is DOTMA.
• neutral co-lipid refers to a lipid which has is usually uncharged at physiological pH.
• An example of such a lipid is cholesterol.
• negative to positive charge ratio for the DNA and cationic lipid refers to the ratio between the net negative charges on the DNA compared to the net positive charges on the cationic lipid.
• the DNA preferably is at least about 80% supercoiled, more preferably at least about 90% supercoiled, and most preferably at least about 95% supercoiled.
• the composition preferably includes an isotonic carbohydrate solution, such as an isotonic carbohydrate solution that consists essentially of about 10% lactose.
• the composition the cationic lipid and the neutral co-lipid are prepared as a liposome having an extrusion size of about 800 nanometers.
• the liposomes are prepared to have an average diameter of between about 20 and 800 nm, more preferably between about 50 and 400 nm, still more preferably between about 75 and 200 nm, and most preferably about 100 nm.
• Microfluidization is the preferred method of preparation of the liposomes.
• the invention features a composition containing: (a) a first component having a plasmid including an interferon alpha coding sequence and a cationic lipid with a neutral co-lipid, wherein the cationic lipid is DOTMA and the neutral co-lipid is cholesterol, wherein the DNA in the plasmid and the cationic lipid are present in amounts such that the negative to positive charge ratio is about 1:3; and (b) a second component including a protective, interactive non-condensing compound, wherein the first component is present within the second component.
• the invention provides a composition having a protective, interactive non-condensing compound, a first plasmid including an interferon alpha coding sequence, and one or more other plasmids independently having an IL-12 p35 or IL-12 p40 subunit coding sequence.
• the invention features a method for making any of the plasmids described above by inserting a CMV promoter transcriptionally linked with an interferon alpha coding sequence, and a growth hormone 3 ' -untranslated region into a plasmid.
• the invention also provides methods of making the compositions described above.
• the method may involve: (a) preparing a DNA molecule having a transcriptional unit, wherein the transcriptional unit contains an interferon alpha coding sequence; (b) preparing a protective, interactive non-condensing compound; and (c) combining the protective, interactive non-condensing compound with the DNA in conditions such that a composition capable of delivering a therapeutically effective amount of an interferon alpha coding sequence to a mammal is formed.
• the DNA molecule is a plasmid, wherein the plasmid includes a CMV promoter transcriptionally linked with an interferon alpha coding sequence, and a human growth hormone 3 ' -untranslated region/poly (A) signal.
• the plasmid includes a CMV promoter transcriptionally linked with an interferon alpha coding sequence, and a human growth hormone 3 ' -untranslated region/poly (A) signal.
• the method may involve the steps of: (a) preparing a DNA having an interferon alpha coding sequence; (b) preparing a mixture of a cationic lipid and a neutral co- lipid, wherein the cationic lipid is DOTMA and the neutral co-lipid is cholesterol; and (c) combining the mixture with the DNA in amounts such that the cationic lipid and the DNA are present in a negative to positive charge ratio of about 1:3.
• the method involves the steps of: (a) preparing a first component having a plasmid containing an interferon alpha coding sequence and a cationic lipid with a neutral co-lipid, wherein the cationic lipid is DOTMA and the neutral co-lipid is cholesterol, wherein the DNA in the plasmid and the cationic lipid are present in amounts such that the negative to positive charge ratio is about 1:3; (b) preparing a second component having a protective, interactive non-condensing compound; and (c) combining the first and second components such that the resulting composition includes the first component within the second component .
• the method involves the steps of: (a) preparing a protective, interactive non-condensing compound, (b) preparing a first plasmid having an interferon alpha coding sequence, (c) preparing one or more other plasmids independently having an IL-12 p35 or IL-12 p40 subunit coding sequence, and (d) combining the protective, interactive non-condensing compound, the plasmid having the interferon alpha coding sequence and the other plasmids.
• the invention provides a method for treatment of a mammalian condition or disease, by administering to a mammal suffering from the condition or disease a therapeutically effective amount of a plasmid as described herein.
• a “therapeutically effective amount” of a composition is an amount which is sufficient to cause at least temporary relief or improvement in a symptom or indication of a disease or condition. Thus, the amount is also sufficient to cause a pharmacological effect. The amount of the composition need not cause permanent improvement or improvement of all symptoms or indications.
• a therapeutically effective amount of a cancer therapeutic would be one that reduces overall tumor burden in the case of metastatic disease (i.e., the number of metasteses or their size) or one that reduces the mass of the tumor in localized cancers.
• the condition or disease preferably is a cancer, such as epithelial glandular cancer, including adenoma and adenocarcinoma; squamous and transitional cancer, including polyp, papilloma, squamous cell and transitional cell carcinoma; connective tissue cancer, including tissue type positive, sarcoma and other (oma's); hematopoietic and lymphoreticular cancer, including lymphoma, leukemia and Hodgkin's disease; neural tissue cancer, including neuroma, sarcoma, neurofibroma and blastoma; mixed tissues of origin cancer, including teratoma and teratocarcinoma.
• a cancer such as epithelial glandular cancer, including adenoma and adenocarcinoma
• squamous and transitional cancer including polyp, papilloma, squamous cell and transitional cell carcinoma
• connective tissue cancer including tissue type positive, sarcoma and other (
• cancerous conditions that are applicable to treatment include cancer of any of the following: adrenal gland, anus, bile duct, bladder, brain tumors: adult, breast, cancer of an unknown primary site, carcinoids of the gastrointestinal tract, cervix, childhood cancers, colon and rectum, esophagus, gall bladder, head and neck, islet cell and other pancreatic carcinomas, Kaposi's sarcoma, kidney, leukemia, liver, lung: non-small cell, lung: small cell, lymphoma: AIDS-associated, lymphoma: Hodgkin's disease, Lymphomas: non-Hodgkin' s disease, melanoma, mesothelioma, metastatic cancer, multiple myeloma, ovary, ovarian germ cell tumors, pancreas, parathyroid, penis, pituitary, prostate, sarcomas of bone and soft tissue, skin, small intestine, stomach, testis, thymus, thyroid,
• the invention provides a method for transfection (i.e., the delivery and expression of a gene to cells) of a cell in si tu, by contacting the cell with a plasmid described herein for sufficient time to transfect the cell.
• Transfection of the cell preferably is performed in vivo and the contacting preferably is performed in the presence of about 5% PVP solution.
• the invention features a method for delivery and expression of an interferon alpha gene in a plurality of cells, by: (a) transfecting the plurality of cells with a plasmid or composition of the invention; and (b) incubating the plurality of cells under conditions allowing expression of a nucleic acid sequence in the vector, wherein the nucleic acid sequence encodes interferon alpha.
• the interferon alpha is human interferon alpha and the cells are human cells and/or the contacting is performed in the presence of an about 5% PVP solution.
• the invention features a method for treating a disease or condition, by transfecting a cell in si tu with a plasmid or composition of the invention.
• the disease or condition can be a localized disease or condition or a systemic disease or condition.
• the invention features a cell transfected with a plasmid or composition of the invention.
• the invention features a method for treatment of a mammalian condition or disease, by administering to a mammal suffering from the condition or disease a therapeutically effective amount of a composition described herein.
• compositions are useful for delivery of a nucleic acid molecule to cells in vivo
• the invention provides a composition at an in vivo site of administration. In particular this includes at an in vivo site in a mammal.
• the nucleic acid molecule includes a sequence encoding a gene product.
• the site of administration is in an interstitial space or a tissue of an animal, particularly of a mammal .
• compositions also provides methods for using the above compositions. Therefore, in further related aspects, methods of administering the compositions are provided in which the composition is introduced into a mammal, preferably into a tissue or an interstitial space.
