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WO2000040273A2 - Traitement de maladies virales a l'aide d'un polynucleotide exprimant l'interferon omega - Google Patents

Traitement de maladies virales a l'aide d'un polynucleotide exprimant l'interferon omega Download PDF

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Publication number
WO2000040273A2
WO2000040273A2 PCT/US1999/030843 US9930843W WO0040273A2 WO 2000040273 A2 WO2000040273 A2 WO 2000040273A2 US 9930843 W US9930843 W US 9930843W WO 0040273 A2 WO0040273 A2 WO 0040273A2
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Prior art keywords
polynucleotide
construct
seq
polypeptide
amino acids
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PCT/US1999/030843
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WO2000040273A8 (fr
WO2000040273A3 (fr
Inventor
Suezanne Parker
Holly Horton
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Vical Incorporated
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Publication of WO2000040273A3 publication Critical patent/WO2000040273A3/fr
Publication of WO2000040273A8 publication Critical patent/WO2000040273A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/215—IFN-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to treatment of infectious diseases in mammals.
  • the present invention provides methods of treating viral infections in a mammal by administering a polynucleotide construct comprising a polynucleotide encoding IFN ⁇ .
  • interferon Treatment of infectious diseases with an interferon (IFN) has traditionally involved repeat injections of large doses of recombinant protein.
  • the interferons (IFNs) have been divided into two classes, type I IFN and type II IFN, on the basis of antigenicity, biochemical properties, and producer cell.
  • IFN ⁇ , IFN ⁇ , IFN ⁇ , and IFN ⁇ are type I interferons and bind to the same ⁇ / ⁇ receptor, whereas IFN ⁇ is a type II interferon and binds to the ⁇ receptor (Petska, S., Seminars in
  • IFN ⁇ and IFN ⁇ are naturally expressed in many cells upon viral infection, whereas the Type II IFN, IFN ⁇ , is produced by activated T lymphocytes and natural killer (NK) cells.
  • IFN ⁇ is a naturally occurring protein which is produced by cells in the body to stimulate the immune system to respond to viral infections.
  • IFN ⁇ systemic protein therapy has been used to treat certain types of cancer and infectious diseases.
  • Recombinant IFN ⁇ polypeptide is approved for use in humans for hairy cell leukemia, AIDS -related Kaposi's sarcoma, malignant melanoma, chronic hepatitis B and C, chronic myleogenous leukemia, and condylomata acuminata (Baron, S. etal.,JAMA 266: 1315 (1991)).
  • IFN ⁇ has never been used for the treatment of infectious diseases, even in the form of a recombinant protein. IFN ⁇ was discovered independently by three different groups in 1985 (Capon, D.J., et al., Mol. Cell. Biol. 5: 768-779 (1985), Feinstein, S. et al, Mol. Cell. Biol 5:510 (1985); and Hauptmann and
  • IFN ⁇ is encoded by a single functional gene. IFN ⁇ genes are believed to be present in most mammals, but have not been found in dogs or mice. The mature IFN ⁇ polypeptide is 172 amino acids and shares 60% sequence homology with the human IFN ⁇ 's. Due to the sequence similarity with IFN ⁇ , IFN ⁇ was originally considered to be a member of a subfamily of IFN ⁇ , and was originally termed EFN ⁇ - ⁇ .
  • IFN ⁇ is a significant component ( « 10%) of human leukocyte derived interferon, the natural mixture of IFN produced after viral infection (Adolf, G., et al., Virology 775:410 (1990)). IFN ⁇ has been demonstrated to bind to the same ⁇ / ⁇ receptor as IFN ⁇ (Flores, I., et al, J. Biol. Chem. 266: 19875-19877 (1991)), and to share similar biological activities with IFN ⁇ , including anti- proliferative activity against tumor cells in vitro (Kubes, M. et al, J. Interferon Research 14:51 (1994) and immunomodulatory activity (Nieroda, et al, Mol. Cell Differentiation 4: 335-351 (1996)). Summary of the Invention
  • the present invention is broadly directed to treatment of an infectious disease by administering into a tissue of a mammal a polynucleotide construct comprising a polynucleotide encoding a cytokine, or an active variant thereof.
  • the polynucleotide construct is incorporated into the cells of the mammal, and a therapeutically effective amount of a cytokine is produced.
  • the present invention provides a method of treating a viral infection in a mammal, comprising administering to said mammal a polynucleotide construct comprising a polynucleotide selected from the group consisting of (a) a polynucleotide that hybridizes under stringent conditions to the nucleotide sequence of SEQ ED NO:l or the complement thereof, wherein said polynucleotide encodes a polypeptide having antiviral activity, (b) a polynucleotide that encodes a polypeptide comprising an amino acid sequence which, except for at least one but no more than 20 amino acid substitutions, deletions, or insertions, is identical to amino acids -23 to 172 of SEQ ED NO:2, wherein said polypeptide has antiviral activity; (c) a polynucleotide that encodes a polypeptide comprising an amino acid sequence which, except for at least one but no more than 20 amino acid substitutions, deletions, or insertions,
  • the present invention provides a method for treating Hepatitis B and Hepatitis C.
  • the present invention provides the first description of treating infectious diseases using EFN ⁇ -encoding plasmid DNA.
  • the procedure described herein has several important advantages.
  • IFN ⁇ plasmid DNA the IFN ⁇ is produced by cells in the body at lower levels for a sustained period.
  • This delivery profile allows for less frequent administration, compared to the frequent high doses required for interferon protein, and thereby minimize the side effects while maintaining the therapeutic benefits of protein delivery.
  • administration of plasmid vectors have not been found to induce significant toxicity or pathological immune responses as demonstrated in mice, pigs and monkeys (Parker, S. E., et al, Human Gene Ther.
  • Figure 1 shows the plasmid map of VR4151 (SEQ ID NO:3).