• compositions are introduced into the tissue or interstitial space by injection.
• the compositions may also be delivered to a variety of different tissues, but in preferred embodiments the tissue is muscle or tumor.
• the invention provides methods for treating a mammalian condition or disease by administering a therapeutically effective amount of a composition as described above.
• the disease or condition is a cancer.
• Figure 1 shows the effects of interferon alpha in two cancer models.
• Figure 2 shows a plasmid map and sequence (SEQ ID NO: 18) for an exemplary IL-12 plasmid of the present invention.
• Figure 3 shows optimal codon usage for highly expressed human genes .
• Figure 4 shows a plasmid map and sequence (SEQ ID NO: 19) for plasmid pIF0836, an exemplary interferon alpha plasmid of the present invention.
• Figure 5 shows a plasmid map and sequence (SEQ ID NO: 20) for pIN096, an exemplary IL-12 plasmid that can be used with the present invention.
• Figure 6 shows the nucleic acid sequence (SEQ ID NO: 21) of plasmid pIF0921, an exemplary interferon alpha plasmid of the present invention.
• Figures 7A and 7B show a plasmid map and sequence (SEQ ID NO:22) for plasmid pIF0921.
• Figure 8 shows an outline of a strategy that can be used to synthesize a pIF0921 plasmid.
• Figure 9 shows interferon alpha and IL-12 gene medicine (combination therapy) in Renca model.
• the present invention relates to gene delivery and gene therapy, and provides novel nucleic acid constructs for expression of interferon alpha in a mammal, formulations for delivery that incorporate a nucleic acid construct for expression, and methods for preparing and using such constructs and formulations.
• this invention relates to plasmid constructs for delivery of therapeutic interferon alpha encoding nucleic acids to cells in order to modulate tumor activity, methods of using those constructs (including combination therapy with other agents, such as cytokines, preferably IL-12) , as well as methods for preparing such constructs.
• this invention concerns expression systems for the delivery and expression of interferon alpha coding sequences in mammalian cells, and formulations and methods for delivering such expression systems or other expression systems to a mammal. Therefore, particular genetic constructs are described which includes nucleotide sequences coding for interferon alpha, preferably human interferon alpha. Such a construct can beneficially be formulated and administered as described herein, utilizing the expression systems of this invention. To allow convenient production of such plasmids, it is generally preferable that the plasmid be capable of replication in a cell to high copy number. Generally such production is carried out in prokaryotic cells, particularly including Esherichia coli (E. coli) cells. Thus, the plasmid preferably contains a replication origin functional in a prokaryotic cell, and preferably the replication origin is one which will direct replication to a high copy number.
• E. coli Esherichia coli
• non-DNA components of the formulation may contribute to an immune system enhancement or activation.
• components of a delivery system can be selected in conjunction with a particular gene product to enhance or minimize the immuno-stimulatory effect.
• the plasmids may also include elements for expression of IL-12 or one or more subunits thereof.
• the treatment may involve administration of an interferon alpha coding sequence and one or more IL-12 coding sequences whether on a single plasmid or on separate plasmids.
• Such plasmids may be incorporated into compositions for delivery with a protective, interactive non-condensing compound, a cationic lipid and neutral co-lipid, or both.
• interferon alpha expression vectors of the present invention may be utilized in formulations containing the interferon alpha expression vectors of the present invention to provide effective delivery and expression of interferon alpha or with other coding sequences for which manipulation of the activation of immune system components is desirable.
• the selection of delivery system components and preparation methods in conjunction with the selection of coding sequences provides the ability to balance the specific effects of the encoded gene products and the immune system effects of the overall delivery system composition.
• This capacity to control the biological effects of delivery system formulation administration represents an aspect of the invention in addition to the interferon alpha encoding constructs and specific formulations of delivery systems.
• Co-selection of the encoded gene product and the delivery system components and parameters provides enhanced desired effects rather than merely providing high level gene expression. In particular, such enhancement is described below for the antitumor effects of the exemplary PVP containing compositions.
• the antitumor effect can be greater than merely additive (i.e., greater than merely the sum of the antitumor effects of interferon alpha alone and IL-12 alone) . Enhancement of immuno-stimulatory effects is also desirable in other contexts, for example, for vaccine applications.
• the immune system activation be minimized for compositions to be delivered to the lung in order to minimize lung tissue swelling.
• a useful approach for selecting the delivery system components and preparation techniques for a particular coding sequence can proceed as follows, but is not limited to these steps or step order.
• Such parameters can, for example, include the amount and ratio of DNA to one or more other composition components, the relative amounts of non-DNA composition components, the size of delivery system formulation particles, the percent supercoiled DNA for circular dsDNA vectors, and the specific method of preparation of delivery system formulation particles.
• the particular parameters relevant for specific types of formulations will be apparent or readily determined by testing. The description below illustrates the selection of components and parameters in the context of interferon alpha encoding constructs. However, it should be recognized that the approach is applicable to constructs containing a variety of other coding sequences.
• Plasmid Construct Expression Systems A. Plasmid Design and Construction For the methods and constructs of this invention, a number of different plasmids were constructed which are useful for delivery and expression of sequences encoding interferon alpha. Thus, these plasmids contain coding regions for interferon alpha, along with genetic elements necessary or useful for expression of those coding regions. While these embodiments utilized interferon alpha cDNA clones or partial genomic sequences from a particular source, those skilled in the art could readily obtain interferon alpha coding sequences from other sources, or obtain a coding sequence by identifying a cDNA clone in a library using a probe (s) based on the published interferon alpha coding and/or flanking sequences. This also applies to the IL-12 coding sequences utilized in the embodiments described herein.
• Coding sequences for interferon alpha were incorporated into an expression plasmid that contains eukaryotic and bacterial genetic elements.
• Eukaryotic genetic elements include the CMV immediate early promoter and 5 ' UTR, and a human growth hormone 3' UTR/poly(a) signal, which influence gene expression by controlling the accuracy and efficiency of RNA processing, mRNA stability, and translation.
• the human growth hormone 3 ' UTR is from a human growth hormone gene, and preferably includes a poly (a) signal. This sequence can be linked immediately following the natural translation termination codon for a cDNA sequence, genomic sequence, modified genomic sequence, or synthetic sequence coding for interferon alpha.
• human growth hormone 3' UTR/poly(a) signal is shown by the human growth hormone 3 ' UTR (nucleotides 1 - 100) and 3 1 flanking sequence (nucleotides 101 - 191; GenBank accession #J03071, HUMGHCSA) below. (SEQ ID NO: 13)
• the 5 ' and 3 ' UTR and flanking regions can be further and more precisely defined by routine methodology, e . g. , deletion or mutation analysis or their equivalents., and can be modified to provide other sequences having appropriate transcriptional and translational functions.
• Plasmid Backbone Plasmid Backbone, human interferon alpha cDNA, Final Construct
• Plasmid pIF0921 was constructed from commercially available plasmids, and contains the TN5 gene encoding the kanamycin resistance gene, the pUC origin of replication, the CMV enhancer and promoter to base +112, a synthetic intron called IVS8, the human IFN-a2b gene, and the human growth hormone 3' UTR.
• the plasmid construction descendancy for pIL0697 is shown in Figure 8.
• pIL0697 was cut with BamHI and Xba I and the hIFN-a2b PCR product, which had been amplified from human genomic DNA with BamHI and Xba I ends, was cloned into the pIL0697 backbone in place of the IL-2 coding region.
• the resulting plasmid was pIF0863.
• pIF0863 was cut with Neo I and intron IVS8 from pCT0828 was cloned in.