  • the cytomegalovirus immediate early gene promoter enhancer and 5' untranslated sequences (5 ' UTR + intron A) drive the expression of the h-LFN ⁇ coding sequence.
  • the transcriptional terminator region includes a polyadenylation and termination signal derived from the rabbit ⁇ -globin gene.
  • Figure 2 shows the pharmacokinetics of hEF ⁇ in nude mice following intramuscular (i.m.) injection of VR4151. Mice were injected i.m. with 100 ⁇ g of plasmid.
  • Figure 3 shows the dose response to a single i.m. administration of NR4151. Mice were injected with either 200 ⁇ g, 100 ⁇ g or 10 ⁇ g bilaterally or 100 ⁇ g unilaterally. Control mice received injections of 100 ⁇ g NR1055.
  • Figure 4 shows the pharmacokinetics of IF ⁇ in nude mice. Mice were i.m. inj ected bilaterally on days 0 and 14 with 100 ⁇ g NR4151. Control mice were injected with 100 ⁇ g NR1055.
  • Figures 5A-B show the pharmacokinetics of IF ⁇ in rats.
  • rats received a single i.m. injection of either 0.1 mg or 1 mg ofNR4151.
  • rats received three consecutive i.m. injections of 1 mg NR4151.
  • the present invention is directed to treating a viral infection by administering in vivo, into a tissue of a mammal, a polynucleotide construct comprising a polynucleotide encoding a cytokine, or an active variant thereof.
  • the polynucleotide construct is incorporated into the cells of the mammal in vivo, followed by production of a therapeutically effective amount of cytokine.
  • Combinations of cytokine-encoding polynucleotides can also be administered.
  • the present invention provides a method of treating a viral infection in a mammal, comprising administering to said mammal, a polynucleotide construct comprising a polynucleotide which encodes IF ⁇ , or an active variant thereof, wherein said polynucleotide is expressed in vivo in an amount effective to treat the viral infection.
  • mammal is intended to encompass a singular "mammal” and plural “mammals,” and includes, but is not limited to, primate mammals such as human, apes, monkeys, orangutans, and chimpanzees; canine mammals such as dogs and wolves; feline mammals such as cats, lions, and tigers; equine mammals such as horses, donkeys, deer, zebras, and giraffes; and bears.
  • the mammal is a human subject.
  • treatment is meant reduction in symptoms, reduction in viral load, reduction in the rate of viral replication, and/or no worsening of symptoms, viral load, or viral replication over a specified period of time.
  • the polynucleotide construct comprising a polynucleotide which encodes IFN ⁇ is delivered along with one or more polynucleotide constructs comprising a polynucleotide which encodes a different cytokine.
  • cytokine refers to polypeptides, including, but not limited to, interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, E -6, IL-7, EL-8, EL-9, IL-10, IL-11, IL-12, EL-13, EL-14, IL-15, IL-16, BL-17, and EL- 18), ⁇ interferons (e.g., IFN ⁇ ), ⁇ interferon (IFN ⁇ ), ⁇ interferons (e.g., EFN ⁇ ), ⁇ interferons (e.g.,
  • EFN ⁇ EFN ⁇
  • FN ⁇ ⁇ interferon
  • CSFs colony stimulating factors
  • GMCSF granulocyte-macrophage colony stimulating factor
  • TGF transforming growth factor
  • IGFs insulin-like growth factors
  • a polynucleotide sequence encoding IFN ⁇ is shown in SEQ ID NO:l .
  • a full length IFN ⁇ amino acid sequence is shown as amino acids -23 to 172 of SEQ ID NO:2.
  • a mature IFN ⁇ amino acid sequence is shown as amino acids 1 to 172 of SEQ ID NO:2.
  • IFN ⁇ polypeptides include the sequences encoding the complete EFN ⁇ and the mature IFN ⁇ proteins set forth in U.S. Patent Nos. 4,530,901; 4,695,543; 4,695,623; 4,748,233; 4,892,743; 4,897,471; 4,973,479; 4,975,276; and 5,098,703; and in Pestka, S., Methods Enzymol. 119: 3-14 (1986); Hughes, A.L., J. Mol. Evol. 41: 539-548 (1995); and
  • cytokines can be assayed according to the antiviral assay described herein.
  • Amino acids that are critical for cytokine activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith etal,
  • the present invention further relates to using variants of the cytokine- encoding polynucleotide, which encode portions, analogs or derivatives of the cytokine.
  • Variants may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the cytokine or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • aromatic amino acids that can be conservatively substituted for one another include phenylalanine, tryptophan, and tyrosine.
  • Hydrophobic amino acids that can be conservatively substituted for one another include leucine, isoleucine, and valine.
  • Polar amino acids that can be conservatively substituted for one another include glutamine and asparagine.
  • Basic amino acids that can be conservatively substituted for one another include arginine, lysine, and histidine.
  • Acidic amino acids that can be conservatively substituted for one another include aspartic acid and glutamic acid.
  • Small amino acids that can be conservatively substituted for one another include alanine, serine, threonine, methionine, and glycine.
  • mutated proteins or muteins
  • Gayle and coworkers conducted an extensive mutational analysis of the human cytokine IL-l ⁇ . They used random mutagenesis to generate over 3,500 individual EL-l ⁇ mutants with an average of 2.5 amino acid changes per mutein over the entire length of the molecule.
  • a polynucleotide sequence used in the present invention can encode a polypeptide having one to twenty amino acid substitutions, deletions or insertions, either from natural mutations or human manipulation, relative to the full length or mature IFN ⁇ or IFN ⁇ . Preferably, no more than one to fifteen substitutions, deletions or insertions are present, relative to the full length or mature IFN ⁇ or IFN ⁇ (excluding the signal sequence). More preferably, no more than one to ten substitutions, deletions or insertions are present. Still more preferably, no more than one to five substitutions, deletions or insertions are present.
  • Polynucleotides used in the present invention also include those that hybridize under stringent conditions to polynucleotides encoding a cytokine, or the complement thereof, which encode a functional variant of the cytokine.
  • stringent conditions is intended a hybridization by overnight incubation at 42 °C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by repeatedly washing the filters (at least three times) in O.lx SSC and 0.1% sodium dodecyl sulfate (w/v) for 20 minutes at about 65 °C.
  • the present invention provides a method for treating a viral infection in a mammal, comprising administering to the mammal a construct comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or the complement thereof, wherein said polynucleotide encodes a polypeptide having antiviral activity; (b) a polynucleotide that encodes a polypeptide comprising an amino acid sequence which, except for at least one but no more than 20 amino acid substitutions, deletions, or insertions, is identical to amino acids -23 to 172 of SEQ ID NO:2, wherein said polypeptide has antiviral activity; (c) a polynucleotide that encodes a polypeptide comprising an amino acid sequence which, except for at least one but no more than 20 amino acid substitutions, deletions, or insertions, is identical to amino acids 1 to
  • a "polynucleotide construct” is a polynucleotide molecule that carries genetic information for encoding one or more cytokines.
  • the polynucleotide material delivered to the cells in vivo can take any number of forms. It can contain the entire sequence or only a functionally active variant of a cytokine gene.
  • active variant is intended a variant of a cytokine that displays the antiviral activity of the mature or full length cytokine.
  • a full length IFN ⁇ is set forth in amino acids -23 to 172 of SEQ ED NO:2.
  • the corresponding mature IFN ⁇ is set forth in amino acids 1 to 172 of SEQ ED NO:2.
  • Useful fragments of IFN ⁇ are shown as amino acids 21 to 172, 41 to 172, 61 to 172 and 86 to 172 of SEQ ID NO:2.
  • Example 3 Assays of antiviral activity in vitro are well known to those of ordinary skill in the art.
  • one antiviral assay that can be used is outlined in Example 3, below. Briefly, plasmids expressing the EFN ⁇ variant are transfected into cells, such as UM449 cells. The supernatant from the transfected cells is added onto cells which have been infected with Murine Encephalomyocarditis Virus (EMCV) at Multiplicity of Infection (MOI) of approximately 0.04. The viral cytopathic effect in infected cells treated with the supernatant is compared to untreated infected cells. A decrease in viral cytopathic effect in treated cells indicates that the IFN ⁇ or a variant thereof is active.
  • EMCV Murine Encephalomyocarditis Virus
  • MOI Multiplicity of Infection
  • the polynucleotide construct comprises at least one polynucleotide (e.g., DNA, RNA, ribozyme, phosphorothioate, or other modified nucleic acid) encoding one or more cytokines.
  • the polynucleotide can be provided in linear, circular (e.g. plasmid), or branched form; and double-stranded or single-stranded form.
  • the polynucleotide can involve a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond as in peptide nucleic acid (PNA)).
  • PNA peptide nucleic acid
  • the choice of polynucleotide encoding a cytokine will depend on the desired kinetics and duration of expression. When long term delivery of the polynucleotide construct is desired, the preferred polynucleotide is DNA.
  • the preferred polynucleotide is mR A.
  • RNA will be rapidly translated into polypeptide, but will be degraded by the target cell more quickly than DNA.
  • circular DNA molecules will persist longer than single-stranded polynucleotides, and they will be less likely to cause insertional mutation by integrating into the target genome.
  • the polynucleotide sequence encoding one or more cytokines is RNA.
  • the RNA is messenger RNA (mRNA).
  • mRNA messenger RNA
  • Methods for introducing RNA sequences into mammalian cells is described in U. S . Patent No. 5,580,859, which is herein incorporated by reference.
  • a viral alphavector a non-infectious vector useful for administering RNA, may be used to introduce RNA into mammalian cells. Methods for the in vivo introduction of alphaviral vectors to mammalian tissues are described in Altman-Hamamdzic, S., et al, Gene Therapy 4: 815-822 (1997), which is herein incorporated by reference.
  • the polynucleotide sequence encoding one or more cytokines is DNA.
  • a promoter is preferably operably linked to the polynucleotide encoding a cytokine.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • Other transcription control elements, besides a promoter, can be included in the polynucleotide construct to direct cell-specific transcription of the DNA.
  • An operable linkage is a linkage in which a polynucleotide sequence encoding a cytokine is connected to one or more regulatory sequence in such a way as to place expression of the cytokine sequence under the influence or control of the regulatory sequence(s).
  • Two DNA sequences are operably linked if induction of promoter function results in the transcription of mRNA encoding the desired polypeptide and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the polypeptide, antisense RNA, or (3) interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably linked to a DNA sequence if the promoter was capable of effecting transcription of that DNA sequence.
  • the polynucleotide construct is a circular or linearized plasmid containing non-infectious, nonintegrating nucleotide sequence.
  • a linearized plasmid is a plasmid that was previously circular but has been linearized, for example, by digestion with a restriction endonuclease.
  • the polynucleotide sequence encoding a cytokine may comprise a sequence which directs the secretion of the polypeptide.
  • Noninfectious means that the polynucleotide construct does not infect mammalian cells.
  • the polynucleotide construct can contain functional sequences from non-mammalian (e.g., viral or bacterial) species, but the construct does not contain functional non-mammalian nucleotide sequences that facilitate infection of the construct into mammalian cells.
  • Nonintegrating means that the polynucleotide construct does not integrate into the genome of mammalian cells.
  • the construct can be a non- replicating DNA sequence, or specific replicating sequences genetically engineered to lack the ability to integrate into the genome.
  • the polynucleotide construct does not contain functional sequences that facilitate integration of the cytokine- encoding polynucleotide sequence into the genome of mammalian cells.