• the resulting plasmid was pIF0890.
• pIF0890 was cut with Nde I and Pac I and an additional region of the CMV 5' UTR to base +112 was cloned in from plasmid pLC0888.
• some or all of the genetic elements can be synthetic, derived from synthetic oligonucleotides, and thus are not obtained directly from natural genetic sequences. These synthetic elements are appropriate for use in many different expression vectors.
• a synthetic intron is designed with splice sites that ensure that RNA splicing is accurate and efficient.
• a synthetic 3' UTR/poly(A) signal is designed to facilitate mRNA 3' end formation and mRNA stability.
• a synthetic 5' UTR is designed to facilitate the initiation of translation. The design of exemplary synthetic elements is described in more detail below.
• Intron 5' splice site sequence matches consensus .
• 5' splice site sequence is exactly complementary to 5 ' end of Ul snRNA.
• Branch point sequence matches consensus .
• Branch point sequence is complementary to U2 snRNA.
• Polypyrimidine tract is 16 bases in length and contains 7 consecutive
• the tract preferably contains at least 5 consecutive
• Bbsl cleaves at the 5'ss
• Earl cleaves at the 3'ss.
• the 5' untranslated region (5'UTR) influences the translational efficiency of messenger RNA, and is therefore an important determinant of eukaryotic gene expression.
• the synthetic 5'UTR sequence (UT6) has been designed to maximize the translational efficiency of mRNAs encoded by vectors that express genes of therapeutic interest.
• the sequence of the synthetic 5' UTR (UT6) is shown below.
• the Kozak sequence is in boldface and the initiation codon is double underlined.
• the location of the intron is shown below.
• the 5' untranslated region (5' UTR), located between the cap site and initiation codon, is known to influence the efficiency of mRNA translation. Any features that influence the accessibility of the 5' cap structure to initiation factors, the binding and subsequent migration of the 43S preinitiation complex, or the recognition of the initiation codon, will influence mRNA translatability.
• An efficient 5' UTR is expected to be one that is moderate in length, devoid of secondary structure, devoid of upstream initiation codons, and has an AUG within an optimal local context (Kozak, 1994, Biochimie 76:815-821; Jansen et al . , 1994).
• a 5' UTR with these characteristics should allow efficient recognition of the 5' cap structure, followed by rapid and unimpeded ribosome scanning by the ribosome, thereby facilitating the translation initiation process.
• the sequence of the synthetic 5'UTR was designed to be moderate in length (54 nucleotides (nts)), to be deficient in G but rich in C and A residues, to lack an upstream ATG, to place the intended ATG within the context of a optimal Kozak sequence (CCACCATGG) , and to lack potential secondary structure.
• the synthetic 5' UTR sequence was also designed to lack AU-rich sequences that target mRNAs to be rapidly degraded in the cytoplasm.
• the synthetic 5' UTR sequence was designed to accommodate an intron with consensus splice site sequences.
• the intron may, for example, be located between residues 48 and 49 (See intron sequence structure below) .
• the CAG at position 46-48 is the exonic portion of a consensus 5' splice site.
• the G at position 49 is the exonic portion of a consensus 3' splice site.
• the synthetic 5' UTR sequence was designed to begin with a Hindlll site and terminate with a Ncol site.
• RNA splicing is required for the expression of most eukaryotic genes. For optimal gene expression, RNA splicing must be highly efficient and accurate.
• a synthetic intron termed OPTIVS8B, was designed to be maximally efficient and accurate.
• the structure of the exemplary synthetic intron, OPTIVS8 is shown below. Sequences for the 5' splice site (5'ss), branch point (bp) , and 3' splice site (3'ss) are double underlined.
• the recognition sequences for the restriction enzymes Bbsl and Earl are overlined. The cleavage site for Bbsl corresponds to the 5'ss, and the cleavage site for Earl corresponds to the 3'ss.
• the polypyrimidine tract (Y ⁇ ) is the major determinant of splice site strength.
• the length of the polypyrimidine tract was extended to 16 bases, and its sequence was adjusted to contain 7 consecutive T residues. This feature was included because Roscigno et al. (1993) demonstrated that optimal splicing requires the presence of at least 5 consecutive T residues in the polypyrimidine tract.
• Splicing in vi tro is generally optimal when introns are >80 nts in length (Wieringa, et al., 1984; Ulfendahl et al., 1985, Nucl . Acids Res . 13:6299-6315). Although many introns may be thousands of bases in length, most naturally occurring introns are 90-200 nt in length (Hawkins, 1988, Nucl . Acids Res . 16:9893-9908). The length of the synthetic intron (118 nts) falls within this latter range.
• 0PTIVS8B was designed with three internal restriction enzyme sites, Bbsl, Nhel, and Earl. These restriction sites facilitate the screening and identification of genes that contain the synthetic intron sequence.
• Bbsl and Earl sites were placed so that their cleavage sites exactly correspond to the 5'ss (Bbsl) or 3'ss (Earl).
• the sequence of the polypyrimidine tract was specifically designed to accommodate the Earl restriction site. Inclusion of the Bbsl and Earl sites at these locations is useful because they permit the intron to be precisely deleted from a gene. They also permit the generation of an "intron cassette" that can be inserted at other locations within a gene.
• the 77 bases between the Bbsl site and the branch point sequence are random in sequence, except for the inclusion of the Nhel restriction site.
• the 3 ' ends of eukaryotic mRNAs are formed by the process of polyadenylation. This process involves site specific site RNA cleavage, followed by addition of a poly (A) tail. RNAs that lack a poly (A) tail are highly unstable. Thus, the efficiency of cleavage/polyadenylation is a major determinant of mRNA levels, and thereby, of gene expression levels.
• 2XPA1 is a synthetic sequence, containing two efficient poly (A) signals, that is designed to be maximally effective in polyadenylation. A poly (A) signal is required for the formation of the 3' end of most eukaryotic mRNA.
• a poly (A) signal directs two RNA processing reactions: site-specific endonucleolytic cleavage of the RNA transcript, and stepwise addition of adenylates (approximately 250) to the newly generated 3' end to form the poly (A) tail.
• a poly (A) signal has three parts: hexanucleotide, cleavage site, and downstream element. The hexanucleotide is typically AAUAAA and cleavage sites are most frequently 3' to the dinucleotide CA (Sheets et al., 1987). Downstream elements are required for optimal poly (A) signal function and are located downstream of the cleavage site.
• Naturally occurring poly (A) signals are highly variable in their effectiveness (Peterson, 1992) .
• the effectiveness of a particular poly (A) signal is mostly determined by the quality of the downstream element. (Wahle, 1992) .
• Poly (A) efficiency is important for gene expression, because transcripts that fail to be cleaved and polyadenylated are rapidly degraded in the nuclear compartment. In fact, the efficiency of polyadenylation in living cells is difficult to measure, since nonpolyadenylated RNAs are so unstable. In addition to being required for mRNA stability, poly (A) tails contribute to the translatability of mRNA, and may influence other RNA processing reactions such as splicing or RNA transport ((Jackson and Standart, 1990, Cell 62:15-24; Wahle, 1992).
• Some eukaryotic genes have more than one poly (A) site, implying that if the cleavage/polyadenylation reaction fails to occur at the first site, it will occur at one of the later sites.
• a gene with two strong poly (A) sites yielded approximately two-fold more mRNA than one with a single strong poly (A) site (Bordonaro, 1995) .
• the sequence of the exemplary synthetic poly (A) signal is shown below.
• the sequence is named 2XPA.