  • the polynucleotide construct is assembled out of components where different selectable genes, origins, promoters, introns, 5' untranslated (UT) sequence, terminators, polyadenylation signals, 3 ' UT sequence, and leader peptides, etc. are put together to make the desired vector.
  • the precise nature of the regulatory regions needed for gene expression can vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non- translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the TATA box, capping sequence, CAAT sequence, and the like, with those elements necessary for the promoter sequence being provided by the promoters of the invention.
  • Such transcriptional control sequences can also include enhancer sequences or upstream activator sequences, as desired.
  • the polynucleotide construct can be an expression vector.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the polypeptide coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRS) from retrovirases, e.g., RS V, HTLVI, HIVI, MPS V and the immediate early promoter of the cytomegalovirus (CMV IEP). However, cellular elements can also be used (e.g.
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), VR1012, VR1055, and pcDNA3 (Invitrogen, San Diego, CA). All forms of DNA, whether replicating or non- replicating, which do not become integrated into the genome, and which are expressible, are within the methods contemplated by the invention.
  • the vector containing the DNA sequence (or the corresponding RNA sequence) which can be used in accordance with the invention can be a eukaryotic expression vector.
  • Techniques for obtaining expression of exogenous DNA or RNA sequences in a host are known. See, for example, Korman et al, Proc. Nat. Acad. Sci. (USA) 54:2150-2154 (1987), which is herein incorporated by reference.
  • Secretion of a cytokine from a cell can be facilitated by a leader or secretory signal sequence.
  • a leader or secretory signal sequence In a preferred embodiment, either the native leader sequence of a cytokine is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the peptide that is operably linked to it.
  • a heterologous mammalian leader sequence, or a functional derivative thereof may be used.
  • the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator or mouse ⁇ -glucuronidase.
  • the natural leader sequence of EFN ⁇ is used.
  • the polynucleotide construct comprises a sequence encoding amino acids -23 to -1 of SEQ ID NO:2 directly 5' to the sequence encoding amino acids 86 to 172 of SEQ ID NO:2.
  • the native leader sequence will direct secretion of the polypeptide comprising amino acids 86 to 172 of SEQ ED NO:2.
  • the leader sequence is cleaved off, leaving only the polypeptide comprising amino acids 86 to 172 of SEQ ED NO:2.
  • a single polynucleotide construct containing more than one polynucleotide sequence encoding one or more molecules may be administered.
  • a single polynucleotide construct containing one polynucleotide encoding an IFN ⁇ and another polynucleotide encoding an additional cytokine or an immunomodulatory molecule, i.e., MHC class I antigen, viral antigen, and co-stimulatory molecule can be administered.
  • more than one polynucleotide construct each containing polynucleotide sequences encoding one or more molecules may be co- administered or sequentially administered.
  • two polynucleotide constructs can be administered where one gene product enhances anti-viral efficacy of the other gene product.
  • An IFN ⁇ -expressing polynucleotide construct can be co-injected with a polynucleotide construct encoding a different cytokine.
  • one or more plasmids could be administered initially and other plasmid(s) could be administered subsequently at various time intervals.
  • each polynucleotide encoding a polypeptide will be operably linked to a separate promoter.
  • the polynucleotides encoding polypeptides may be operably linked to the same promoter in order to form a polycistronic transcription unit wherein each sequence encoding a polypeptide is separated by translational stop and start signals. Transcription termination is also shared by these sequences.
  • the single polynucleotide construct containing more than one polynucleotide encoding a polypeptide is RNA
  • the polynucleotide construct is delivered as naked polynucleotide.
  • naked is meant that the polynucleotide construct is free from association with any delivery vehicle known in the art that can act to facilitate entry into cells, for example, from transfection- facilitating proteins, viral particles, liposomes, cationic Hpids, and calcium phosphate precipitating agents.
  • the polynucleotide construct is complexed with a cationic vehicle, which comprises a cationic compound, such as cationic lipids, cationic peptides, cationic proteins, cationic polymers, and mixtures thereof.
  • a cationic compound such as cationic lipids, cationic peptides, cationic proteins, cationic polymers, and mixtures thereof.
  • a preferred cationic compound is a cationic lipid.
  • One or more cationic lipids can be complexed with the polynucleotide construct by ionic interaction. Generally, the complex then contacts the cell membrane and is transfected into the cell. This transfection mechanism is referred to as "lipofection,” and is a highly efficient transfection procedure (Feigner et al, Proc. Natl Acad. Sci. USA 54: 7413 -7417, (Nov. 1987); and Feigner et al,
  • lipid refers to a synthetic or naturally occurring compound that possesses both a lipophilic region and a polar region, commonly referred to as a head group.
  • Preferred cationic compounds are cationic lipids. Cationic lipids are described in U.S. Pat. Nos. 4,897,355,
  • cationic lipids comprise structural features that may be present in a variety of core molecular classes.
  • U.S. Patent No. 5,676,954 reports on the injection of genetic material, complexed with cationic lipid carriers, into mice.
  • Examples of cationic lipids are 5-carboxyspermylglycine dioctadecylamide
  • Cationic cholesterol derivatives are also useful, including ⁇ 3 ⁇ -[N- N',N'-dimethylamino)ethane]-carbomoyl ⁇ -cholesterol (DC-Choi).
  • Dimethyldioctdecyl-ammonium bromide (DDAB), N-(3 -aminopropyl)-N,N-(bw- (2-tetradecyloxyethyl))-N-methyl-ammonium bromide (PA-DEMO), N-(3- aminopropyl)-N,N-(bw-(2-dodecyloxyethyl))-N-methyl-ammonium bromide (PA- DELO),N,N,N-tr/5-(2-dodecyloxy)ethyl-N-(3-amino)propyl-ammonium bromide (PA-TELO), and N 1 -(3-aminopropyl)((2-dodecyloxy)ethyl)-N 2 -(2- dodecyloxy)ethyl-l-piperazinaminium bromide (GA-LOE-BP) can also be employed in the present invention
  • Non-diether cationic lipids such as DL-l,2-dioleoyl-3- dimethylaminopropyl- ⁇ -hydroxyethylammonium (DORI diester), l-O-oleyl-2- oleoyl-3-dimethylaminopropyl- ⁇ -hydroxyethylammonium(DORI ester/ether), and their salts promote in vivo gene delivery.
  • Preferred cationic lipids comprise groups attached via a heteroatom attached to the quaternary ammonium moiety in the head group.
  • a glycyl spacer can connect the linker to the hydroxyl group.
  • Preferred cationic lipids are 3, 5-(N,N-dilysyl)-diaminobenzoyl-3-(DL-l,2- dioleoyl-dimethylaminopropyl- ⁇ -hydroxyethylamine) (DLYS-DABA-DORI diester), 3,5-(N,N-di-lysyl)diamino-benzoylglycyl-3-(DL-l,2-dioleoyl- dimethylaminopropyl- ⁇ -hydroxyethylamine) (DLYS-DAB A-GLY-DORI diester), and ( ⁇ )-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-l- propaniminium bromide (DMREE).
  • DLYS-DABA-DORI diester 3,5-(N,N-di-lysyl)diamino-benzoylglycy
  • GAP-DLRIE ( ⁇ )-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-l-propaniminium bromide
  • DMRIE-derived cationic lipids that are useful for the present invention are ( ⁇ )-N-(3 -aminopropyl)-N,N-dimethyl-2, 3 -(bw-decyloxy)- 1 - propanaminium bromide (GAP-DDREE), ( ⁇ )-N-(3-aminopropyl)-N,N-dimethyl- 2,3-(b/5-tetradecyloxy)-l-propanaminium bromide (GAP-DMREE), ( ⁇ )-N-((N H - methyl)-N'-ureyl)propyl-N,N-dimethyl-2,3-bis(tetradecyloxy)- 1 -propanaminium bromide (GMU-DMRIE), ( ⁇ )-N-(2-hydroxyethyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-l -propanaminium bromide (
  • the lipids of the lipid-containing formulation can comprise a cationic lipid alone, or further comprise a neutral lipid such as cardiolipin, phosphatidylcholine, phosphatidylethanolamine, dioleoylphosphatidylcholine, dioleoylphosphatidyl- ethanolamine,l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), sphingomyelin, and mono-, di- or tri-acylglycerol.
  • a neutral lipid such as cardiolipin, phosphatidylcholine, phosphatidylethanolamine, dioleoylphosphatidylcholine, dioleoylphosphatidyl- ethanolamine,l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), sphingomyelin, and mono-, di- or tri-acylgly
  • lipid-containing formulations can further comprise a lyso lipid (e.g. , lyso-phosphatidylcholine, lysophosphatidyl-ethanolamine, or a lyso form of a cationic lipid).
  • a lyso lipid e.g. , lyso-phosphatidylcholine, lysophosphatidyl-ethanolamine, or a lyso form of a cationic lipid.
  • the cationic lipid is ( ⁇ )-N-(2-hydroxyethyl)-N,N-dimethyl-2,3 - bis(tetradecyloxy)- 1 -propaniminium bromide (DMREE) and the neutral lipid is 1,2- dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) such that the mass ratio of polynucleotide construct to lipid is from about 10: 1 and about 0.5: 1. More preferably, the mass ratio of polynucleotide construct to lipid is from about 5:1 and about 1:1. Still more preferably, the mass ratio of polynucleotide construct to lipid is about 5:1.
  • DMRIE/DOPE has been shown to be effective for both in vitro (Feigner et al.,J. Biol. Chem.269:2550-2561, 1994) and in vivo transfection (Stopeck et al., J. Clin. Oncol. 15:341-349, 1997 and Rubin et al, Gene Ther.
  • Lipid-containing pharmaceutical compositionforuse in a complex with the polynucleotide construct of the present invention can also comprise cationic lipid together with an effective amount of a lysophosphatide.
  • the lysophosphatide can have a neutral or a negative head group. Lysophosphatidylcholine and lysophosphatidyl-ethanolamine are preferred, and 1 -oleoyl lysophosphatidylcholine is particularly preferred.
  • Lysophosphatide lipids are advantageously present in the lipid-containing formulation in a 1 :2 ratio of lysolipid to cationic lipid.
  • Lyso forms of a cationic lipid can also be used to promote polynucleotide delivery. These lyso forms are advantageously present in effective amounts up to about one-third of the total cationic lipid in the lipid-containing formulations.
  • the cationic lipid can be present at a concentration of between about 0.1 mole % and about 100 mole %, preferably about 5 mole % and 100 mole %, and most preferably between about 20 mole % and 100 mole %, relative to other compounds present in the formulation.
  • the neutral lipid can be present in a concentration of between zero and about 99.9 mole %, preferably zero and about 95 mole %, and most preferably zero and about 80 mole %.
  • the quantity of the positively charged component must exceed that of the negatively charged component.
  • the negatively charged lipid can be present at between zero and about 49 mole %, and preferably between zero and about 40 mole %.
  • Cholesterol or a similar sterol can be present at between zero to about 80 mole %, and preferably zero and about 50 mole %.
  • the polynucleotide to be delivered can be solubilized in a buffer prior to mixing with lipid vesicles.
  • Suitable buffers include, for example, phosphate buflfered saline (PBS), normal saline, Tris buffer, and sodium phosphate vehicle ( 100- 150 mM preferred).
  • Insoluble polynucleotides can be solubilized in a weak acid or base, and then diluted to the desired volume with a neutral buffer such as PBS.
  • the pH of the buffer is suitably adjusted, and moreover, a pharmaceutically acceptable additive can be used in the buffer to provide an appropriate osmolarity within the lipid vesicle.