• the hexanucleotide sequences and downstream element sequences are double underlined, and the two poly (A) sites are labeled as pA#l and pA#2. Convenient restriction sites are overlined.
• the entire 2XPA unit may be transferred in cloning experiments as a Xbal-Kpnl fragment. Deletion of the internal BspHI fragment results in the formation of a 1XPA unit. (SEQ ID NO. 17)
• sequence of the synthetic poly (A) site shown above is based on the sequence of the rabbit D-globin poly (A) signal, a signal that has been characterized in the literature as strong (Gil and Proudfoot, 1987, Cell 49:399- 406; Gil and Proudfoot, 1984, Na ture 312:473-474) .
• One of its key features is the structure of its downstream element, which contains both UG- and U-rich domains.
• a double-stranded DNA sequence corresponding to the 1XPA sequence was constructed from synthetic oligonucleotides. Two copies of the 1XPA sequence were then joined to form the 2XPA sequence. The sequences were joined in such as way as to have a unique Xbal site at the 5' end of the first poly (A) signal containing fragment, and a unique Kpnl site at the 3' end of the second poly (A) signal containing fragment.
• interferon alpha instead of the natural sequence coding for interferon alpha, it is advantageous to utilize synthetic sequences which encode interferon alpha.
• Such synthetic sequences have alternate codon usage from the natural sequence, and thus have dramatically different nucleotide sequences from the natural sequence.
• synthetic sequences can be used which have codon usage at least partially optimized for expression in a human. The natural sequences do not have such optimal codon usage. Preferably, substantially all the codons are optimized.
• Optimal codon usage in humans is indicated by codon usage frequencies for highly expressed human genes, as shown in Fig. 3.
• the codon usage chart is from the program "Human_High. cod” from the Wisconsin Sequence Analysis Package, Version 8.1, Genetics Computer Group, Madison, WI.
• the codons which are most frequently used in highly expressed human genes are presumptively the optimal codons for expression in human host cells, and thus form the basis for constructing a synthetic coding sequence.
• An example of a synthetic interferon alpha coding sequence is shown as the bottom sequence in the table below.
• interferon alpha encoding sequence which has optimized codon usage except in areas where the same amino acid is too close together or abundant to make uniform codon usage optimal.
• other synthetic sequences can be used which have substantial portions of the codon usage optimized, for example, with at least 50%, 70%, 80% or 90% optimized codons as compared to a natural coding sequence.
• Other particular synthetic sequences for interferon alpha can be selected by reference to the codon usage chart in Fig. 3. A sequence is selected by choosing a codon for each of the amino acids of the polypeptide sequences. DNA molecules corresponding to each of the polypeptides can then by constructed by routine chemical synthesis methods. For example, shorter oligonucleotides can be synthesized, and then ligated in the appropriate relationships to construct the full-length coding sequences.
• interferon alpha amino acid sequence SEQ ID NO: 9; interferon alpha wild type nucleic acid sequence, SEQ ID NO: 10; interferon alpha synthetic nucleic acid sequence with optimized codon usage, SEQ ID NO: 11; interferon alpha nucleic acid sequence with additional/semi- optimized codon usage, SEQ ID NO: 12; IL-12 p40 subunit amino acid sequence, SEQ ID NO:l; IL-12 p40 wild type nucleic acid sequence, SEQ ID NO: 2; IL-12 p40 synthetic nucleic acid sequence with all codons optimized, SEQ ID NO: 3; IL-12 p40 subunit nucleic acid sequence with all codons optimized except when same nucleic acids were too close/abundant, SEQ ID NO: 4; IL-12 p35 amino acid sequence, SEQ ID NO: 5; IL-12 p35 wild type nucleic acid sequence, SEQ ID NO: 6; IL-12 p35
• nucleic acid sequences with optimized codon usage can be constructed, for example based on the various combinations shown below, wherein optimal usage for each codon is shown below the IL-12 p35 and p40 subunit wild type sequences and the interferon alpha wild type sequence.
• First line natural sequence (SEQ ID NO. 6)
• Second line all codons optimized (SEQ ID NO. 7)
• Third line all codons optimized except when same nucleic acids were too close/abundant (changes between second and third lines bolded) (SEQ ID NO. 8)
• TCT CAA AAT TTA TTA CGT GCT GTT TCT AAT ATG TTA CAA AAA GCT CGT CAA ACT TTA GAA TCC CAG AAC TTG TTG CGC GCC GTC TCC AAC TTG CAG AAG GCC CGC CAG ACC TTG GAG TCA CTT CTT CGA GCA GTA TCA CTT GCA CGA ACA CTT TCG CTC CTC CGG GCG GTG TCG CTC GCG CGG ACG CTC AGT CTA CTA AGA AGT CTA AGA CTA
• First line natural sequence (SEQ ID NO. 2)
• Second line all codons optimized (SEQ ID NO. 3)
• Third line all codons optimized except when same nucleic acids were too close/abundant (changes between second and third lines bolded) (SEQ ID NO. 4)
• GTG GCC ATA TGG GAA CTG AAG AAA GAT GTT TAT GTC GTA GAA TTG GAT TGG TAT CCG GAT GTG GCC ATC TGG GAG CTG AAG AAG GAC GTG TAC GTG GTG GAG CTG GAC TGG TAC CCC GAC GTG GCC ATC TGG GAG CTG AAG AAA GAC GTG TAC GTG GTC GAG CTG GAC TGG TAC CCC GAC
• AAA GAA CCC AAA AAT AAG ACC TTT CTA AGA TGC GAG GCC AAG AAT TAT TCT GGA CGT TTC
• CGT AAA AAT GCT TCT ATT TCT GTT CGT GCT CAA GAT CGT TAT TAT TCT TCT TGG TCT CGC AAG AAC GCC TCC ATC TCC GTC CGC GCC CAG GAC CGC TAC TAC TCC TCC TCC TCC CGA GCA TCA ATA TCA GTA CGA GCA CGA TCA TCA TCA CGG GCG TCG TCG GTG CGG GCG CGG TCG TCG TCG TCG AGA AGT AGT AGT AGT AGT AGT AGT
• GCA CTT ACA GCA CTT CTT GTA GCA CTT CTT GTA CTT TCA TCA TCA
• GCG CTC ACG GCG CTC CTC GTG GCG CTC CTC GTG CTC TCG TCG TCG
• GCC GTC CGC AAG TAC TTC CAG CGC ATC ACC TTG TAC TTG AAG GAG AAG AAG TAC TCC CCC
• nucleic acids in many formulations is limited due to degradation of the nucleic acids by components of organisms, such as nucleases.
• protection of the nucleic acids when delivered in vivo can greatly enhance the resulting expression, thereby enhancing a desired pharmacological or therapeutic effect.
• certain types of compounds which interact with a nucleic acid e.g., DNA
• do not condense the nucleic acid provide in vivo protection to the nucleic acid, and correspondingly enhance the expression of an encoded gene product .
• PINC systems are non-condensing systems that allow the plasmid to maintain flexibility and diffuse freely throughout the muscle while being protected from nuclease degradation. While the PINC systems are primarily discussed below, it will be understood that cationic lipid based systems and systems utilizing both PINCS and cationic lipids are also within the scope of the present invention.
• a common structural component of the PINC systems is that they are amphiphilic molecules, having both a hydrophilic and a hydrophobic portion.
• the hydrophilic portion of the PINC is meant to interact with plasmids by hydrogen bonding (via hydrogen bond acceptor or donor groups), Van der Waals interactions, or/and by ionic interactions.
• PVP and N-methyl-2-pyrrolidone (NM2P) are hydrogen bond acceptors while PVA and PG are hydrogen bond donors .