  • a lipid solution comprising at least one amphipathic lipid can spontaneously assemble to form primary lipid vesicles, heterogeneous in size. Therefore, according to a preferred method, the lipids of the lipid-containing formulation, comprising at least one cationic lipid, are prepared by dissolution in a solvent such as chloroform and the mixture is evaporated to dryness as a film on the inner surface of a glass vessel. On suspension in an aqueous solvent, the amphipathic lipid molecules assemble themselves into primary lipid vesicles. These primary lipid vesicles are reduced to a selected mean diameter by means of a freeze-thaw procedure.
  • Vesicles of uniform size can be formed prior to drug delivery according to methods for vesicle production known to those in the art; for example, the sonication of a lipid solution as described by Feigner et al, Proc. Natl. Acad. Sci. USA 54:7413-7417 (1987) and U.S. Pat. No. 5,264,618, which are herein incorporated by reference.
  • the constructs may be delivered to any tissue, including, but not limited to, muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, or connective tissue.
  • the construct is delivered to muscle.
  • the muscle may be skeletal or cardiac. Most preferably, the construct is delivered to skeletal muscle.
  • the polynucleotide construct is delivered to the interstitial space of tissues.
  • Interstitial space comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels.
  • the polynucleotide construct of the present invention can be administered by any suitable route of administration, including intramuscularly, subcutaneously, intravenously, transdermally, intranasally, by inhalation, or transmucosally (i.e., across a mucous membrane).
  • the pharmaceutical composition of the present invention can by administered by any suitable route, including intramuscularly, into a cavity (e.g., intraperitoneally), subcutaneously, intravenously, transdermally, intranasally, by inhalation, or transmucosally (i.e., across a mucous membrane).
  • Any mode of administration can be used so long as the mode results in the expression of one or more cytokines in an amount sufficient to decrease the viral infection of a mammal.
  • This includes needle injection, catheter infusion, biolistic injectors, particle accelerators (i.e., "gene guns”, pneumatic "needleless” injectors, e.g., Med-E-Jet (Vahlsing, H. et al, J. Immunol. Methods 171:11-22 (1994)),
  • Pigjet (Schrijver, R. etal, Vaccine 15: 1908-1916 (1997)), Biojector (Davis, H. etal, Vaccine 12: 1503-1509 (1994); Gramzinski, R. etal, Mol. Med. 4: 109-118 (1998))), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery.
  • osmotic pumps e.g., Alza minipumps
  • oral or suppositorial solid (tablet or pill) pharmaceutical formulations e.g., suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery.
  • the preferred mode is injection.
  • Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the mammal, the precise condition requiring treatment and its severity, and the route of administration.
  • the polynucleotide construct is administered as a pharmaceutical composition.
  • the pharmaceutical composition can be formulated according to known methods for preparing pharmaceutical compositions, whereby the substance to be delivered is combined with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their preparation are described, for example, in Remington's Pharmaceutical Sciences, 16 th Edition, A. Osol, Ed., Mack Publishing Co., Easton, PA (1980), and Remington's Pharmaceutical Sciences, 19 th Edition, A.R. Gennaro, Ed., Mack Publishing Co., Easton, PA (1995).
  • the pharmaceutical composition can be in the form of an emulsion, gel, solution, suspension, or other form known in the art.
  • it can contain one or more lipids as described above.
  • the pharmaceutical composition can also contain pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives.
  • Administration of pharmaceutically acceptable salts of the polynucleotides described herein is preferred.
  • Such salts can be prepared from pharmaceutically acceptable non-toxic bases including organic bases and inorganic bases. Salts derived from inorganic bases include sodium, potassium, lithium, ammonium, calcium, magnesium, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, basic amino acids, and the like.
  • aqueous pharmaceutical compositions used in vivo sterile pyrogen- free water is preferred.
  • Such formulations will contain an effective amount of the substance together with a suitable amount of vehicle in order to prepare pharmaceutically acceptable compositions suitable for administration to a human or animal.
  • a pharmaceutical composition can be in solution form, or alternatively, in lyophilized form for reconstitution with a suitable vehicle, such as sterile, pyrogen- free water. Both liquid and lyophilized forms will comprise one or more agents, preferably buffers, in amounts necessary to suitably adjust the pH of the injected solution.
  • the container in which the pharmaceutical formulation is packaged prior to use can comprise a hermetically sealed container enclosing an amount of the lyophilized formulation or a solution containing the formulation suitable for a pharmaceutically effective dose thereof, or multiples of an effective dose.
  • the pharmaceutical formulation is packaged in a sterile container, and the hermetically sealed container is designed to preserve sterility of the pharmaceutical formulation until use.
  • the container can be associated with administration means and or instruction for use.
  • the polynucleotide constructs are delivered with additional, non-cytokine antiviral agents.
  • Antiviral agents include, but are not limited to, protease inhibitors, nucleoside RT inhibitors, non-nucleoside RT inhibitors, fusion/binding inhibitors, and pyrophosphate analogue RT inhibitors.
  • Viral diseases which can be treated using the method of the present invention include chickenpox, shingles, rubella, influenza, rubeola, mumps, yellow fever, AEDS, mononucleosis, rabies, acute viral gastroenteritis, poliomyelitis, subacute sclerosing panencephalitis, encephalitis, Colorado tick fever, pharyngitis, croup, bronchiolitis, viral pneumonia, pleurodynia, aseptic meningitis, keratitis, conjunctivitis, viral leukemias, rabies, polio, myocarditis, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E; and any infections caused by adenoviruses, coxsackieviruses, parainfluenza viruses, respiratory syncytial virus, reovirus, cytomegalovirus, Epstein-Barr virus, herpes simple
  • the method of the present invention is used to treat either Hepatitis B or Hepatitis C.