• Hyper-osmotic formulation i.e., 340 mOsm/kg H 2 0
• Luminescence Plasmid/PVP binding decreased Spectroscopy in salt and/or at pH 7
• the structure-activity relationship described above can be used to design novel co-polymers that will also have enhanced interaction with plasmids. It is expected that there is "an interactive window of opportunity" whereby enhanced binding affinity of the PINC systems will result in a further enhancement of gene expression after their intramuscular injection due to more extensive protection of plasmids from nuclease degradation. It is expected that there will be an optimal interaction beyond which either condensation of plasmids will occur or "triplex" type formation, either of which can result in decreased bioavailability in muscle and consequently reduced gene expression.
• the PINC compounds are generally amphiphilic compounds having both a hydrophobic portion and a hydrophilic portion. In many cases the hydrophilic portion is provided by a polar group.
• polar groups can be provided by groups such as, but not limited to, pyrrolidone, alcohol, acetate, amine or heterocyclic groups such as those shown on pp. 2-73 and 2-74 of CRC Handbook of Chemistry and Physics (72nd Edition) , David R. Lide, editor, including pyrroles, pyrazoles, imidazoles, triazoles, dithiols, oxazoles,
• hydrophobic groups which, in the case of a polymer, are typically contained in the backbone of the molecule, but which may also be part of a non- polymeric molecule.
• hydrophobic backbone groups include, but are not limited to, vinyls, ethyls, acrylates, acrylamides, esters, celluloses, amides, hydrides, ethers, carbonates, phosphazenes, sulfones, propylenes, and derivatives of these groups.
• the polarity characteristics of various groups are quite well known to those skilled in the art as illustrated, for example, by discussions of polarity in any introductory organic chemistry textbook.
• Targeting Ligands In addition to the nucleic acid/PINC complexes described above for delivery and expression of nucleic acid sequences, in particular embodiments it is also useful to provide a targeting ligand in order to preferentially obtain expression in particular tissues, cells, or cellular regions or compartments.
• a targeted PINC complex includes a PINC system (monomeric or polymeric PINC compound) complexed to plasmid (or other nucleic acid molecule) .
• the PINC system is covalently or non-covalently attached to (bound to) a targeting ligand (TL) which binds to receptors having an affinity for the ligand.
• TL targeting ligand
• Such receptors may be on the surface or within compartments of a cell.
• Such targeting provides enhanced uptake or intracellular trafficking of the nucleic acid.
• the targeting ligand may include, but is not limited to, galactosyl residues, fucosal residues, mannosyl residues, carnitine derivatives, monoclonal antibodies, polyclonal antibodies, peptide ligands, and DNA-binding proteins.
• Examples of cells which may usefully be targeted include, but are not limited to, antigen-presenting cells, hepatocytes, myocytes, epithelial cells, endothelial cells, and cancer cells.
• a targeting method for cytotoxic agents is described in Subramanian et al., International Application No. PCT/US96/08852, International Publication No. WO 96/39124, hereby incorporated by reference.
• This application describes the use of polymer affinity systems for targeting cytotoxic materials using a two-step targeting method involving zip polymers, i.e., pairs of interacting polymers. An antibody attached to one of the interacting polymers binds to a cellular target. That polymer then acts as a target for a second polymer attached to a cytotoxic agent.
• other two-step (or multi-step) systems for delivery of toxic agents are also described.
• nucleic acid coding sequences can be delivered and expressed using a two-step targeting approach involving a non-natural target for a PINC system or PINC- targeting ligand complex.
• a PINC-plasmid complex can target a binding pair member which is itself attached to a ligand which binds to a cellular target (e.g., a MAB) . Binding pairs for certain of the compounds identified herein as PINC compounds as identified in Subramanian et al.
• the PINC can be complexed to a tareting ligand, such as an antibody. That antibody can be targeted to a non-natural target which binds to, for example, a second antibody.
• S.C. VII/SF is a cell line derived from murine squamous cell carcinoma (S.C). Squamous cell carcinoma of the head and neck begins with the cells lining the oral and pharyngeal cavities. Clinical disease progresses via infiltration and spreads into the underlying tissues and lymphatics. The undifferentiated, in vivo passage tumor line S.C. VII/SF displays this typical growth pattern. In addition, its rapid growth rate provides a relatively short test period for individual experiments.
• Other murine tumor cell lines include another SCC line KLN-205, a keratinocyte line 1-7, and a colon adenocarcinoma line MC-38.
• An optimal model system preferably satisfies the criteria based on having tumor growth rate in vivo (i . e . , tumors are ready for treatment in 4-10 days post implant) , invasiveness, and local spread similar to those observed in clinical disease, and providing accessibility for experimental treatment.
• the SCC VII/SF cell line was utilized as the primary model system cell line. This cell line typically grows rapidly, resulting in death of untreated syngeneic mice 14-17 days after tumor cell implantation. This cell line can be utilized in a variety of ways to provide model system suitable for a variety of different tests. Four such possibilities are described below.
• SCCVII cells can be utilized in cell culture to provide an in vi tro evaluation of interferon alpha expression construct and formulation characteristics, such as expression levels and cellular toxicities.
• the cells can be implanted subcutaneously in mice.
• This system can be utilized in tests in which accessibility of the implant site is beneficial.
• the method was utilized in evaluations of expression efficiencies based on the expression of chloramphenicol acetyltransferase (CAT) .
• CAT chloramphenicol acetyltransferase
• the cells can be implanted transcutaneously into the fascia of digastric muscle.
• the cells can be implanted transcutaneously into digrastric/mylohyoid muscles.
• the important features of models 3 and 4 are shown in the table below.
• the tumor size treated in the mouse models is generally 20-50 mm 3 .
• a 50 mm 3 mouse tumor is approximately equivalent to 150 cc 3 human tumor having an average diameter of about 6.6 cm. This tumor size is approximately 10-fold larger than the size proposed to be treated in the phase I clinical trials. This indicates that the mouse models are strongly biased towards over estimating the expected tumor burden in human patients.
• expression systems such as those described above provide the potential for expression when delivered to an appropriate location
• this invention also provides particular formulations which include one or more expression system constructs (e.g., DNA plasmids as described above), and a protective, interactive non-condensing compound.
• plasmid construct An additional significant factor relating to the plasmid construct is the percentage of plasmids which are in a supercoiled (SC) form rather than the open circular (OC) form.
• a variety of delivery methods can be used with the constructs and formulations described above, in particular, delivery by injection to the site of a tumor can be used.
• the submandibular tumor models utilized injection into four quadrants of the tumor being treated.
• the formulations were delivered by injection.
• the exemplary formulations do not show high cellular toxicity at the concentrations tested, suggesting that the formulations do not significantly kill cells by direct toxic action in vivo .
• the anti-tumor activity induced by IFN ⁇ gene therapy is dependent upon activation of the immune system, which is demonstrated by depletion studies in vivo. Removal of a specific T lymphocyte population (CD8 + ) abrogates the anti-tumor activity elicited by IFN ⁇ gene therapy.
• Administration refers to the route of introduction of a plasmid or carrier of DNA into the body.
• the expression systems constructs and the delivery system formulations can be administered by a variety of different methods .
• Administration can be directly to a target tissue or by targeted delivery to the target tissue after systemic administration.
• the present invention can be used for treating disease by administration of the expression system or formulation to the body in order to establishing controlled expression of any specific nucleic acid sequence within tissues at certain levels that are useful for gene therapy.
• the preferred means for administration of vector (plasmid) and use of formulations for delivery are described above.
• the preferred embodiments are by direct injection using needle injection.