  • the polynucleotide construct encoding IFN ⁇ is administered with a polynucleotide encoding another cytokine to treat either Hepatitis B or Hepatitis C.
  • the polynucleotide encoding another cytokine may be part of the same construct encoding BFN ⁇ , or it may be part of a different construct.
  • the other cytokine is IFN ⁇ , EL-2, or IL-12.
  • other antiviral agents are administered with the polynucleotide construct encoding EFN ⁇ to treat either Hepatitis B or Hepatitis
  • antiviral agents include levamisole, thymosin, thymus humoral factor- gamma 2, phosphonoformic acid trisodium, prednisone, interferon gamma, thymosin, levamisole, vidarabine, acyclovir, suramin, foscarnet, didanosine, and fialuridine; and nucleoside analogs such as penciclovir, ganciclovir, zidovudine, ribavirin, famciclovir, lamivudine (BioChem Pharma, Epivir/HB V), lobucavir, and adefovir dipivoxil.
  • nucleoside analogs such as penciclovir, ganciclovir, zidovudine, ribavirin, famciclovir, lamivudine (BioChem Pharma, Epivir/HB V), lobucavir, and adefovir dipivoxil.
  • the present inventors have evaluated the therapeutic use of IFN- ⁇ expressing plasmid DNA injected intramuscularly for the treatment of infectious disease. Described herein are 1) the in vitro characterization of the anti-viral biological activity of IFNs delivered by plasmid DNA; 2) in vivo expression (serum levels) of IFN ⁇ following intramuscular administration of EFN ⁇ plasmid
  • IFN ⁇ plasmid DNA vectors used herein have been demonstrated to have potent anti- viral activity in vitro.
  • the intramuscular injection of IFN ⁇ plasmid DNA result in systemic levels of the IFN protein.
  • therapeutic levels of IFN can be achieved following intramuscular administration of an EFN plasmid DNA in a very aggressive disease setting. Therefore, IFN plasmid DNA therapy is useful in the treatment of infectious disease. Examples
  • Two basic eukaryotic expression plasmid vectors termed VR1012 and VR1055, can be used in the construction of interferon plasmids. These two blank plasmids differ only in transcriptional termination sequences.
  • the backbone of both plasmids is derived from pUC19, with the beta-lactamase (ampicillin resistance) gene replaced by the aminoglycoside acetyltransferase (kanamycin resistance) gene from pET9a (Novagen, Madison, WI). Both plasmids direct eukaryotic gene expression from a cassette containing the human cytomegalovirus immediate early I gene promoter/enhancer, 5' untranslated sequence, and intron A.
  • a cloning polylinker for insertion of polypeptide coding sequences.
  • the transcriptional terminator region from the bovine growth hormone gene. This region contains 3 ' untranslated sequences and polyadenylation and termination signals.
  • the transcriptional terminator region is derived from the rabbit beta-globin gene, and contains the polyadenylation and termination signals, but lacks any 3 ' untranslated sequences.
  • Plasmid VR4101 (murine interferon a (mIFN ⁇ )) was constructed by cloning the murine interferon a cDNA into the vector VR1012 vector.
  • the cDNA was obtained by amplifying the coding sequence from the plasmid RSV-al (Kelly,
  • Plasmid VR4111 was constructed by transferring the coding sequences from VR4101 to the VR1055 cloning vector.
  • the oligonucleotide primers used for polymerase chain reaction (PCR) were 5'- AACTGCAGATGGCTAGGCTCTGTGCT-3' (SEQ ID NO:4) and 5'-
  • Plasmid VR4102 human interferon a (hlFN ⁇ ) was constructed by cloning the human interferon a gene into the VR1012 vector.
  • the cDNA was obtained by amplifying the coding sequence from human genomic DNA prepared from a fresh blood sample.
  • Plasmid VR4112 was constructed by transferring the coding sequence sequencesfromVR4102to theVR1055 cloningvector. GenomicDNA was isolated using the QIAamp Blood Kit (Qiagen, Inc.).
  • the oligonucleotide primers used for PCR were 5'-AACTGCAGATGGCCTC-GCCCTTTGCT-3' (SEQ ID NO:6) and 5'-CGGGATCCTTATTCCTTC-CTCCTTAATC-3' (SEQ ED NO:7). Reaction conditions were 30 cycles of 94EC for 1 minute (denaturing), 58EC for 2 minutes (annealing), and 72EC for 1 minute (amplification).
  • Plasmid VR4150 (human interferon T (hEFN ⁇ )) was constructed by cloning the human FN ⁇ gene into the VR1012 cloning vector.
  • the cDNA was obtained by amplifying the coding sequence from human genomic DNA prepared from a fresh blood sample.
  • Plasmid VR4151 (SEQ ED NO:3) was constructed by transferring the coding sequence sequences from VR4150 to the NR1055 cloning vector.
  • the oligonucleotide primers used for PCR were 5'- GCTCTAGATGGCCCTCCTGTTCCCT-3 ' (SEQ ID NO: 8) and 5'-GCGG- ATCCTCAAGATGAGCCCAGGTC-3 ' (SEQ ID NO:9). Reaction conditions were 30 cycles of 94EC for 1 minute (denaturing), 58EC for 2 minutes (annealing), and 72EC for 1 minute (amplification).
  • pDNA was transformed into Escherichia coli DH1 OB-competent cells and grown in Terrific Broth (Sambrook, J. et al, in: Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. A.2 (1989)) complemented with 50 mg ml kanamycin in a 1 Liter shaker flask. Cells were harvested by centrifugation at the end of the exponential growth phase (approximately 16 hr), typically yielding 10 grams of biomass net weight per liter. Covalently closed circular pDNA was isolated by a modified lysis procedure (Horn, N.A.
  • the lipid DMREE/DOPE consists of the cationic lipid ( ⁇ )- ⁇ -(2-hydroxyethyl)- ⁇ , ⁇ - dimethyl-2,3-bis(tetradecyloxy)-l-propanaminium bromide (DMREE) and the neutral lipid dioleoylphosphatidylethanolamine (DOPE) at a 1: 1 mohmol ratio
  • An antiviral assay was performed to evaluate the ability of the supernatants from the interferon plasmid DNA-transfected cells to protect murine L929 cells or human A549 cells from infection by murine encephalomyocarditis (EMC) virus (Assay performed at IIT Institute, Chicago, IL). In vitro transfections were performed as described above and supernatants were collected from cells transfected with either VR4151 (hEFN ⁇ ), VR4112 (hlFN ⁇ ), VR4111 (mIFN ⁇ ) or VR1055 (control). Antiviral activity of the supernatants was performed by IIT Research Institute (Chicago, EL).
  • Samples with interferon activity protected the cells from virus infection, resulting in darkly stained cell monolayers.
  • Supernatants from UM449 cells transfected with VR4151, VR4112, or VR4111 had antiviral activity of 30,000, 3,000 or 30 Units ml, respectively, on human A549 cells.
  • supernatants from UM449 cells transfected with VR4151, VR4112, or VR4111 had antiviral activity of 300, 1000 and 30,000 Units/ml, respectively (Table 1) showing species specificity of the hEFNs for human cells and mIFNs for mouse cells.
  • Intramuscular injections and serum analysis were performed using a 300 ⁇ l sterile tuberculin syringe fitted with a 28G 1/2 needle (Becton Dickenson) and a plastic collar cut from a 200 ⁇ l micropipette tip. The collar length was adjusted to limit the needle from penetrating further than 2 mm into the rectus femoris muscle. Serum samples were collected at the indicated time-points and analyzed using a hEFN ⁇ ELISA kit (Alexis, San Diego, CA) which was sensitive to 2 pg/ml.
  • PK single dose pharmacokinetics.
  • nude mice received intramuscular (i.m.) injections on day 0 with 100 ⁇ g of VR4151 or VR1055 (50 ⁇ g/50 ⁇ l per leg, bilateral) in the rectus femoris.
  • Serum samples were collected daily over a two week period and analyzed in the hEFN ⁇ ELISA kit. Serum samples were collected from 4-5 mice per day. Serum levels of IFN ⁇ were found as early as one day after injection (133 pg/ml). Peak serum levels were found on day 7 (648 pg/ml) and expression continued out to day 14 (134 pg/ml), the final time point of the study ( Figure 2).
  • interferon could be detected in the serum after a single i.m. injection of an interferon- encoding plasmid DNA.
  • mice received i.m. injections on day 0 with 200, 100, or 10 ⁇ g of VR4151 or 100 ⁇ g of VR1055 (vector control) (bilaterally, 50 ⁇ g/50 ⁇ l per leg) in the rectus femoris.
  • An additional group of mice received a single injection of 100 ⁇ g of VR4151 (100 ⁇ g/100 ⁇ l) into one rectus femoris muscle. No significant differences in the serum levels of IFN ⁇ were observed following the intramuscular administration of 200 ⁇ g vs. 100 ⁇ g bilaterally vs. 100 ⁇ g administered to a single muscle.
  • Serum samples were collected every 3-4 days and assayed in a hEFN ⁇ ELISA. Cohorts of 5 mice per timepoint were bled from the VR4151 -injected mice. All of the VR1055 -inj ected mice were bled at each timepoint.
  • Rats received a single i.m. injection of either 0.1 mg (in 100 ⁇ l, 50 ⁇ l bilateral) or 1 mg (in 500 ⁇ l, 250 ⁇ l bilateral) of VR4151.
  • An additional group of rats received 3 consecutive i.m. injections 1 mg of VR4151 (in
  • VR4151 was diluted in 150 mM sodium phosphate buffer, pH7.2 for the i.m. injections. Serum samples were collected every 2-3 days and assayed in a hlFN ⁇ ELISA (Alexis, San Diego). Prebleeds of the rats (prior to i.m. injection) were used to determine background hEFN ⁇ levels and were subtracted from the final hlFN ⁇ values obtained at each timepoint after i.m. injection. Each group consisted of 4 rats.
  • NR4151 for 3 consecutive days had an average of 68 pg/ml MF ⁇ in the serum by day 5 after the first i.m. injection and 36 pg/ml hlF ⁇ 4 days later.
  • Example 6 Treatment Regimen with IFN ⁇ Plasmid DNA for Patients with Chronic Hepatitis B or C
  • 1-50 mg, preferably 10-30 mg of EFN ⁇ plasmid DNA in a pharmacologically acceptable carrier is injected to the patients biweekly or monthly.
  • the therapy regimen is continued for a minimum of 24 weeks during which time the patients are monitored for levels of serum alanine aminotransferase, and serum HB s Ag and
  • HBV DNA for HBV patients or HCV RNA in the case of HCV patients.
  • liver biopsies are performed at the end of the treatment period.
  • a successful outcome of the therapy is indicated by a normalization of serum alanine aminotransferase levels, a disappearance or decrease in detectable virus in the patient's serum, and histological improvement in the liver. En some cases, this therapy is used in conjunction with anti-virals such as lamivudine for HBV and ribavirin for HCV.

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Abstract

L'invention concerne un procédé permettant de traiter une maladie virale, consistant à administrer à un mammifère une construction polynucléotidique qui comprend un polynucléotide codant pour IFNφ. La construction polynucléotidique de l'invention peut être administrée sans être associée à des agents facilitant la transfection ou sous forme d'un complexe renfermant au moins un ou plusieurs lipides cationiques.
PCT/US1999/030843 1999-01-08 1999-12-28 Traitement de maladies virales a l'aide d'un polynucleotide exprimant l'interferon omega WO2000040273A2 (fr)

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