• the route of administration of any selected vector construct will depend on the particular use for the expression vectors. In general, a specific formulation for each vector construct used will focus on vector uptake with regard to the particular targeted tissue, followed by demonstration of efficacy. Uptake studies will include uptake assays to evaluate cellular uptake of the vectors and expression of the DNA of choice. Such assays will also determine the localization of the target DNA after uptake, and establishing the requirements for maintenance of steady- state concentrations of expressed protein. Efficacy and cytotoxicity can then be tested. Toxicity will not only include cell viability but also cell function.
• Muscle cells have the unique ability to take up DNA from the extracellular space after simple injection of DNA particles as a solution, suspension, or colloid into the muscle. Expression of DNA by this method can be sustained for several months.
• DNA vectors Delivery of formulated DNA vectors involves incorporating DNA into macromolecular complexes that undergo endocytosis by the target cell.
• complexes may include lipids, proteins, carbohydrates, synthetic organic compounds, or inorganic compounds.
• the complex includes DNA, a cationic lipid, and a neutral lipid in particular proportions.
• the characteristics of the complex formed with the vector determines the bioavailability of the vector within the body.
• Other elements of the formulation function as ligand which interact with specific receptors on the surface or interior of the cell. Other elements of the formulation function to enhance entry into the cell, release from the endosome, and entry into the nucleus.
• DNA transporters refers to molecules which bind to DNA vectors and are capable of being taken up by epidermal cells. DNA transporters contain a molecular complex capable of noncovalently binding to DNA and efficiently transporting the DNA through the cell membrane. It is preferable that the transporter also transport the DNA through the nuclear membrane. See, e . g. , the following applications all of which (including drawings) are hereby incorporated by reference herein: (1) Woo et al., U.S. Serial No. 07/855,389, entitled "A DNA Transporter System and Method of Use,, filed March 20, 1992, now abandoned; (2) Woo et al., PCT/US93/02725, International Publ.
• a DNA transporter system can consist of particles containing several elements that are independently and non- covalently bound to DNA. Each element consists of a ligand which recognizes specific receptors or other functional groups such as a protein complexed with a cationic group that binds to DNA. Examples of cations which may be used are sper ine, spermine derivatives, histone, cationic peptides and/or polylysine. One element is capable of binding both to the DNA vector and to a cell surface receptor on the target cell.
• a second element is capable of binding both to the DNA vector and to a receptor on the nuclear membrane.
• the nuclear ligand is capable of recognizing and transporting a transporter system through a nuclear membrane.
• An example of such ligand is the nuclear targeting sequence from SV40 large T antigen or histone.
• a third element is capable of binding to both the DNA vector and to elements which induce episomal lysis. Examples include inactivated virus particles such as adenovirus, peptides related to influenza virus hemagglutinin, or the GALA peptide described in the Szoka patent cited above.
• the lipids may form liposomes which are hollow spherical vesicles composed of lipids arranged in unilamellar, bilamellar, or multilamellar fashion and an internal aqueous space for entrapping water soluble compounds, such as DNA, ranging in size from 0.05 to several microns in diameter.
• Lipids may be useful without forming liposomes. Specific examples include the use of cationic lipids and complexes containing DOPE which interact with DNA and with the membrane of the target cell to facilitate entry of DNA into the cell.
• Gene delivery can also be performed by transplanting genetically engineered cells.
• immature muscle cells called myoblasts may be used to carry genes into the muscle fibers.
• Myoblast genetically engineered to express recombinant human growth hormone can secrete the growth hormone into the animal's blood. Secretion of the incorporated gene can be sustained over periods up to 3 months.
• Myoblasts eventually differentiate and fuse to existing muscle tissue. Because the cell is incorporated into an existing structure, it is not just tolerated but nurtured. Myoblasts can easily be obtained by taking muscle tissue from an individual who needs gene therapy and the genetically engineered cells can also be easily put back with out causing damage to the patient's muscle.
• keratinocytes may be used to delivery genes to tissues.
• keratinocytes can be generated by cultivation of a small biopsy.
• the cultures can be prepared as stratified sheets and when grafted to humans, generate epidermis which continues to improve in histotypic quality over many years.
• the keratinocytes are genetically engineered while in culture by transfecting the keratinocytes with the appropriate vector. Although keratinocytes are separated from the circulation by the basement membrane dividing the epidermis from the dermis, human keratinocytes secrete into circulation the protein produced.
• the chosen method of delivery should result in expression of the gene product encoded within the nucleic acid cassette at levels which exert an appropriate biological effect.
• the rate of expression will depend upon the disease, the pharmacokinetics of the vector and gene product, and the route of administration, but should be in the range 0.001-100 mg/kg of body weight /day, and preferably 0.01-10 mg/kg of body weight/day. This level is readily determinable by standard methods. It could be more or less depending on the optimal dosing.
• the duration of treatment will extend through the course of the disease symptoms, possibly continuously. The number of doses will depend upon the disease, delivery vehicle, and efficacy data from clinical trials.
• mIFN- gene medicine reduces the growth of tumors in syngeneic murine tumor models.
• Lipid formulations of mIFN- gene medicine display anti tumor activity in both SCC-VII and MC-38 tumor models.
• PINC and peptide formulations of mIFN- gene medicine display anti tumor effects in the MC-38 tumor model.
• the anti tumor effects of mIFN- gene medicine are dose dependent.
• the examples demonstrate that treatment of tumors with the combination of IFN ⁇ and IL-12 gives an unanticipated more than additive (synergystic) anti-tumor activity using either a PINC or a lipid formulation.
• a plasmid expression system encoding murine IFN ⁇ 4 and formulated in a polymeric delivery system was used for in vivo immunotherapeutic activity against an immunogenic murine renal cell carcinoma, Renca, and a non-immunogenic mammary adenocarcinoma, TS/A.
• Mice bearing established tumors were treated with IFN ⁇ /polyvinyl-pyrrolidone (PVP) expression complexes via direct intra-tumoral injection. Up to 100 % tumor growth inhibition was observed in the treated mice.
• PVP polyvinyl-pyrrolidone
• mice that remained tumor-free following IFN- ⁇ gene therapy displayed immune resistance to a subsequent challenge of tumor.
• cytokines have limited effect on tumor proliferation directly but are capable of activating a rapid and potent anti-tumor immune response, which impedes tumor progression.
• Established parental tumors are difficult to eradicate with ex vivo cytokine-transduced tumor cells because efficacy of vaccination is highly dependent on the size, growth rate and invasiveness of the tumor.
• cytokine-based gene therapy approaches which can deliver transgenic cytokines locally and induce an anti-tumor immune response
• have been recently evaluated by a number of investigators (Forni et al., "Cytokine-Induced Immunogenicity: From Exogenous Cytokines to Gene Therapy", Journal of Immunotherapy, 14, 253-257, (1993); Pericle et al., "An Efficient Th2-type Memory followss Cd8+ Lymphocyte-driven and Eosinophil-mediated Rejection of a Spontaneous Mouse Mamary Adenocarcicoma Engineered to Release 11-4", The Journal of Immunology, 153, 5660-5673. (1994); Pardoll et al .
• PINC polymeric interactive non-condensing
• hIGF-1 human insulin growth factor-1
• PINC complex a plasmid that encodes human insulin growth factor-1 (hIGF-1) and formulated as a PINC complex
• production of biologically active h IGF-1 in vivo following intra-muscular injection has been shown (Alila et al., "Expression of Biologically Active Human Insulin-Like Growth Factor-1 Following Intramuscular Injection of a Formlated Plasmid in Rats", Human Gene Therapy, 8, 1785-1795 (1997)).
• the specific objective of this study was to determine whether a plasmid expression system encoding murine IFN ⁇ 4 and formulated as a complex with PVP could induce an anti-tumor immune response following direct injection into subcutaneous murine tumors.
• the IFN family consists of three major glycoproteins, IFN ⁇ , IFN ⁇ and IFN ⁇ .
• IFNs were first developed as antiviral agents, it is now clear that they also control cell growth and differentiation, and modulate various aspects of host immunity (Gresser et al., "Antitumor effects of interferon", Acta Oncol. 28, 347-353 (1989)).
• Clinical data concluded that systemic chronic administration of IFN ⁇ could produce regression of vascular tumors, including Kaposi's sarcoma, pulmonary hemangiomastosis, and he angiomas (Singh et al., "Interferons A and B Down- regulate the Expression of Basic Fibroblast Growth Factor in Human Carcicomas", Proc. Natl. Acad.
• IFN ⁇ was the first cytokine to be used in clinical trials that proved to be effective against certain types of human cancer, only recently has this cytokine been considered as a candidate for gene therapy (Ogura et al. 1993, Belldegrun et al., "Human Renal Carcinoma Line Transfected With Interleukin-2 and/or Interferon ⁇ Gene(s): Implications for Live Cancer Vaccines, Journal of the National Cancer Institute, 85, 207-216 (1993) .
• a plasmid expression system containing an expression cassette for mIFN-l ⁇ 4 was constructed as follows.
• the coding sequence of the murine IFN- ⁇ 4 gene (Genebank X01973 M15456 M23830 X01967) was amplified by PCR from mouse genomic DNA.
• the amplified mIFN- ⁇ 4 sequence was then subcloned into a plasmid backbone, and the sequence fidelity was verified by DNA sequence analysis (data not shown) .
• the coding sequence for mIFN- ⁇ 4 was then subcloned as an XbaI-BamHIfragment into the expression plasmid pIL0697 to create the mIFN- ⁇ 4 expression system pIF0836.
• Plasmid pVC0612 (empty plasmid, EP) contains expression elements including the cytomegalovirus immediate early promoter and the 3 ' UTR/poly(A) signal from the bovine growth gene in the pVC0289 backbone described by Alila et al. (1997) . Plasmid pVC0612 was used as a control plasmid in all in vivo experiments. Plasmids for intra-tumoral injection were grown under kanamycin selection in E. coli host strains DH5 ⁇ and purified using conventional alkaline lysis and chromatographic methods.
• Purified plasmid utilized for intra-tumoral injections had the following specifications: endotoxin ( ⁇ 500 Eu/mg plasmid); protein ( ⁇ 1%) ; and chromosomal DNA ( ⁇ 20 %) .
• Purified pIF0836 and control plasmids were formulated at a concentration of 3 g DNA/ml in a solution of 5 % w/v polyvinyl-pyrrolidone (Plasdone C-30, ISP
• HeLa cells were plated in 6 well plates at 3 x 10 5 cells per well, and transfected using 1 ⁇ g of mouse IFN ⁇ 4 plasmid pIF0836C and 3 ⁇ g of Lipofectamine (Life Technologies, Inc., Gaithersburg, MD) in serum-free DMEM. Same supernatants were harvested 24 hours later and immunoprecipitated using anti-mouse interferon ⁇ / ⁇ polyclonal antibody (BioSource International, Camarillo, CA) and protein A and G agarose (Boehringer Mannheim, Indianapolis, IN) . Samples were run on a 12% Tris-glycine gel and electroblotted to Millipore PVDF membrane. Anti-mouse interferon ⁇ / ⁇ polyclonal antibody was used at 1:1000, followed by anti-sheep Ig HRP
• mice Normal 8-week-old female BALB/c mice were purchased from Harlan Laboratories, Houston, TX. Mice were maintained on ad libitum rodent feed and water at 23° C, 40% humidity, and a 12-h/12-h light-dark cycle. Animals were acclimated for at least 4 days before the start of the study.
• TS/A is a tumor cell line established by Dr. P. Nanni, University of Bologna, Italy, from the first in vivo transplant of a moderately differentiated mammary adenocarcinoma that spontaneously arose in a BALB/c mouse (Nanni et al . , 1983) .
• a number of pre-immunization- challenge experiments suggested that TS/A does not elicit a long-lasting anti-tumor immunity (Forni et al . , 1987).
• TS/A was generously provided by Dr. Guido Forni, University of Turin, Italy.
• Renca a spontaneously arising murine renal cell carcinoma
• CT-26 a colon adenocarcinoma
• Tumor cell cultures were maintained in sterile disposable flasks from Corning
• mice were challenged s.c. in the middle of the left flank with 30 ⁇ l of a single-cell suspension contained the specified number of cells. Seven days later when the tumor size reached approximately 10 mm 3 , treatments with IFN ⁇ /PVP or EP/PVP started and were repeated at 1-2 day intervals for 2 weeks (total of 8 treatments: 4/week) . Tumor volume was measured with electronic caliper in the two perpendicular diameters and in the depth. Measurements of the tumor masses (mm 3 ) were performed twice a week for 40-50 days. All mice bearing tumor masses exceeding 1 cm3 volume were sacrificed for humane reasons.
• mice When depletion of immunocompetent cells in vivo was required, a group of mice received i.v 0.5 ml of ⁇ -CD4 (GK1.5 hybridoma, 207-TIB, ATCC, Rockville, MD) ascite (1:10), or ⁇ -CD8 (2.43 hybridoma, 210-TIB, ATCC) ascite (1:100) or i.p. 100 ⁇ g ⁇ -GRl (RB6-8C5 hybridoma, Pharmingen, San Diego, CA) .
• ⁇ -CD4 GK1.5 hybridoma, 207-TIB, ATCC, Rockville, MD
• ⁇ -CD8 (2.43 hybridoma, 210-TIB, ATCC
• i.p. 100 ⁇ g ⁇ -GRl RB6-8C5 hybridoma, Pharmingen, San Diego, CA
• mice received i.v. 0.5 ml isotype control IgG
• Antibody treatments were performed twice: first injection 1 day before starting the gene therapy treatment and the second injection (i.p at the same dosage) 7 days later.
• a standard 6-hour 51-chromium ( 51 Cr) -release assay was performed following 5 days of in vitro effector cell stimulation.
• Single cell suspensions of splenocytes were prepared 3 weeks following tumor challenge by mashing the spleens in RPMI 1640 medium (Life Technologies) and passing the cells through 70 ⁇ m nylon mesh cell strainers (Falcon, Becton Dickinson, Lincoln Park, NJ) into 50ml centrifuge tubes (Falcon) . After centrifugation, red blood cells were lysed with ACK Lysing Buffer (Biofluids, Inc., Rockville, MD) and the splenocytes washed twice with RPMI.
• In vitro stimulation cultures contained 3 X 10 6 splenocytes/effectors per ml with 6 X 10 5 mitomycin-C-treated Renca/stimulator cells per ml and 10 Units per ml recombinant murine IL-2 (Genzy e, Cambridge, MA) in RPMI containing 10% FBS, 22mM HEPES buffer (Research Organics Inc., Cleveland OH), Penn- Streptomycin, 5 X 10 "5 M 2- ⁇ -mercapto-ethanol (Life Technologies), OPI media supplement (Sigma, St. Louis, MO), and essential and non-essential amino acids (Life Technologies) (for a 5 : 1 responder : stimulator ratio) .
• Stimulators were prepared by incubating Renca cells at 3 X 10 7 per ml in RPMI with 30 ⁇ g per ml mitomycin-C (Sigma) at 37° C for 60 minutes, followed by four washes in HBSS with
• effector cells were pelleted, resuspended in complete RPMI, counted, and mixed with 51 Cr -labeled targets in a 96 well V-bottomed plate
• Renca and CT26 targets were labeled by incubating them at 2 X 10 6 cells per ml in complete RPMI with 150 uCi 51 Cr (Amersham) for 2.5 hours. Targets were washed 3 times in HBSS with 2.5% FBS and resuspended in complete RPMI before addition to the assay. After mixing effectors and targets (in triplicate wells) and a brief pelleting, plates were placed at 37° C for 6 hours. Approximately 90% of the supernatants were then collected from each well with the Skatron Harvesting Press and
• Murine IFN- ⁇ expression plasmid (pIF0836) was transfected into Cos-1 cells, and the resulting conditioned media was assayed for mIFN- ⁇ by Western blot and by bioassay.
• Western blot analysis of conditioned media indicated that the recombinant mIFN- ⁇ expressed from pIF0836 template was present as a single band with an approximate molecular weight of 23 kDa. This band was not observed in conditioned media from mock-transfected cells and likely represents a glycosylated form of mIFN- ⁇ .
• Recombinant mlFN- ⁇ ran with an approximate molecular weight of 18 kDa, which corresponds to the predicted molecular weight of non- glycosylated mIFN- ⁇ .
• Assay of conditioned media using an anti-viral bioassay for mIFN- ⁇ indicated that approximately 175 x 10 3 IU/ml mIFN- ⁇ were present.
• IFN- ⁇ gene therapy Anti-tumor activity of IFN- ⁇ gene therapy.
• the antitumor effect of murine IFN ⁇ 4 plasmid formulated as a complex with PVP (IFN ⁇ /PVP) was tested in a syngeneic murine renal cell carcinoma (Renca) and a mammary adenocarcinoma (TS/A) tumor model.
• Renca syngeneic murine renal cell carcinoma
• TS/A mammary adenocarcinoma
• BALB/c mice were challenged subcutaneously with 7 X10 5 Renca or 1 X10 5 CT26 cells, and IFN ⁇ /PVP injections were initiated 7 days later when tumors reached approximately 10 mm 3 size.
• mice received at interval of 1-2 days 8 treatments (4 injections/week) of IFN ⁇ /PVP at scalar doses (from 12 to 96 ⁇ g/mouse) , EP/PVP (96 ⁇ g/mouse) or no treatments for control (ctrl) .
• Tumor size increased progressively in mice injected with EP/PVP (Renca, TS/A) or low doses of IFN ⁇ /PVP (TS/A)
• tumors in mice injected with each dose of IFN ⁇ /PVP (Renca) or high dose of IFN ⁇ /PVP (TS/A) showed marked growth inhibition.
• Tumor growth inhibition is associated to systemic immune response
• mice with no tumors for 40-50 days following IFN ⁇ treatments were re-challenged with a greater number of fresh tumors in the right flank. All mice that rejected primary tumors displayed protection against the second tumor challenge whereas mice used as the control group and injected for the first time with same number of tumor cells (1 X 10 6 Renca or 2 X 10 5 TS/A) developed tumors.
• Renca and TS/A were injected into BALB/c rendered immunosuppressed by treatment with anti-CD4, anti-CD8 or anti-polymorphonuclear cells (PMN) .
• PMN anti-polymorphonuclear cells
• mice depleted of CD4 + T cells and treated with IFN ⁇ /PVP demonstrated potent CTL activity against Renca cells.
• little CTL activity against Renca cells was evident from splenocytes isolated from mice treated with EP/PVP.
• Our approach to this problem is to use a non-viral delivery system to modify tumor cells in vivo with cytokine cDNAs so that the tumor cells can supply the cytokine of interest in a paracrine fashion to the antitumor responder cells present within the tumor.
• CD4 + T cells in tumor-bearing mice treated with IFN ⁇ gene therapy significantly enhanced the therapeutic effect of IFN ⁇ , resulting in tumor regression and prolonged survival of up to 80% of treated mice.
• a CD4- mediated suppression during tumor progression has been previously reported and it has also been shown that depletion of CD4 + T cells in tumor-bearing mice results in augmentation of anti-tumor therapy with either IL-2 or IL-12 (Rack ilevich et al., 1994 and Martinotti et al., "Cd4 T
• CD4 suppression The mechanism driving CD4 suppression is poorly understood, although Th2 type cytokines, directly or through B cell activation, may inhibit cell-mediated immunity (Mossman et al., 1989; Powrie et al., Eur-J- Immunol, 23 (11) : 3043-9 (1993)).
• CTL can be generated in both CD4-depleted and non-depleted mice from lymphocytes obtained from spleens by in vitro re-stimulation with mitomycin-treated Renca cells and rIL-2.
• CD4-mediated suppression appears to be exerted on CD8 expansion and not priming.
• Example 2 Pharmacology of mIFN - Gene Medicine in Syngeneic Tumor Models Gene expression systems encoding either mIFN-2 or mlFN- 4 were tested for anti tumor activity when formulated in either cationic lipid, peptide, or PINC delivery systems and injected intratumorally into subcutaneous squamous cell carcinoma (SCC-VII) or adenocarcinoma (MC-38) tumors.
• SCC-VII subcutaneous squamous cell carcinoma
• MC-38 adenocarcinoma
• IFN ⁇ /PVP syngeneic murine renal cell carcinoma
• TS/A mammary adenocarcinoma
• BALB/c mice were challenged subcutaneously with 7 X10 5 or 1 X10 5 CT26, and IFN ⁇ /PVP injections were initiated 7 days later when tumors reached approximately 10 mm 3 size.
• Each group of mice received 8 treatments (4 injections for 2 weeks) of IFN ⁇ /PVP at scalar doses (from 12 to 96 ⁇ g/mouse) , empty plasmid/PVP (EP/PVP, 96 ⁇ g/mouse) or no treatments for control (ctrl) .
• Tumor size increased progressively in mice injected with EP/PVP (Renca, TS/A) or low doses of IFN ⁇ /PVP
• IFN ⁇ /PVP Rhca
• TS/A high dose of IFN ⁇ /PVP
• Example 3 mIFN- Gene Medicine Formulated in Cationic lipid Reduces the Growth of SCC-VII Tumors
• the formulations for the IFN-a are: (1) PVP 4 vial, (2) PVP three vial, (3) PVP two vial. The details are listed below:
• Method Add in order of water, plasmid, 25% PVP and 5 M NaCl into a vial to make a plasmid in 5% PVP in saline formulation.
• the final concentration of PVP and NaCl are fixed (5% and 150 mM) and plasmid concentration could be varied (but based on the IGF-1 data, 3 mg DNA/ml in 5% PVP in saline should be the best formulation) .
• the quality of the formulation is characterized by pH, DNA concentration, osmolality, and gel electrophoresis.
• the DNA concentration could be varied from 0.1-5 mg/ml.
• the pH may be varied from 3-5, osmolality may be 250 - 400 mOsm.
• the murine studies are predictive of the response of Human tumors to therapy using a plasmid construct encoding the human IFN alpha gene sequence such as that depicted in SEQ ID NO: 10, 11 or 12.
• a patient in need of anti-cancer therapy is injected with up to 3mg of plasmid formulation at daily intervals.
• the plasmid formulation may contain INF alpha plasmid alone or optionally a mixture of IFN-alpha encoding plasmid and an additional plasmid coding for a cytokine.
• the preffered cytokine is IL-12.
• the treatments are continued and the patient monitored as is the usual practice for anti-cancer chemotherapeutic regimes.

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